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Introduction
1. INTRODUCTION
"Let food be thy medicine and
medicine be thy food", - Hippocrates
Developing countries are being encouraged to diversify their food experts by
developing new products and adding more value to existing ones. Adding value to and
diversifying food exports depends not only on changing production but also processing
systems (John & Antje, 2000). The traditional Indian fruits are very rich in nutritional
parameters and a variety of by-products can also be prepared from them (Singh et al.,
2009).
Fruits play an important role in food and nutritional security as well as
sustainable income to farmers. Fruits account for about 30% of the total production of
horticulture crops. India is the second largest producer of fruits in the world, it account
for 13.0 % of the total world production of fruits. Nevertheless, during the past few
decades, phenomenal developments have taken place in the production technology of
fruits. Fruit production increased 14 times from 5.5 million tonnes in 1952-53 to 76.4
million tonnes in 2011-12. Presently, area under fruit crops is at 6.7 million hectare
with a production of 76.4 million tonnes, which contributes to about 30% share in total
production of horticulture produce. Production of fruit is expected to reach about 115
million tonnes by 2017 (Singh, 2012).
Fruits form an important part of the diet and are usually regarded as good
foods. They are major sources of vitamin C, folic acid and dietary fibre (Brian &
Cameron, 1995). Fruit seeds, common byproducts of the food industry, are generally
discarded despite their potential use as a source of nutrients in the human diet (Lima
et al., 2014). The food industry generates large volumes of wastes and byproducts
from processing fruits to make products such as juices, jellies and other candies. Much
of these materials are composed by seeds, which are normally discarded or used to
make animal feed. Recently, more attention has been focused on the utilisation of food
processing byproducts and wastes, as well as underutilized agricultural products.
Obviously, such use would contribute to maximizing available resources and result in
the production of various new foods (El-Adawy & Taha, 2001).
A seed is considered the propagative portion of plant. It contains a fertilized and
ripen ovule comprising a miniature plant usually accompanied by a supply of food as
endosperm or perisperm. A protective coat is another feature of a seed. Under suitable
conditions, the seed will develop into a plant similar to the one that produced it. The
synthesis of proteins by seeds is important because the utilization of seeds can depend
Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein
Introduction
largely upon the quantity and kinds of protein produced. The nutritive value of such
seed protein is virtually equivalent to milk proteins where as ordinary proteins are
deficient in the essential amino acids, lysine and tryptophan (Inglett, 1972).
Due to their nutritional value, some seeds can be employed as part of the
human diet. Researchers have confirmed the nutritional usefulness of seeds and many
different interpretations and views have been discussed about their nutritional
benefits (Al-Jassir, 1992; Barampama & Simard, 1993), such as their carbohydrate
content (Oates & Powell, 1996; Tavares et al., 2003), antioxidant activity (Einbond et
al., 2004; Soong & Barlow, 2004; Wang et al., 2010), fatty acid content (Nagy et al.,
1977; Akpata & Akubor, 1999; Ennouri et al., 2005) and protein content (Kaur et al.,
1988; Rashwan et al., 1993; Sogi et al., 2002). But most seeds are at present not well
known and thus may be grossly underutilized in relation to their potential.
Plant seeds are important unconventional sources of proteins which when
incorporated in food products would improve the functional properties such as
absorption of water or oil and formation of stable foam. They are also good nutritional
supplements. Numerous studies were reported earlier on screening various seed
meals (Rao et al., 2011). Oilseeds comprise those seeds that contain reasonably high
percentages of oil and about 20-25% protein. After removal of the oil they contain 5060% protein (Altschul, 1958).
Among plant protein sources, oilseeds have been proved to be interesting fish
meal substitutes, in particular soy protein concentrates or isolates (Burel et al., 2000;
Drew et al., 2005, 2007; Gatlin et al., 2007; Brown et al., 2008; Burel & Kaushik, 2008;
Peres & Lim, 2008). However, soy protein concentrates and isolates are expensive and
the use of less purified soy products as toasted soybean meals is limited in aquatic
animals by anti-nutritional factors, higher crude fibre and unavailable carbohydrate
concentrations (Brown, 2008; Brown et al., 2008). Therefore, alternative protein
sources are needed to reduce the current dependence on fish meal and soybean meal
as the primary protein sources for aquatic animal diets (Reigh, 2008). Oilseeds are
highly valued for their sensory and nutritional attributes. They are used as snack foods
in various processed forms in a variety of foods to impart the desirable quality
attribute that include flavour and texture (Prachi & Jyothi, 2012).
In the recent years, many plants have attracted a great deal of interest as a
source of low-cost protein to supplement human diets. Also, proteins are important in
food processing and food product development, because they are responsible for many
Introduction
functional properties that influence the consumer acceptance of food products

(Ogunwolu et al., 2009).
Plant proteins play significant roles in human nutrition, mainly in developing
countries where the average protein intake is less than required. Increase in
population at a high rate should match the food supply, which is usually difficult. In the
third world countries, especially in developing countries, it is essential to find new
sources of edible protein and other nutrients to overcome the population problem.
Proteins from seeds of non-conventional plants should be explored. These types of
plants, which are found in abundance, are not yet utilised. Such seeds may have good
quality proteins that can be used to meet the increasing demand for dietary proteins
(Samanta & Subrata, 2009).
Plant proteins are used in foods as functional ingredients to improve stability
and texture as well as the nutritional quality of the product (Makri et al., 2005).
Numerous studies were reported earlier on screening various seed meals. Most of the
studies were related to determination of proximate composition, mineral content,
physico-chemical and functional characteristics and protein extractability. They were
based on soy flour (Surekha & Jamuna, 2006), pumpkin seed (Lazos, 1992), cashew nut
(Fagbemi, 2008), erythrina seed flour (Jyothirmayi et al., 2006) and beach pea protein
isolate (Chavan et al., 2001).
Seed dietary proteins have started to assume a key position in the interests
among nutritionists not solely for their nutrient role, but also for a number of both
adverse and beneficial effects that they may exert on the human body, including food
intolerance, allergies, inhibition of endogenous hydrolytic enzymes and hypolipidemic,
hypoglycemic, hypotensive, anti-carcinogenic and anti-obesity activities, respectively.
In addition, plant proteins can play important techno/functional roles, as witnessed by
their increased use as food ingredients (Duranti et al., 2008).
Proteins in the form of isolates or concentrates are important ingredients in
many food products, where they perform specific functions (Kinsella & Melachouris,
1976). Protein isolates or concentrates are important in food product development
and processing due to their functional and nutritional properties (Gamage et al., 2011).
Protein isolates are the most refined form of protein products containing the greatest
concentration of protein but unlike flour, concentrates contains no dietary fibre.
Isolates are originated from United States around 1950s. They are very digestible and
easily incorporated into different food products (Jay & Michael, 2004).
Introduction
Protein isolates are nowadays believed to have played a major role in the
development of new class of formulated foods. Its high concentration of protein with
the advantage of colour, flavour and functional properties make it an ideal raw
ingredient used in beverages, infant foods and children milk food, textured protein
products and certain types of specialty foods (Olaofe et al., 1998). Protein isolates have
been developed from a variety of legumes among which are soy bean, peanut, canola,
cashew nut, almonds, sesame, pinto and navy beans (Seyam et al., 1983).
Proteins are known as the functional molecules and play fundamental roles in
the cells (Ingaver & Lennart, 2007). As proteins are the main structural and functional
molecules, molecular characterization of proteomes is the cornerstone for any
comprehensive understanding of any biological system (Giorgianni, 2003).
The ongoing development in protein extraction, purification and identification
methods has significantly advanced our ability to address an increasing number of
biological questions using proteomic approaches. However, the ideal extraction
method should not only capture the greatest possible number of proteins from a
biological sample, it should also be compatible with protein analysis by subsequent
protein identification methods like SDS-PAGE (Sheoran et al., 2009).
The study of the food proteome at any specific time is extremely complex and
diverse. The use of proteomics approach is a powerful tool in Food Science in terms of
process optimization and monitoring, quality, traceability, safety and nutritional
assessment (Pedreschi et al., 2010).
Inadequate supply of protein food, especially in the context of increasing
population, has been an important factor in malnutrition in third world countries.
Serious protein deficiencies and the high costs of animal protein sources have
stimulated research on developing new sources of protein from unexploited sources or
wastes and by-products of fruit seeds (Perumal et al., 2001). The papaya seed is
currently a waste product as it is often discarded after eaten or processing, whereas
seeds constitute 22% of the waste from papaya puree plants (Marfo et al., 1986).
Papaya seeds are recently gaining importance due to its medicinal value, since it
recently had been used in curing sickle cell diseases, poisoning related renal disorder
(Imaga et al., 2009) and as anti-helminthes (Okeniyi et al., 2007). Also, increasing
growth of orange processing industries result in producing large quantities of orange
seeds as by-product necessitate the determination of the potential of orange seed
utilization in human and/or animal diets (Akpata & Akubor, 1999). In this context, ElDin & Yassen (1997) used guava seeds as an additional source of fiber in cookies.
Introduction
New sources of food proteins are needed because of increasing global demand.
The lack of information on many basic aspects of underutilized crops hinders their
development and their sustainable utilization. These types of plants, which are found
in abundance, are not yet utilized. Such seeds may have good quality proteins that can
be used to meet the increasing demand for dietary protein. Protein from seeds of nonconventional plants, which are found in abundance, should be explored (Tlili et al.,
2011).
Under-utilized crops can play a crucial role in food and livelihood security for
the poor. Underutilized or neglected crops are those plant species traditionally used
for their food, fibre, fodder, oil or medicinal properties, but which have been
overlooked by scientific research and development workers. These plants risk falling
into disuse, yet they often play a crucial role in food security, income generation and
the culture of the rural poor. They have a great potential to provide income to rural
micro-entrepreneurs. Unfortunately the lack of attention has meant that their potential
value is under-exploited and they are in danger of continued genetic erosion,
ultimately leading to disappearance. One such non-conventional plant seed is wood
apple seed and their composition was reported as 28% protein (Ramakrishna et al.,
1979); Protein and fat together accounts for 3/4th the weight of the seeds and is
grouped under oilseed, but it has received less attention as an oilseed.
Wood apple (Limonia acidissima) is the only species of its genus, in the family
Rutaceae. The fruits of L. acidissima are well documented for its use in Indian
traditional medicine and are commonly known as wood apple, elephant-apple, monkey
fruit, vilampazham, kathbel and kaitha (Allen, 1967; Khare, 2007; Teepica et al., 2013).
The fruits are spherical in shape with greyish-white rind and a woody hard outer shell.
L. acidissima fruit pulp is sweet, sticky, brown, resinous, with peculiar aroma and
consists of white seeds (Vidhya & Narain, 2011). The raw fruit pulp is helpful in the
treatment of gastro intestinal problems like flatulence, diarrhoea, dysentery and piles.
The plant is reported to have adaptogenic activity against blood impurities,
leucorrhoea, dyspepsia and is used in case of snake bites, throat infections (Yusuf et al.,
1994; Ratnayake et al., 2009; Muniappan & Pandurangan, 2012). The original home of
wood apple is South India and Sri Lanka (Lande et al., 2010). The wood apple is mainly
found in forest and dry plain area of Indian subcontinents (Harsh et al., 2014). The
various parts of this plant (leaf, stem, bark, fruit and seed) have been used for curing
various diseases (Joshi et al., 2011; Muthulakshmi et al., 2013; Rahman & Gray, 2002;
Introduction
Sharma et al., 2012). Wood apple has got high medicinal value. Every part of the fruit
has got its medicinal property (Mondal, 2002).
India is one of the leading countries in Asia in terms of the wealth of traditional
knowledge systems related to the use of plant species. India is also known to harbor a
rich diversity of higher plant species (about 17000 species) of which 7500 are known
as medicinal plants (Shiva, 1996). One of the medicinally important plants is Limonia
acidissima, contains a number of phyto constituents, which are the key factors in the
medicinal value of this plant. Almost all parts of this plant such as leaf, fruit, seed, bark
and root are used to cure a variety of diseases. A systemic research and development
work should be undertaken for the development of products for their better economic
and therapeutic utilization (Vijayvargia & Vijayvergia, 2014). The pulp surrounding the
seeds of some fruits is rich in mucilage, carbohydrates, proteins and polyphenols.
Wood apple grows in abundance throughout Indias drier regions and is
cultivated along both peninsulas of the country. Along with arid conditions, the fruit
requires monsoons to thrive. Wood apples grow along the plains in the north, south,
east and central areas of India. States growing the fruit include Maharashtra, Andhra
Pradesh, Tamil Nadu, Kerala, Karnataka, Madhya Pradesh and the Western Himalayas.
In the cooler northern regions, the fruit grows well up to 450 meters
(http://theindianvegan.blogspot.in/all-about-wood-apple-fruit.html, 2013). In India,
the fruit was traditionally a poor mans food until processing techniques were
developed in the mid-1950s (Singh et al., 2009).
Wood apple fruit is considered to be one of the natural sources of anti-oxidants
due to its potential radical scavenging activity of various phytochemicals (Nithya &
Saraswathi, 2010). The diverse pharmacological properties of the fruit include antidiabetic (Gupta et al., 2006; Gupta et al., 2009), anti-ulcerative (Mishra et al., 2009),
hepatoprotective (Kangralkar et al., 2010; Jain et al., 2011), wound healing (Ilango &
Chitra, 2010), anti-tumour (Saima et al., 2000), anti-microbial activity (Senthilkumar &
Venkatesalu, 2013), larvicidal (Rahuman et al., 2000) and antimicrobial activity (Metha
et al., 1983; Rahman & Gray, 2002). Fruit pulp showed anti-inflammatory, antipyretic
and analgesic activity (Ahamed et al., 2008). People consume the raw fruit pulp as such
with or without sugars, or as a beverage after blending it with other ingredients
(Ilaiyaraja et al., 2015).
The wood apple fruit contains 47-58% shell (average 53%), 32-38% pulp
(average 36%) and 11% seed and fiber component. Wet seed constitutes calcium 1%
and dry seed 6% of the weight of the whole seed. The seed oil is yellow in colour. The
Introduction
wood apple seed contains 34% of moisture, 0.7% of ash and 19% of crude fibre. The
fatty acid composition of wood apple seed oil contains 19.3% of palmitic acid, 7.3% of
stearic acid, 27.7% of oleic acid, 19.8% linoleic acid and 26.4% of linolenic acid
(Ramakrishna et al., 1979).
Rao et al., (2011) executed a preparation of wood apple (Feronia limonia L.)
seed protein concentrate and evaluated its nutritional and functional characteristics.
Wood apple (Feronia limonia L.) seed protein concentrate (WSPC) was prepared and
its properties were compared with the wood apple seed meal (WSM). The protein
content was found to be 33.79 and 77g/100 g in WSM and WSPC respectively. WSPC
was good source of essential amino acids leucine, phenylalanine, valine, iso-leucine
and threonine. Higher water absorption, lower oil absorption capacity, stable foam and
presence of essential minerals of WSPC favour its industrial application.
The defatted Citrullus lanatus and Limonia acidissima seed flours are good
source of protein and nutrients such as minerals. These flours could be appropriately
used as a matrix or included in different preparations for food fortification, due to its
very good functional properties that were observed in these flours. Thermal stability
and presence of spherical morphology and amorphous nature present in the defatted
flours will be useful in the elaboration or inclusion in food systems (Sonawane et al.,
2016).
.
In view of this, effort was made for identifying and evaluating underutilized non-
convectional cheap protein sources like wood apple seed as an alternative. The
development of a protein concentrate from defatted wood apple seed flour would also
provide the food industry with a new high protein food ingredient for product
formulation and protein fortification. The latter is critically needed in many developing
countries where protein deficiencies remain a major health problem, especially among
children.
Need for the study
1. Wood apple seed is a rich source of protein and oil has not been widely
studied for its functional and nutritional quality. The literature on utilization
of wood apple seed is very limited.
2. The extraction of protein and its purification are critical for the
determination of protein quality and depend on the food samples used for
protein extraction. Since wood apple seed is rich in protein, it remains a
challenge to isolate both abundant and less abundant protein using
Introduction
conventional extraction methods. Hence there is a need to identify the best

extraction protocol for the utilization of non-conventional protein source.
3. The protein concentrate and protein isolate from wood apple seed can be a
valuable source and a good alternative to soyabean derivatives but better
understanding and knowledge on in vitro and in vivo effect of wood apple
seed protein in comparison with soy protein is very much essential.
Scope of the Study
1. Identification of industrially exploitable method of extraction of protein from
wood apple seed enhances the utility of plant based proteins in dietary
supplement formulations.
2. Exploration of the functional characteristics of wood apple seed flour,
protein concentrate and isolate will provide the platform for its
incorporation in various processed food as an alternative protein source.
OBJECTIVES
Broad Objective
The present study was aimed to explore the characteristics of whole seed flour,
protein concentrate and protein isolate of wood apple (Limonia acidissima) seed.
Specific objectives
To determine the physical and morphological characteristics of wood apple

seed.
To identify the best method of isolation of protein from defatted wood apple
seed flour.
To compare the quality of whole seed flour, protein concentrate and protein
isolate of wood apple seed.
Literature Review
2. LITERATURE REVIEW
The review of literature pertaining to the present study entitled,
Extractability and Characteristics of Wood apple (Limonia acidissima) Seed
Protein, are discussed under the following headings.
2.1 Origin of wood apple
2.2 Distribution and ecology of wood apple
2.3 Botanical description of wood apple
2.4 Plant production and availability of wood apple
2.5 Health benefits of wood apple
2.6 Product development and value addition of wood apple
2.7 Plant Protein Resources
Fruits are undoubtedly very important for nutrition security with high potential
of value addition and foreign exchange earnings. Fruits are now considered as an
important item of commerce as they have gained enormous market potential. India
accounts for 12.5% of the total world population of fruit crops and ranks second with
the production of 75 million tons in 2013 (FAO, 2014). Post-harvest losses of fruits are
more serious in developing countries than those in well-developed countries. The total
losses from harvest to the consumer point are as high as 30-40%, which is worth
thousands of crores rupees. Tropical fruits, which are at present underutilized, have an
important role to play in satisfying the demand for nutritious, delicately flavoured and
attractive natural foods of high therapeutic value. Today, consumers are becoming
increasingly conscious of the health and nutritional aspects of their food basket. The
tendency is to avoid chemicals and synthetic foods and preference for nutrition
through natural resources. The underutilized fruits like aonla, bael, jamun, karonda,
passion fruit, phalsa, pomegranate, pumpkin, tamarind, wood apple are the main
sources of livelihood for the poor and play an important role in overcoming the
problem of malnutrition (Gajanana et al., 2010). They are in general accepted as being
rich in vitamins, minerals and dietary fibre and therefore, are an essential ingredient of
a healthy diet.
2.1 ORIGIN OF WOOD APPLE
Wood apples (Limonia acidissima) are native to India, parts of Sri Lanka,
Bangladesh and Pakistan. The history of wood apple is slightly blurry because its often
referenced as bael, which may also be a similar fruit, Aegle marmelos (Picture 2.1).
Literature Review
Picture 2.1: Bael (Aegle marmelos); Wood apple (Limonia acidissima)

However, wood apples Sanskrit term, kapittha, receives several mentions
in a number of ancient texts. According to the book, Hinduism: an Alphabetical
Guide, the Puranas-Sanskrit texts dating between 1BC and 1,000 AD-referenced
wood apple seed as being like great cosmic egg holding the origin of creation.
Buddhist scholar Xuanzang (602 and 664 AD) mentioned wood apple as an Indian
fruit and military commander and poet Chauvundaraya (940-989AD) listed wood
apple in numerous medicinal remedies. The original home of wood apple is South
India and Sri Lanka (Lande et al., 2010).
2.2 DISTRIBUTION AND ECOLOGY OF WOOD APPLE
Limonia acidissima (L.) of family Rutaceae (Citrus family) belongs to the

monotypic genus Limonia, is native to India and also cultivated in Bangladesh,
Pakistan and Sri Lanka (Bakshi et al., 2001). The wood apple is native and common
in dry plains. It prefers a monsoon climate with a distinct dry season. The tree
grows up to an elevation of 450 m in the western Himalayas. It is apparently
drought tolerant and best adapted to light soils (Vaidayaratnam et al., 1995). In
India and Sri Lanka, it is cultivated along roads and edges of fields and
occasionally in orchards. In Malaysia and Indonesia, it is cultivated in villages
and parks (Verheij & Coronel, 1992).
2.3 BOTANICAL DESCRIPTION OF WOOD APPLE
Limonia acidissima is a moderate sized deciduous tree grown throughout

India. It is an aromatic, slow growing up to 9metre tall, grows all over India in dry
and warm areas up to 450metre elevation, often polygamomonoecious tree with
rough, spiny bark. The spines are axillary, short, straight, 2-5 cm long on some of
the zigzag twigs. The leaves are deciduous, alternate, dark-green, leathery, 3 to 5
inch long. The minutely toothed, blunt or notched at the apex, dotted with oil glands
and slightly lemon scented when crushed (Picture 2.2B). Flowers are small
10
Literature Review
numerous, dull- red or greenish, born in small, loose, terminal or lateral panicles
(Picture 2.2A). The fruit is berry, round to oval, globose, large, 2 to 5 inch wide, with
a hard, woody rind, which is grayish-white, scurfy rind about 6 mm thick (Picture
2.2C). Seeds embedded in an edible pulp (Kirtikar & Basu, 1998). The pulp is sticky
brown, aromatic odorous, resinous, astringent, acid or sweetish, white seeds
scattered through it. There are two varieties of fruits, one with large, sweet fruits
and the other with small, acid fruits. The age of the plant varies from 13 to 70 years
with yield potentiality in mother plants varying from 650 to 1085 kg of fruit/plant
having the fruit weight between 130 and 225 g. Fruits length varies between 7.3 to
8.9 cm while breadth between 7.2 and 8.4 cm. Fruit size (lengthbreadth) varies in
relation to fruit weight (Ghosh et al., 2012).
Picture 2.2: (A) Flowers of wood apple; (B) Wood apple tree;
(C) Wood apple fruit
2.3.1 Taxonomy Classification (Bhandari, 1978)
Kingdom
: Plantae
Sub-kingdom
: Tracheobionta
Super division
: Spermatophyta
Division
: Magnoliophyta
Class
: Magnoliospida
Subclass
: Rosidae
Order
: Sapindales
Family
: Rutaceae
Genus
: Limonia L.
Species
: L. acidissima
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Literature Review
Synonyms
Feroniaelephantum Correa,
Feronialimonia (L.)Swingle,
Schinuslimonia L.
2.4 PLANT PRODUCTION AND AVAILABILITY OF WOOD APPLE
Flowering tends to start in July and the harvesting season is from August to
September. The fruit is tested for maturity by dropping onto a hard surface from a
height of 1 feet (30 cm). Immature fruits bounce, while mature fruits do not. After
harvest, the fruit is kept in the sun for two weeks to fully ripen (Morton, 1987). Ripe
fruits drop from trees and as they have a hard shell outside, the inner pulp keeps
well for some days (Bose & Mitra, 1989).
In Malaya, the leaves are shed in January, flowering occurs in February and
March and the fruit matures in October and November. In India, the fruit ripens
from early October through March (Morton, 1987).
The wood apple is generally grown from seeds though seedlings, will not
bear fruit until at least 15 years old. But efforts were made for early budding and
production of fruit within five years through different methods of propagation.
Propagation is done by seed and vegetative method (Parajapati et al., 2003; Hiwale
et al., 2008). But high rate of seedling mortality and outbreeding nature of this plant
account for poor regeneration and inferior germplasm. To overcome this, in vitro
propagation through axillary bud proliferation has been developed (Anonymous,
2002 and Hiregoudar et al., 2003). Studies on softwood grafting in wood apple
(Feronia limonia L.) was conducted in the Department of Horticulture, UAS,
Dharwad during the year 2007-08. Wood apple grafting has been done for early
growth and production of fruits in plants. Considering the wide gene diversity in
wood apple, studies on growth of wood apple plants by budding (Angadi et al.,
2011) and commercial multiplication may also be by root cuttings were studied by
several authors. The results indicate grafting was the better as compared to
budding and 85 to 95% success in grafting was recorded when operation was
performed in September (Ghosh et al., 2012). The wood apple plant grown through
grafting was shown in pictures 2.3 (A-C).
There are no specific government efforts targeting wood apple nurseries at
the moment but private nurseries had been identified of selling wood apple
plantings during the survey.
12
Literature Review
Picture 2.3: (A)- Wood apple seeds used for raising the seedling; (B)- Grafted
wood apple plants; (C)- Budded plants in wood apple on 5 to 10
months old rootstock
According to Agri Statistics 2010, there is no availability of prominent data on
production of wood apple in Tamil Nadu, since there is no created orchard or
plantation of these plants under the Department of Horticulture. While surveying the
sustainable production of wood apple inpar with current research work, more than
thousands of wood apple trees were seen in forest areas of Yercaud and Kolli hills,
roads/edges of fields and occasionally in orchards of Salem and Krishnagiri districts.
As it is a wild tree fruit, many trees were seen in Colleges and Universities of Salem,
Dharmapuri, Namakkal, Krishnagiri and Coimbatore districts. The availability of fruits
in the market is plenty during the months of September to March in Salem district. But
the surplus quantity produced is wasted due to unavailable process or technology on
preservation of this nutritional poor mans fruit. According to Barry (2008) there
seems to be a genuine lack of co-ordination along the supply chain with the absence of
any superior varieties being key issues and major problem related to production and
transportation of wood apple; due to the geographic spread of the crop transport cost
incurved by intermediaries are very high and direct market access to urban and periurban markets by growers is limited. No socio-economic research were conducted for
wood apple and exporting of wood apple has been constrained to ethenic markets;
13
Literature Review
although wood apple products especially drinks and jam are very popular
domestically, there is no or only limited export. Hence efforts should be concentrated
on improving the functioning of domestic markets, encouraging smaller and medium
sized entrepreneurs into gaining access to international markets and research
identifying new potential export markets. Accordingly the effort taken and situation in
India especially in Salem district of Tamil Nadu was picturaised (Picture 2.4. A D)
Picture 2.4 (A): Wood apple trees grown in Tamil Nadu Forest Department and
medicinal orchard (Salem)
Picture 2.4 B: Wood apple sapling grown in nursery in Salem for plantation
at Yercaud Adivaaram
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Literature Review
Picture 2.4 C: Wood apple available at local market of Salem

(Near old bus stand)
Picture 2.4 D: Evidence of Poor Mans Fruit and cheap availability

2.5 HEALTH BENEFITS OF WOOD APPLE
The number of traditional remedies attributed to wood apple is

unsurprising. According to Khare (2007), wood apples are an antiscorbutic,
15
Literature Review
carminative and digestive stimulant. The leaves possess a number of health

benefits, as they are used for indigestion, diarrhea, dysentery and haemorrhoids. A
number of medicinal concoctions included the fruit. Pullaiah (2006) reported that
the pulp mixed with cardamom, honey and cumin seeds are a remedy for liver
cirrhosis of malnourished children, piles and diarrhoea. Traditional healers have
also
prescribed
the
fruit
to
fight
breast
and
uterine
cancer
(http://theindianvegan.blogspot.in/2013/03/all-about-wood-apple-fruit.html,
2014).
2.5.1 Nutritional Properties of Wood Apple
The nutritive value of fruit reported by Gopalan et al (2007), that the fruit
contains 64.2g of moisture, 7.7g of protein, 3.7g of fat, 1.9g of minerals, 5g of fibre,
18.1g of carbohydrate, 0.13g of calcium, 0.11g of phosphorous and 0.08g of iron per
100g of fruit. The wood apple is high in oxalic, malic, citric and tannic acids. It also
has a good mixture of vitamins and minerals including calcium, iron, phosphorus,
carotene, thiamine, riboflavin and niacin (Jayakumar & Geetha, 2012). The
composition of wood apple and seed were given in following Table (2.1).
TABLE 2.1: COMPOSITION OF WOOD APPLE (PER 100g OF EDIBLE PULP AND SEED)
Constituents
Pulp (ripe) (%)
Seeds (%)
Moisture
74.00
4.00
Protein
8.00
26.18
Fat
1.45
27.00
Carbohydrates
7.45
35.49
Ash
5.00
5.03
Calcium
0.17
1.58
Phosphorus
0.08
1.43
Iron
0.07
0.03
Tannin
1.03
0.08
Source: Singh et al (2009)
Joshi & Jain (2008) reported that the ripe fruits contain sweet aromatic pulp
(70%) which have protein (2.2%), carbohydrate (22.1%), fat (3.3%) and ash (1.3%)
and provides 127 Kcal of energy. Its nutritive value is due to ascorbic acid. Ca, Fe,
Na, P, Zn, Cu and Mn contents of fruit pulp were 15.9, 3.5, 8.5, 46.5, 386.3, 0.8, 0.5
and 0.7 (mg/100g pulp) respectively.
Adikaram et al (1989) said that the content of reducing sugar increased by
about five times during ripening. The fully ripe fruit contained reducing sugar of
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Literature Review
about 1.25mg per 100g fresh weight. The weight of the fruit decreased by about
25% during ripening.
Ratnayake et al (2009) reported that the wood-apple is a nutrient rich fruit
which contains a surprisingly high amount of protein (3-7%) and low levels of
sugar and carbohydrates compared with many other fruits. It also contains high
amounts of vitamins and minerals and the fruit is especially rich in ascorbic acid,
additionally the pectin content of the fruit pulp is 3-8%.
Kumar et al (2012) analyzed seventeen different common fruits for their
contents of nutrients such as carbohydrate, protein, lipids, vitamins, -carotene,
thiamine, riboflavin, ascorbic acid, macro and micro nutrients such as sodium,
potassium, calcium, magnesium, phosphorous, iron, zinc and copper. The wood
apple contained the highest amount of riboflavin (0.15 mg %), phosphorous (98.9
mg %) and calcium (29 mg %).
2.5.2 Medicinal Properties of Wood Apple
Qureshi et al (2010) reported that the wood apple fruit contains fruit acids,
vitamins and minerals. The dried pulp contains 15% of citric acid and a small
quantity of deliquescent ash consisting of potassium, calcium and iron salt. Seeds
and fruits contained oil and protein; oil composed of palmitic, oleic, linoleic and
linolenic acids besides traces of palmitoleic and stearic acids; -sitosterol, -amyrin,
lupeol and stigmasterol from unsaponifiable matter of seed oil.
Adikaram et al (1989) showed that the presence of three antifungal
compounds such as psoralene, xanthin, 2,6-dimethoxy benzoquinone on the shells
of wood apple have been to inhibit the in vitro growth of Aspergillus sp., Penicillium
sp. (isolated from ripe wood apple), Colletirichum sp. and Curvularia sp. The overall
effect of antifungal activity in the unripe fruit shell, therefore, may be more in
favour of supression of saprophytic species such as Aspergillus and Penicillium.
Upadhyay et al (2010) identified that the Feronia limonia extracts fed to
such carbon tetra chloride treated rats reduced the enhanced level of serum
glutamate
oxaloacetate
transaminase
(SGPT);
serum
glutamate
pyruvate
transaminase (SGPT); alkaline phosphatase (ALP) and serum bilirubin by which

seems to alter the protection and maintain the functional integrity of hepatic cells.
Illango & Chitra (2009) reported that the methanolic extract of Limonia
acidissima pulp proved to have a hypoglycemic and hepatoprotective effect on
alloxan induced diabetic rats, a fact that there was a repair/regeneration of the beta
cells of islets of langerhans. As a result, there was an increase in insulin level, which
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Literature Review
brought about homeostasis in the biochemical parameters such as cholesterol, urea,

creatinine, total protein and in the enzyme activities.
Phapale & Thakur (2010) indicated that the ripe pulp of Feronia limonia
having varying total phenolic contents i.e. based on their free or bound form. The
phenolic glycosides (bound form) released after acid hydrolysis showed higher
total phenolic contents than ester and free form. The Feronia limonia ripe fruit can
be considered as an important source of phenolic compounds which exhibit their
antioxidant and antimutagenic effect. A bound form of phenolic glycosides in fruit
pulp is supporting its potential use as an antioxidant and antimutagenic
phytochemicals. The level of blood glucose was significantly increased in alloxan
alone treated rats as compared to control animals. The level of blood glucose was
returned to near normal concentrations in diabetic rats treated with Feronia
elephantum pulp extract and Glibenclamide. Feronia elephantum pulp extract
showed comparable effect to that of Glibenclamide (Kangralkar et al., 2010).
Thomas & Ponnammal (2005) reported that the preliminary phytochemical
analysis of Limonia acidissima plant parts such as bark, leaf, rind, pulp and seed
showed the presence of alkaloids, flavonoids, steroids, saponins, glycosides, phenols,
gum and mucilage, fixed oils and fats, resins and tannins. Among the five plant parts,
pulp possessed high amount of protein and the carbohydrate content was more in
seeds and the rind is rich in amino acid. The methanolic extracts of L. Acidissima
plant parts were tested against Escherichia coli and Staphylococcusaureus using disc
diffusion method. The extracts from different parts showed varying degrees of
antimicrobial activity.
Preliminary qualitative chemical tests of fruit pulp of Feronia elephantum
corr. done by Mahour et al (2008) revealed the presence of flavanoids, triterpenoids,
tannin, phytosterol, amino acid and carbohydrates. According to literature survey,
the flavanoids and triterpenoid are responsible for various biological activities like
immunomodulator, antiulcer and antioxidant tentatively. It can be said that
flavanoid and triterpenoid are the active constituents in fruit pulp of Feronia
elephantum corr. and are responsible for its pharmacological activities.
Wood apple has anti-diabetic and antioxidant potential by reducing the level
of blood glucose and malondialdehyde (Patel et al., 2012). Fruit is much used in
India as a liver and cardiac tonic and when unripe, as a means of halting diarrhoea
and dysentery and for effective treatment for high cough, sore throat and disease of
the gums. In addition to this, wood apple also have hypoglycemic activity, antitumor,
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Literature Review
larvicidal and antimicrobial activity and hepatoprotective activity (Vidhya & Narain,
2011).
2.5.2.1 Anti-cancer Activity
The fruit extract of L. acidissima Linn. showed anticancer effect. Fruit
extracts from fractions 1 to 4 and also the crude extract (ethanolic extract) were
used to determine the ED50 value (50% inhibition of cancer cell growth) in two
different breast cancer cell lines, SKBR3 and MDA-MB-435. The bio-assays of
extracts from L. acidissimaLinn.showed that a fraction (fraction 3) from an ethanolic
extract had an anticancer effect on SKBR3 and MDA-MB-435 human breast cancer
cells. After 48 h of exposure, this fraction at a concentration of 100 g/ml,
significantly reduced cell proliferation in both cancer cells. In MDA-MB-435 cells,
cell cycle analysis showed that the fruit extract fraction 3 induced the accumulation
of cells in G2/M phase, whereas no significant change in cell cycle was detected in
SKBR3 cells. L. acidissima Linn. fraction 3 could inhibit the proliferation of human
breast cancer cell lines, SKBR3 and MDA-MB-435. Our results demonstrated that
the cell cycle via G2/M blockage plays some roles in L. acidissima Linn. which
induced antiproliferative activities in MDA-MB-435 (Pradhan et al., 2012).
An acidic heteropolysaccharide has been isolated from the ripe wood apple
fruit which shows antitumor activity against ascites carcinoma cell growth. Tumor
growth was estimated in terms of Ehrlichascites carcinoma (EAC) cells in the intraperitonealfluid of the inoculated adult female Balb:C mice (20 24gm) on day 4
after being treated with an isolated pectic polysaccharide, from Feronialimonia (FL1a-I) (50 100mg) from day 1 to day 3 after intraperitoneal inoculation. The study
showed significant in vivo EAC cell growth inhibition. The effect of FL-1a-I on
normal Balb/C mice has also been suggested that the enhanced macrophage might
be responsible for the strong antitumor activity of the pectic polysaccharide, FL-1aI (Saima et al., 2000).
The methanolic extract of Limonia acidissima L. fruits when administered
at a dose of 570mg/kg shows significant antitumor activity against Daltons
Ascitic Lymphoma (DAL) induced tumour genesis in mice. The antitumor effect can
be attributed to its cytotoxic and antioxidant properties. The fruits are known to
contain flavonoids and coumarins which might reveal the antitumor potential. The
result showed that the Tumour bearing + Fluorouracil (5-FU) showed significant
antitumor activity than LAME (Limonia acidissima L. methanolic extract) fruits
(Jagadeeshet al., 2015). A study was carried out to evaluate the toxicity and in-vivo
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Literature Review
anti-breast cancer activity of methanolic extract of Limonia acidissima (MELA) on

MCF-7 bearing rats. The MELA possess flavonoids as exhibited in the phytochemical
screening. These flavonoids may be the major contributors for the anti-breast
cancer activity which exhibited significant correlation. Both the extracts restored
the mean survival time, decrease tumor volume, cancer cell count in treated rat.
Thus MELA fed at 400 mg/kg possess significantly higher anti breast cancer activity
against MCF-7 breast cancer cell line and increases the life span of the treated
animals as compared to the other test groups (Gitanjali & Debasish, 2015).
2.5.2.2 Anti-oxidative Property
The wood apple fruit is considered to be one of the natural sources of antioxidants due to its potential radical scavenging activity of various phytochemicals
(Nithya & Saraswathi, 2010). The methanolic extract of Limonia fruit was also
screened for their free radical scavenging properties by Ferric reducing antioxidant
power (FRAP) assay and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging
assay (Phapale & Thakur, 2010; Nanasombat et al., 2012). The effect of extraction
conditions on total polyphenols content and antioxidant activities were studied by
response surface methodology in wood apple fruit. The results showed that ethanol
concentration and incubation temperature affected the measured responses
significantly. Under the optimal condition of 62.7% ethanol, 49.7C temperature
and 39.4 (ml/g) of solid-to-solid ratio, the predicted values were found to be at 7.37
g GAE/g, 83.8% and 84.2%, respectively for TPC (Total phenol content), DPPH and
ABTS (2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging
activity. The study suggested that the model obtained can be applied for large scale
production of polyphenols for further use in pharma/food industries. (Ilaiyaraja et
al., 2015).
The Limonia acidissima an underutilized edible fruit was evaluated for its
antioxidant properties and amino acid analysis by Teepica et al (2013). The highest
phenol and flavonoid contents were present in methanol and the lowest in
chloroform extract. Also, water extract of the fruit exhibited potentially high nitric
oxide radical scavenging activity than other solvent extracts. Moreover, phenolic
and flavonoid contents of these fruit extracts strongly correlated with antioxidant
capacity. Amino acid analysis revealed that among all essential amino acids, the
concentrations of isoleucine, phenylalanine and tryptophan were found to be
present in higher amounts. Obviously, this fruit may be used as a potential natural
20
Literature Review
antioxidant and in the development of functional food and raw materials of

medicine.
Antioxidant capacity of the wood apple (Kaitha) fruit extracts was evaluated
by Pandey et al (2014) against ascorbic acid as percent inhibition of ABTS free
radicals. ABTS radical is a blue chromophore produced by the reaction between
ABTS and potassium persulfate. The antioxidant activity (IC50 value) as determined
by ABTS assay was found to be 0.7 mg/ml and 0.8mg/ml for methanolic extracts of
the pulp and rind of kaitha, respectively. FRAP activity was found to be much higher
in the rind (46.03 g BHTE/ mg) as compared to the pulp (42.95 g BHTE/ mg) of
the fruit.
2.5.2.3 Anti-mutagenic Property
The protective action of free phenolic, phenolic esters and phenolic glycosides
extractions of Feronia limonia ripe fruit pulp against mutagenicity of sodium azide was
evaluated by Phapale & Thakur (2010). Total phenolic content was evaluated following
Folin-Ciocalteu method; antioxidant activity by the DPPH assay and antimutagenic
potential by the Ames test. The phenolic glycoside extract presented higher (229 mg/g,
GAE) total phenolic contents followed by phenolic ester (37.5 mg/g) and free phenolics
(11.0 mg/g), where as the antioxidant activity was 88.7%, 11.8% and 3.8%
respectively. Phenolic glycoside extract showed antioxidant higher than that of
commercial antioxidant Trolox (64.6%) and Butylatedhydroxytoluene (83.2%). At
2500 l/plate, a significant antimutagenic effect was shown by phenolic glycoside
extract and the order of antimutagenic activity was found as phenolic glycosides >
phenolic esters > free phenolics. Thus it indicated good correlation between total
phenol content, antioxidant activity and animutagenic effect. Phenolic glycosides in
Feronia limonia ripe fruit pulp showed promising antioxidant activity and
antimutagenic effect.
2.5.2.4 Anxiolytic Property
The anxiolytic effect of hydroalcohol extract of Feronia limonia was
investigated by Ilaiyaraja & Khanum (2014). The extract was orally administered to
the Balb/C mice for a period of 7 days at the graded dose of 50, 100 and 200 mg/kg
body weight. The results obtained in this study indicate that extract of wood apple
fruit has peak anxiolytic-like activity at the dose of 100mg/kg body weight.
2.5.2.5 Antiulcer Activity
The wood apple (Feronia elephantum) fruit pulp was extracted with ethanol
and the anti-ulcer activity of the extract in indomethacin-induced gastric ulceration in
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Literature Review
Swiss albino rats was evaluated. Ranitidine was used as the reference anti-ulcer drug.
Acute toxicity studies were also carried out. The results showed that the extract (500
mg/kg, per oral) inhibited indomethacin-induced gastric ulceration by decreasing
acid concentration of gastric fluid while elevating its pH (p < 0.01), and compared well
with the standard drug, ranitidine (p < 0.001). Acute toxicity studies showed that
there was no mortality following the administration of the extract in a dose range of
250 - 5000 mg/kg, per oral. Thus the study concluded, wood apple fruit pulp extract
has potent antiulcer activity with low toxicity. Its anti-ulcer property probably acts
via a reduction in gastric acid secretion. The results obtained support the use of this
herbal material in folk medicine (Mishra et al., 2009).
2.5.2.6 Antifungal Activity
The different extracts (petroleum ether, chloroform, methanol and aqueous)
of Feronia limonia Linn fruit pulp exhibited antifungal activity against four bacteria
strains namely Salmonella typhimurium (MTCC 98), Klebsiella pneumonia (MTCC 432),
Escherichia coli (MTCC 45), Pseudomonas aeruginosa (MTCC 647) and two fungal
strains Aspergillusniger (MTCC 282), Aspergillusflavus (MTCC 277). The pulp extracts
of F. limonia has shown a great potential for antimicrobial activity against the tested
microorganisms.
The
maximum
inhibition
zone
and
minimum
inhibitory
concentration (MIC) values, which were sensitive to the methanolic extract, were in
the range of 1521 mm and 3.125-12.5 mg/ml, respectively. On the basis of zone of
inhibition and MIC values, the Pseudomonas aeruginosa was more sensitive to the
methanolic extract than all other organisms with inhibition zone of 21 mm and MIC
value of 6.25 mg/ml respectively (Jayashree & Londonkar, 2014).
2.5.2.7 Antibacterial activity
The antibacterial activity of wood apple fruit can be attributed to the phenolic
content of the sample extracts. The samples having higher phenolic content were
found to be better in inhibiting the growth of bacteria hence were giving zone of
clearance of greater diameter. The GC-MS screening confirms the presence of
hydrocarbons, sterols, aldehydes, carboxylic acids and their esters, phenolic acids,
and flavonoids which might be contributing towards these antimicrobial properties.
The alkaloid and saponin contents of the crude methanol extract were found to be
38.47 g/100 g and 0.13 g/100 g dry matter respectively. The wood apple fruit
methanol extract showed total phenol contents of 33.38 g/mg and the flavonoids
was found to be 33.80g/mg extract. The assay was performed against gram positive
(S. aureus and B. cereus) and gram negative (E. coli, P. aeruginosa, S. typhi, S.
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Literature Review
dysentriae, V. cholerae and K.pneumoniae) at varying concentration (100 to 500

mg/ml). The study showed that the inhibition of pathogens was dose dependent and
highest inhibition was observed in 500 mg/ml concentration. However, the
antibacterial efficacy of Limonia fruit extract was similar at the tested concentrations
(100 mg/ml to 500 mg/ml) against S. typhi and V. Cholera. It concluded, that the
phytochemical and spectral analysis of Limonia fruit methanol extract confirms the
presence of secondary metabolites, helpful in treatment of a variety of illness. The
antibacterial activity elucidates that the fruit could be used as an alternative source
for treatment against antibiotic resistant pathogens (Ponnuraj et al., 2015).
2.5.2.8 Anti-spermatogenic Activity
The ethanolicextract of fruit pulp of F. limonia impair reproductive activities
in male rats possibly by inhibiting spermatogenesis. It was found that administration
of this extract to male rats brought about a significant weight loss of the reproductive
organs of the rats, alterations in motility, viability and morphology of spermatozoa. It
was concluded that Feronia limonia fruit pulp may have reversible antispermatogenic
activity and could then partially support the scientific rationale for the traditional use
of this plant in inducing sterility in male (Dhanapal et al., 2012).
2.5.2.9 Anti-diabetic activity
The anti-diabetic activity was performed on the alloxan induced wistar rats by
using methanolic extract of fruit pulp of Limonia acidissima. It has been shown that
Limonia acidissima extract markedly improved the glucose tolerance in alloxan
induced diabetes in rats as compared to control (p<0.01). Extract showed dose
dependent effect, 200 and 400 mg/kg dose shows reduction in glucose level. More
over Limoniaacidissima extract showed significant reduction in blood urea and
creatinine in treated rats but significantly increased total protein level (Ilango &
Chitra, 2009).
Gupta et al (2009) studied wood apple for its anti diabetic activity, oral
administration of 250mg/kg body weight of 95% ethanolic extract of unripe fruits
were given and significantly lowered the blood glucose levels of fasted, fed and
streptozocin-induced diabetic male albino rats. It also depressed the peak value in
blood glucose model. Further, study on histopathology of pancreatic -cells
granularity of normal rats was also done. Marked degranulation in -cells of extract
treated rats, associated with the blood glucose lowering was observed. Extract
probably lowered the blood glucose concentrations by stimulating insulin
secretogogue activity.
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Literature Review
2.5.2.10 Antimicrobial Activity

The antimicrobial activity of methanol pericarp extract of FeronialimoniaL.
(Rutaceae) was studied by agar well diffusion method in vitro. Methonolic pericarp
extract of Feronialimoniashowed presence of flavonoids, volatile oils, unsaturated
sterols and free fatty acids except alkaloids, glycosides, saponins, tannins, reducing
sugars and steroids. The effect of antimicrobial potential was examined against
Salmonella typhi, Vibrio cholerae, Shigelladysenteriae and Enterococcus faecalis. The
methanol extract of the fruit pericarp has showed consistently significant inhibitory
activity on different bacterial species tested and found the significance of
antimicrobial activity of Feronia limonia (Putta & Kilari, 2015).
2.5.2.11 Wound Healing and Hepatoprotective Activity
Albino rats of either sex were used to check the wound healing activity by
screening with methanol extract of fruit pulp of L. acidissima (MELA). In incision
wound model, woundbreaking strength and epithelization period were evaluated,
while in excision wound model, wound contraction was studied. In dead-space wound
model, granulation tissue dry weight, hydroxyproline levels in dry granulation tissue,
as well as superoxide dismutase (SOD) and catalase levels in wet granulation tissue
were estimated. In the excision wound model, the wound contracted progressively
when treated with the extracts and required a mean period of 16.0 0.8 days for
optimum healing. Incision wound model showed increased wound breaking strength
and decreased epithelization period when treated with MELA. The methanol extract
of L. acidissimaat the doses of 200 and 400 mg/kg showed significantly increased
healing by wound contraction, when compared to the control group. Increase in the
breaking strength of the wound is indicative of improved collagenation which
contributes to healing, enhances epithelization and promotes wound contraction by
increasing granulation tissue weight due to infiltration of macrophages. An increase
in the levels of anti-oxidant enzymes (SOD and catalase) was observed in the
granulation tissue of the extract-treated groups may also have contributed to the
wound healing effect of the extract. The methanol extract of L. acidissima possesses
significant dose-dependant wound healing and anti-oxidant activities; this supports
traditional claims for the plant as a wound healer (Ilango & Chitra, 2010).
Hepatoprotective activity of the methanolic extract of fruit pulp of L.
acidissima (MELA) was investigated against carbon tetra chloride (CCl4) induced
hepatic injury in rats. The 200 and 400 mg/kg per oral doses of MELA were
administered to group of animals for 10 days. Silymarin (100mg/kg) served as a
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Literature Review
standard and carbon tetrachloride at a dose of 0.5 ml/kg intraperitoneal was used to
induce liver damage. The various hepatic biochemical parameters were evaluated.
MELA exhibited significant dose dependant protective effect against CCl4 induced
liver damage which can be mainly attributed to the antioxidant property of the
extract. This study rationalized the ethno-medicinal use of the plant for curing hepatic
injuries (Ilango & Chitra, 2009).
2.5.2.12 Immunostimulant Effect and Anti-infection Property
Srinivasan et al (2015) evaluated the possible effects of Limonia acidissima L.
fruit (wood apple) supplemented diets on the growth, innate immunity and disease
resistance against Aeromonas hydrophila in Catla fish. Catla catla fingerlings (mean
weight 5.00.5 g) were separated into four groups and cultivated in 100-L tank. Each
group was fed with diets supplemented with 0 g, 1.5 g, 3 g and 6 g per 100 g feed
twice daily. Fish were examined for growth and innate immune parameters at 30 days
interval up to 120 days. Results revealed that wood apple supplemented diets
enhanced the growth and innate immune responses of Catla during the feeding trial.
Growth performance, haematological parameters, biochemical parameters and
immunological indices significantly (p<0.05) increased in fish fed with experimental
diets.
The relative survival percentage after Aeromonas hydrophila challenge
increased in fish fed with Limonia acidissima diet. Thus, the result suggested that fish
fed with Limonia acidissima fruit supplemented diet enhanced growth, improved
immune system and increased survival rate in C. catla fingerlings.
2.5.2.13 Anti-asthmatic Activity
The pulp of Limonia acidissima showed a potent relaxant effect on tracheal
chains of guinea pigs which were lower than the ophyl line at concentrations used;
but it was found that due to the presence of flavanoids, glycosides, saponins, tannins,
alkaloids, polyphenol and sterols in ethanolic extracts of the plants Limonia acidissima
(pulp) might be responsible bronchodilatory and antiasthmatic effect on tracheal
chains of guinea pig (Mahapatra & Pradhan, 2014).
2.5.2.14 Cosmetic Uses
Essential oil from the fruit pulp of F. limonia can be considered as a new and
potential source of natural antioxidant and antimicrobial agent for not only in the
food industry but also in cosmetics and pharmaceutical preparations. However, toxic
study of the essential oil is warranted before its industrial application (Senthilkumar
& Venkatesalu, 2013). Example of such usage in Himalaya cosmetic cream is shown in
picture 2.5.
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Literature Review
Picture 2.5: Wood apple pulp extract used in cosmetics as face scrub
2.6 PRODUCT DEVELOPMENT AND VALUE ADDITION OF WOOD APPLE
India enjoys a prominent position on the pomological map of the world. The
varying weather conditions of this country provide suitable environment for growing
a variety of fruits. These fruits are available in abundance and also in different
seasons. This has resulted in limited scope for expansion of other minor fruits, though
they are nutritious and are the main source of livelihood for the poor. Most of the
underutilized fruits of the tropics are often available only in the local markets and are
practically unknown in other parts of the world. A large number of these fruits can
grow under adverse conditions and are also known for their therapeutic and nutritive
value and can satisfy the demands of the health-conscious consumers. However, some
of these fruits are not acceptable in the market in fresh form due to their acidic nature
and astringent taste. Hence, there is a need to concentrate on research efforts in
diversification and popularization of such underutilized fruit crops. To achieve this,
there is a need to create demand for such fruit crops in the domestic and international
markets. This, to some extent, can be achieved through developing suitable
processing and marketing strategies for these underutilized fruits (Ravani & Joshi,
2014).
Fruit processing is necessary where it ensures fair returns to the growers to
improve their economic condition. It also helps to mitigate the problem of underemployment during off-season in the agricultural sectors. The perishable fruits are
available as seasonal surpluses during certain parts of the year in different regions
and are wasted in large quantities due to absence of facilities and know-how for
proper handling, distribution, marketing and storage. Furthermore massive amounts
of the perishable fruits produced during a particular season results in a glut in the
market and become scarce during other seasons. Food preservation has an important
role in the conservation and better utilization of fruits in order to avoid the glut and
utilize the surplus during the off-season. It is necessary to employ modern methods to
extend storage life for better distribution and also processing techniques to preserve
26
Literature Review
them for utilization in the off-season in both large and small scale (Bhattacharyya &
Bhattacharjee, 2007; Jena et al., 2013).
There are many reasons for processing fruits besides the development of a
business with a good return on investment for the owners such as to prevent post
harvest losses, to eliminate waste, to preserve quality, to preserve the nutritive value
of the raw materials, to make seasonal horticultural produce available throughout the
year, to put them in convenient form for the user, to safely put the food away for
emergencies and to develop new products, to increase the value of the product and
also better return to the farmers. So, ultimately it will be beneficial to producer,
processors and consumers. Proper packaging and storage of processed/preserved
products are also important aspects of agro-processing to retain quality of fresh
horticultural produce which could be adversely affected by physical damage, chemical
reactions, microbiological changes and attack by insects and rodents. There is great
scope for processing and value addition to the underutilized fruits into various
products like jam, jelly, preserve, candy, confectionery, pickle, fruit drinks, dried
products etc. (Ravani & Joshi, 2014).
Value addition is the way to enhance the acceptability and profitability of a
crop. A good number of byproducts from the wood apple have been reported
(Seidemann, 2002; Joshi & Jain, 2008). The wood apple is a cheap, highly nutritious
and seasonally available fruit that can be preserved for human consumption
throughout the year. Young leaves are used in salads in Thailand. Indonesians eat the
pulp of the ripe fruit with palm sugar at breakfast. Indians eat the pulp of the ripe fruit
with sugar or jaggery. It is used for making chutney and pickles or is blended with
coconut milk and palm-sugar syrup and drunk as a beverage. Wood apple pulp is
excellent for making jelly (Jayakumar & Geetha, 2012). The pectin content of the pulp
is 3-5% (16% on dry basis) and forms an excellent material for making jelly. Wood
apple jelly resembles black currant or apple jelly. It is clear and bright purple in colour,
with firm consistency and agreeable flavour (Bose & Mitra, 1989).
Fruit pulp of wood apple is eaten raw with or with-out sugar or is blended with
coconut milk and palm-sugar syrup and drunk as a beverage or frozen as an ice cream
(Panda, 2000). Especially, wood apple jam, jelly and ready-to-serve drinks are
becoming popular in India and Sri Lanka. The demand for wood-apple fruit has
increased remarkably in the last few decades (Ratnayake et al., 2009).
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Literature Review
In Indonesia, wood apple is mixed with honey and eaten in breakfast. In

Thailand, leaves are eaten in salads while in India the pulp is used in savory chutneys.
The wood serves as fuel (Vijayvargia & Vijayvergia, 2014).
Wood apple can be converted into value added products like jam and fruit bar
in order to avoid glut and utilize the surplus during the season, it is necessary to
employ methods to extend storage life, for better distribution, to preserve them for
utilization in the off-season both in large scale and home scale. Jam is more or less a
concentrated fruit processing which has fairly thick consistency and body. It is also rich
in flavor (MacLeod & Pieris, 1981), because ripe fruits which have developed full
flavour are used in its preparation.
Fruit bar is a nutritious product, has a chewy texture, similar to dried raisins
and is a good source of dietary fibre and natural sugar. Wood apple juice when mixed
with other fruits can serve as an excellent beverage. Realizing the importance of wood
apple fruit as a significant contributor to human well being, as a cheaper and better
source of protective foods; its perishable nature and seasonality in production calls for
preservation of it, to be supplied throughout the year for human consumption (Ravani
& Joshi, 2014).
The wood apple pickle was developed by Manthena & Mythili (2014) and
evaluated for its organoleptic characteristics during and after storage of three months.
Microbiological analysis was also done to assess shelf life. The findings revealed that
the product has not changed in sensory properties and no visible signs of colonies were
noticed.
Hiwale (2006) prepared the wood apple syrup and evaluated its
characteristics. The fruits were deshelled by hand and the pulp with seed were mixed
with required quantity of water and boiled. Two extractions from the pulp are made.
Brix is raised to 13Brix by addition of sugar from its original brix. The prepared juice
after boiling for 30 minutes is filled into cans while it is still hot. The cans are sealed
and stored at room temperature for more than 1.5 years.
Peter (2008) advocated that the wood apple fruits may be utilized for
making an excellent quality of jelly, chutney, ready to serve beverage and
squash.
Investigations were carried out to develop a process for the preparation of
wood apple ready-to-serve (RTS) beverage and to study the effect of storage period
on its composition and quality. Preliminary trials were conducted to find out the
optimum levels of juice and other ingredients for preparing RTS. RTS with two
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Literature Review
treatments viz. T1 (sodium benzoate), T2 (Potassium metabisulphite) and T3

(control) was prepared and stored at ambient temperature (3542C). The samples
were periodically evaluated for its quality at intervals of 7 days. Results of the sensory
evaluation of RTS showed that wood apple RTS beverage with 19% juice, 11.2% TSS
and 0.31% acidity gave the best results in terms of overall acceptability. During the
storage of RTS beverage, the TSS and acidity of the samples increased while the pH of
the samples decreased as the storage period increased. Beverage preserved by using
potassium metabisulphite and sodium benzoate remained above minimum overall
acceptability level up to storage period of 14 weeks (Zambare et al., 2009).
Various factors impacting to wood apple beverage production was
investigated by Minh (2015) and examined the effect of mixture formula and
pasteurization to product shelf-life. The results showed that the mixture formula
was 10% fruit juice, 3% honey and final dry matter 20Brix to get the best product
sensory characteristics. The pasteurization strongly affects the vitamin C loss.
Owing to the low pH (2.9-3.5) and low pasteurization temperature (85C) with the
pasteurization unit of 8.2 minutes, it received the good product quality in 4 weeks
of preservation. Thus, it concluded wood apple fruits can be consumed as ripe fruits
or in juice-form. The wood apple fruit is sweet and can also be used for making a
tasty drink known as wood apple milk.
The wood apple squash was prepared using xanthan gum and red chili
powder. The product could be stored for 150 days in an acceptable condition. The
wood apple squash with 15 % TSS, 1 % acidity, 35 % pulp and 0.35 % xanthan gum
scored the highest for overall acceptability (Ghosh et al., 2012).
Joshi & Jain (2008) revealed that the large
sized fully ripe fruit with slight brown colour and
sweetish taste was selected for the preparation of
jelly. Jelly was prepared by boiling the pulp in
water and citric acid for 35min and the juice was
strained with the help of muslin cloth, sugar and
citric acid were added and boiled till it become
thick. Removed from fire and poured in sterilized
bottles. The jelly prepared out of wood apple pulp
is expected to behave like jelly from other fruit and hence is recommended to be
stored under refrigerated conditions. The proximate composition in 100g of wood
apple jelly was 1.3% of protein, 1.4% of fat, 0.8% of ash, 0.2% of fibre and 72.4% of
29
Literature Review
carbohydrate while the energy content of 307kcal/100g. The 100g of jelly provided
9.8mg of ascorbic acid, 2.1mg calcium and 5.0 mg of iron.
Peter (2008) stated that the wood apple toffee can be prepared by boiling the
pulp with sugar. It is also possible to prepare syrup and chutney from wood apple.
Vidhya & Narain (2011) was planned to utilize the wood apple by preserving
them as a jam and fruit bar. The jam was prepared by weighing the pulp (500g) and
equal amount of sugar (500g) was added. The pulp was slow cooked with occasional
stirring for 15min. Then added one teaspoon of citric acid and salt (1pinch), by judging
the end point (TSS-68.5% using refractometer), the jam was packed, cooled and
refrigerated. Fruit Bar is a nutritional product, has a chewy texture similar to dried
raisins and is a good source of dietary fiber and natural sugars. The pulp was weighed
(500g) and boiled for 10 minutes. The sugar (500g) was added and continued boiling
with stirring, then milk power (100g), hydrogenated fat (50g), citric acid (1tsp), salt
(1pinch) was added. By judging the end point (TSS- 71.5% using refractometer), the
sample was poured in greased tray & cooled at room temperature. Organoleptic
evaluation shows that storage stability was good in both jam and fruit bar with respect
to flavor and consistency. Nutritive analysis shows reduction in vitamin C, calcium and
phosphorus in both jam and fruit bar during 90th day of storage. The acidic content of
the preserved products decreased in both jam (2.5%) and fruit bar (1.66%). No
significant change was observed in TSS, pH, pectin and ash value for both jam and fruit
bar during storage. The total sugar was increased up to 0.68 and 0.89% and reducing
sugar increased to 2.59% and 1.53% in both jam and fruit bar respectively. The
microbial load of both jam and fruit bar was under the limit at the end of 90 days.
Hence the prepared jam and fruit bar was safe and fit for consumption.
Bhatt & Jha (2015) developed wood apple mango mix fruit bar and determined
its quality and storage life. The control sample (wood apple fruit bar) prepared by 100
% pulp and wood apple mango bar (50:50, wood apple pulp: mango pulp) was
preferred by panelists. Physico-chemical, sensory, microbial and textural changes were
studied in wood apple fruit bar and wood apple mango bar at room temperature. Wood
apple fruit bars developed in the study had good shelf life stability features with high
consumer appeal.
Wood apple is chosen to produce wine because of its particular flavour and
aroma. The quantitative analysing methods are used to determine sensory and
chemical characteristics. The results showed that the fermentation culture for wood
apple wine is strain of yeast Saccharomyces cerevisae; products quality from wood
30
Literature Review
apple is fermented after 4 days of preservation, wood apple pure is added with water
proportion 20%, Brix 18, starting yeast proportion 1.5%, pH = 4, fermentation
temperature 28-32C and fermentation time 9 days. It was concluded that wood apple
serve as a natural fruit for wine fermentation with good quality and specific aroma
(Tham & Minh, 2014).
Vijayakumar et al (2013) surveyed the drying characteristics and quality
evaluation of wood apple (Limonia acidissima L.) fruit pulp powder. The wood apple
pulp with seed was dried by using hot air oven, tray dryer and solar drier, powdered
and compared for its drying characteristics, fruit powder yield, physical and functional
properties, least gelation concentration, nutritional composition, non-nutritional
qualities and sensory profile. The wood apple pulp gets completely dried within 5-6
hours in all drying methods. The overall drying rate was significantly (p<0.05) high in
hot air oven dried sample; dehydration ratio, rehydration ratio and co-efficient of
rehydration were significantly high in tray dried sample at p<0.01. The total
polyphenol content and antioxidant activity was significantly higher (p<0.01) in sun
dried wood apple pulp powder. Nutrients get concentrated except vitamin C which was
lost on drying. The titrable acidity and pH revealed the medium acidic nature of wood
apple pulp powder. Organoleptically, the hot air oven dried sample was liked very
much in terms of its appearance, color and flavor. Hence the wood apple pulp could be
dried effectively using solar dryer, preserved as dried powder and value added for its
industrial exploitation.
Vijayakumar et al (2015) prepared and dried the wood apple pulp in hot air
oven at 50C, 60C, 70C and 80C and also in solar drier. The experimental moisture
ratio during drying was fitted to the most widely investigated mathematical models.
The yield of wood apple powder was ranged between 28 to 48%. Though the drying
time was lengthier (8 hours) in solar drying, the overall drying rate, dehydration ratio,
moisture ratio, fruit powder yield, bulk density, porosity, swelling power and retention
of nutrients were found to be greater than hot air oven drying at various temperatures.
Authors approximation model could adequately describe hot air oven drying behaviour
at 50C, 60C and 70C; Page, Modified Page and Modified Page Equation models
together could describe the hot air oven drying behaviour at 80C equally and
adequately; and Two-term Exponential model could adequately describe solar drying
behaviour.
The L. acidissima Linn fruit powder was studied for development of phenolic
enriched herbal biscuit by Patel & Pandey (2014) to supplement scarce phytochemicals
31
Literature Review
having greater antioxidant activity. The panelists showed positive responses toward
the sensory attributes of developed herbal biscuits. Nutritional and antioxidant
potential of developed herbal biscuits will be beneficial for all age group of peoples.
The Limonia acidissima L. pulp was supplemented to Cyprinus carpio L. The fruit
enhances the immunological parameters of Cyprinus carpio L. (Darsini et al., 2014). The
fishes were divided into four groups CTc (0%), CT1 (1.5%), CT2 (3%) and CT3 (6%)
based on the percentage of the Limonia fruit supplemented with the basal feed. The
experimental trial was carried out for a period of 60 days. However, at 60 days only CT2
showed significant (p <0.001) difference as compared to control group. The results
proved that Limonia acidissima L. fruit enhanced the immune parameter of C. carpio
fingerlings at 30 and 60 days feeding trial. It also been studied that dietary Limonia
acidissima fruit supplementation improved the enzymatic antioxidant capacity such as
SOD, GST, GPx activities and reduced the MDA levels. The lipid peroxidation of liver and
muscle was prevented by the enhancement of free radical-scavenging ability in C.
carpio. Supplementing L. acidissima pulp powder in fish diets stimulated the growth
performance of fresh water carps Cyprinuscarpio and Cirrhinusmrigala (Darsini et al.,
2013).
The post-harvest losses of wood apple were very low, almost negligible, as the
crops are extremely resistant to pests, diseases, high temperatures and dry spells due
to the outer shells are hardy and rough. The amount of produce which remained
unsold and unconsumed within the homestead was estimated by respondents was to
be less than 5%. Research efforts should be broadened to take into account the
constraining factors faced by a wider spectrum of beli and wood-apple growers.
Department of Agriculture can encourage growers to adopt the new varieties. An
analysis of production, processing, marketing channels and upgrading strategies for
fresh and processed fruit with development of niche markets for high-value produce
creates new opportunities for developing countries producers and exporters that can
meet the required standards. A value chain perspective is used to identify various
routes by which the value of food exports can be increased (Barry, 2008).
Products of underutilized fruits were successful in markets of UK, Canada, USA,
Australia and Dubai. The demand for these fruits is increasing yearly as there is a
change in the consumption trends of people, change of life style and exploration of
novel foods, which are not consumed extensively in India for many reasons aforesaid.
Even though sales for these kind of products continued, profits are marginal due to
32
Literature Review
small scale production at regional level which calls for further development of products
and market promotion of the same (Thillakawardane, 2009).
2.7 PLANT PROTEIN RESOURCES
World demand for proteins is increasing and so more food protein is

required from both conventional and new source of protein. Accepting that all
proteins will have nutritional value, then in both cases successes in food industry
requires that the protein have good functional properties to be acceptable as a
food ingredient. Market potential for new proteins is great both for supplementation
of existing foodstuff and fabrication of new food-stuff (Eltayeb et al., 2010).
Proteins are among the most important nutrients required by the body and
should be available in adequate amounts in the diet. At all-India level protein intake
has fallen from 60.2g to 55.0g per person per day in the rural sector and from 57.2g to
53.5g
in
the
urban
sector
over
the
period
1993-94
to
2009-10
(http://www.indiaenvironmentportal.org.in/files/file/nutrition%20intake%20in%20
india.pdf, 2016).
Protein deficiency is the prevalent form of malnutrition in developing countries. One of
the ways to increase the protein supply is to make more plant proteins available for
human consumption and to develop the production of unconventional protein for
animal food (Mauron, 1973). The constantly rising cost of conventional protein foods,
particularly animal proteins, makes it likely that new sources of proteins will be
increasingly exploited in developing countries. This search for nutritionally balanced
foods to make available to a substantial proportion of the population has stimulated
investigation into many unusual sources of protein (Altschul, 1994). Indeed, protein
isolates have been extensively studied as food and dietary supplements from several
oil seeds such as sesame, soy beans, castor lequerella and even wheat-plantain
composite flour.
One of the goals of nutrition for both developing and industrialized societies is
to provide the population with adequate amounts of food proteins to meet the
physiological and or nutritional requirements of the population (Ahmed, 1998). The
same workers classified protein sources into three groups, the first group, named
conventional protein sources which include products from plant growing, animal
husbandry, poultry farming, fishing and fish breeding. Non-conventional protein
sources, the second group, includes extracted isolates from seeds of different plants,
waste from milling plants and hulling. The third group, new protein sources from
plants, legumes, fruits and seeds.
33
Literature Review
There is a growing interest in the utilization of plant proteins for the

formulation of new food products. Maximal utilization of proteins requires the
matching of variety of functional and nutritional characteristics with the complex
needs of different food products (Adebowale et al., 2009). The varieties of products can
be obtained from soy-proteins (Kinsella, 1976) and oil seeds (Ige et al., 1984).
Novel or un-conventional protein sources are continually developed. Due to
increasing market demands on protein ingredients, novel proteins have been purified
from various sources (El-Nasri & El-Tinay, 2007; Lamsal et al., 2007; Lokra et al.,
2007). However, for a novel protein to be useful for food processing application, it
should possess desirable functional and nutritional qualities (Mu et al., 2009).
In many cultures animal proteins are the preferred forms of dietary protein
for esthetic and nutritional reasons. However, their expenses make it difficult to
increase dietary protein supplies by producing more animal products. Consideration of
alternate protein sources therefore directs attention to plant protein together with
microbial proteins. Alternate protein sources should be available in large supply, low
cost, have good nutritional value, safe to humans and have good functional properties
(Ahmed, 1998).
Plant proteins, in general are deficient in one or more amino acids, specifically,
cereals are mainly deficient in lysine while legumes and leaf protein are deficient in
methionine. The primary deficiency of an amino acid, namely, lysine or methionine, in
many instances is further intensified by a secondary deficiency of amino acid(s), e.g.,
threonine and tryptophan. Many plants contain substances such as phytic acid,
saponins, phenolic compounds, various sugars and metals which may complex with
proteins (Kakade, 1974). Substances that have the ability to inhibit the proteolytic
activity of certain enzymes are found throughout the plant kingdom, particularly
among the legumes (Ahmed, 1998).
Of the plant proteins, soybeans are the clear choice of an alternative source of
proteins for use as food ingredients. The industry is well established and a wide range
of products, isolates, concentrates and flour is available, the industry also has
considerable technical information on application of soy proteins in a wide array of
foods moulding baked goods, confections, processed meat, infant formulas, milk
replacers and beverages.
Other plant proteins used commercially are peanuts,
cottonseed, sunflower seed, peas and lupines are produced on a much smaller scale. Of
the remaining plant protein sources, rapeseed, leaf protein, coconut, winged bean and
other legumes are potential sources. In contrast to the low level of protein in most
34
Literature Review
cereal grains (6-14%), the oilseeds and legumes contain 20-25% protein, they are
adapted to grow under a wide variety of climatic conditions. Oilseeds and legumes are
relatively cheap and the fact that they are already a part of the diet in many parts of the
world greatly simplifies efforts to increase their consumption in such countries
(Ahmed, 1998).
Proteins that are utilised in food processing are of various origins and can
roughly be classified into animal proteins (gelatins), vegetable proteins (e.g. peanut
protein, soy protein, wheat protein, almond protein, canola meal protein etc.) and
animal derived protein (e.g. milk proteins). However, many vegetable proteins require
processing to provide food material having acceptable functional properties such as
emulsification, oil and water absorption, texture modifications, colour control and
whipping properties, which are primarily attributed to the protein characteristics.
Many plants have attracted a deal of interest as a source of low-cost protein to
supplement human diet, this include among others soybeans and peanut (Ogunwolu et
al., 2009).
2.7.1 Extraction and Isolation of Proteins
Among new and developing sources for food applications, proteins from
oilseeds deserve particular consideration. The type of protein isolation technique
directly influences its quality and composition as well as functional properties by
acting directly on their conformation and on the types of proteins being extracted. A
milder extraction method is required, which could solubilize proteins without
damaging their native conformation, maintain their activity and at the same time
would give high percentage of extracted proteins (Gerzhova et al., 2015).
Proteomics is the branch of science dealing with the isolation, identification
and characterization of proteins (Jyoti & Yadav, 2013). A wide variety of extraction and
fractionation tools for proteins and peptides are available based on their
physicochemical and structural characteristics such as solubility, hydrophobicity,
molecular weight, isoelectric point (pI) (Toldra & Nollet, 2013). Plant protein
extraction is the first step in proteomic studies. The solubility of plant proteins is
closely associated with their intracellular localization, and proteins are classically
extracted by aqueous buffer, detergents, or direct precipitation (Michaud & Asselin,
1995).Besides the most commonly used trichloroacetic acid (TCA)/acetone
precipitation method (Damerval et al., 1988), phenol extraction followed by
methanol/ammonium acetateprecipitation was reported by Hurkman & Tanaka
(1986) for proteomic studies.
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Literature Review
The main organic solvents and additives used to extract proteins from food
sources are shown in Table 2.2. Many studies performed in the last few years aimed to
compare different protein solubilization methods suitable for proteomic analysis
(Jiang et al., 2004; Natarajan et al., 2005; He & Wang, 2008).
TABLE 2.2: EXAMPLES OF ORGANIC SOLVENTS AND ADDITIVES USED TO EXTRACT
PROTEINS FROM FOOD SOURCES
Solvent (s)
Acetic acid/ urea/
acetyltrimethyl
ammonium
bromide
Food
Tissue
Reference
Rice
Bran
Hamada (1997)
Soybean
Seed
Natarajan et al (2009)
Rapeseed
Seed
Barbin et al (2011)
Tomato
Pollen grain
Sheoran et al (2009)
Potato
Tuber
Delaplace et al (2006)
Aloe vera
Leaf
He &Wang (2008)
Soybean
Seed
Avocado/tomato/
orange/banana
Fruit
Saravanan & Rose

(2004)
Grape
Fruit
Vincent et al (2006)
Pear
Fruit
Pedreschi et al (2007)
Apple/Strawberry
Fruit
Zheng et al (2007)
Coniferous
Seed
Zhen & Shi (2011)
Banana/apple/
potato
Tissues
Carpentier et al (2005)
Sodium dodecyl
sulphate/
Acetone
Coniferous
Seed
Zhen & Shi (2011)
TCA
Bean
Anther
Wu & Wang (1984)
Citrus
Leaf
Maserti et al (2007)
Soybean
Seed
Soybean
Leaf
Xuet al (2006)
Coniferous
Seed
Zhen & Shi (2011)
Tomato
Pollen grain
Aloe vera
Leaf
He & Wang (2008)
Apple/banana
Tissues
Song et al (2006)
Tomato
Pollen grain
Aqueous
isopropanol
Phenol
Phenol/ammonium
acetate
Phenol/methanolammonium acetate
TCA/acetone
TrisHCl buffer
The most common method used for the extraction of plant proteins is
tricholoracetic acid (TCA)/acetone precipitation as proposed by Damerval et al (1986).
36
Literature Review
This method has been used to extract proteins from different tissues of cereals,
legumes and fruits. The extreme pH and negative charge of TCA and the addition of
acetone realizes an immediate denaturation of the protein, along with precipitation,
thereby instantly arresting the activity of proteolytic and other modifying enzymes.
Use of additives, such as TCA or carboxymethyl cellulose is generally accepted
(Massoura et al., 1998). Extraction and further formation of protein micelles have also
been proposed (Krause et al., 2002; Murray 2003; Ser et al., 2008; Green et al., 2010).
TCA method has been demonstrated to reduce the concentration of problematic antinutritional or toxic factors, including the glucosinolates and their degradation
products during canola protein extraction (Tan et al., 2011). TCA-acetone protocol is
known to be effective with tissues from young plantsand was found not to be the best
choice for more complex tissues (Wang et al., 2003; Saravanan & Rose, 2004;
Carpentier et al., 2005).
The most frequently used protocols for extraction of plant proteins involve a
precipitation step, which should separate proteins from interfering compounds.
Chemicals frequently used for this purpose are acetone, trichloroacetic acid
(TCA)/acetone and Tris-buffered phenol; this protein extraction is generally followed
by methanol/ammonium acetate precipitation (phenol) (Saravanan & Rose, 2004;
Pavokovicet al., 2007; Faurobert et al., 2007).
It is important to isolate the proteins of seeds to harness their availability as
protein supplements in food formulation and for other applications in the food
industry. The most commonly used method for preparing plant protein concentrates
or isolates is alkali extraction followed by precipitation of the extracted protein either
by decreasing the pH to the iso-electric point or by heating (Aremu et al., 2007).
Seven different methods (Miller et al., 1972; Damania et al., 1983; Arulsekar &
Parfitt, 1986; Sammour, 1991; Jha & Ohri, 2002; Hameed et al., 2009 and Naushad et
al., 2010) were compared for their protein extraction efficiency from the seeds of four
different leguminous plants (Pisumsativum, Vignaradiata, Cicerarientum and
Vignamungo). The efficiency was determined by the yield of protein as well as the
clarity in the resolution of protein bands separated on Sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS PAGE). Results showed that the method used
by Naushad et al (2010), the extraction of protein buffer contained 0.5 M Tris-HCl (pH
8.0), 0.2% SDS, 5M urea and 1% 2-mercaptoethanol was found to be the best
performing method as compared to other six methods under study (Ranjan et al.,
2012).
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Literature Review
Zhen & Shi (2011) compared three protein extraction methods such as TCAacetone precipitation, SDS extraction/acetone precipitation and phenol extraction
methanol/ammonium acetate precipitation in coniferous seeds. The result showed
that TCA-acetone precipitation was the most effective method for protein extraction; it
gave the highest yield of total protein (8.9 mg protein per g seed weight) and the
greatest number of proteins spots (1,034 spots) on the 2-DE gel. The data
demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a
suitable method for proteomic analysis of coniferous species, such as Chinese fir and
provides a valuable starting point for similar proteomic analysis of other coniferous
tree species.
Three protocols viz. Trichloroacetic acid (TCA)-acetone, phenol and multidetergent were compared for the extraction of proteins from horsegram (Macrotyloma
uniflorum). The quality of protein from phenol method was superior to that from other
protocols. Bands in One-Dimensional (1-D) and Two-Dimensional Electrophoresis (2DE) of phenol protocol were reproducible and better (Jyoti & Yadav, 2013).
Sheoran et al (2009) evaluated four protein extraction methods, i.e.,
trichloroacetic acid (TCA)acetone, phenol, direct iso-electric focusing (IEF) buffer and
TrisHCl buffer, using tomato pollen for proteome analysis. The TCAacetone and
phenol protein extraction methods are superior to the other two tested methods for
tomato pollen proteome analysis, in terms of two-dimensional gel electrophoresis (2DE) gel separation, mass spectrometric analysis and identification of proteins by
peptide mass fingerprinting (PMF).
Protein extraction methods can vary widely in reproducibility and in
representation of the total proteome, yet there are limited data comparing protein
isolation methods. The aphid Schizaphisgraminum, an agricultural pest, was the source
of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol,
or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a
multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and
compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE).The TCAacetone extraction yielded the greatest amount of protein from aphid tissues and
highly reproducible 1-D gel-banding profiles were observed using the TCA-acetone
and the phenol extractions. Thus it concluded that the TCA-acetone method is a
suitable method for quantitative aphid proteomics (Cilia et al., 2009).
Li et al (2012) developed an efficient protein extraction method for Undifilum
Oxytropisby comparing five different protein extractions such as trichloroacetic acid
38
Literature Review
(TCA) precipitation, acetone preciptation, precipitation with ammonium acetate in

methanol following phenol extraction, precipitation with TCA in acetone and
ammonium sulfate precipitation. Of the five procedures assessed, trichloroacetic acid
(TCA) in acetone was shown to be the best method for the fungus. The U. oxytropis
proteins extracted using the TCA-acetone method were further characterized using
two dimensional gel electrophoresis (2-DE) followed by mass spectrometry. The high
resolution of the 2-DE reference map provided an useful approach for proteomic
analysis of slow growing fungi.
2.7.2 Protein Isolates
Protein isolates are currently of special interest to processors and consumers
due to low fat content and high protein content. Therefore it makes a useful ingredient
for several food products, including baked foods, extruded high-protein foods,
nutritional bars etc. (Hassan et al., 2010). Protein isolate was prepared from defatted
plant materials by several researchers (Nielsen et al., 1973; Hettiarachchyet al., 1996;
Karkiet al., 2009 and Hassan et al., 2010). The common method of alkaline extraction
of proteins from legumes is given in flow chart 2.1.
Deffated Legumes + Water (1:10)
Adjustment of the pH (9.5)
Centrifugation
Precipitatation (Protein Isolate)
Neutralization (using Alkali)
Freeze drying
Storage
Flow chart 2.1: Systematic flow sheet for legume protein isolates recovery
(Qayyum et al., 2012)
Protein isolates shows a lot of potential to combat the problem of malnutrition.
The underutilised plant and animal sources can be exploited in order to extract the
proteins and make them available for used as food supplements. Production of high
protein food from under-exploited sources isone response to the growing protein
malnutrition in developingcountries. Several new protein sources have been identied,
such ascashew nut protein isolate (Ogunwolu et al., 2009; Deng et al., 2011) and
milkweed seed protein isolate (Mila et al., 2009). The protein content of some isolates
are presented in Table 2.3.
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Literature Review
TABLE 2.3: PROTEIN CONTENT OF SOME PROTEIN ISOLATES
Protein Isolate
% Protein
Method
Reference
Whey
>90
Ion- exchange
chromatography and
membrane filtration
Chickpea flour
84.8-87.8
Isoelectric precipitation
Paredes-Lopez et al
(2006)
Lentil
80.47
Qayyum et al (2012)
Kidney bean
52.83
Qayyum et al (2012)
Blue lupin Defatted

flour
93.9
Alkaline and isoelectric

precipitation
Lqaria et al (2002)
Cowpea
75 and 76
and micellization
Khalid et al (2012)
Guava seed flour
96.4 and
93.5
Alkaline(pH 10 and
11.5) and isoelectric
precipitation
Fontanari et al (2007)
Foegeding et al (2011)
Protein isolates are the acceptable ingredients for dairy application due to
their fine particle size and dispersibility, Emulsification, emulsion stability, colour and
flavour are critical in dairy application. Isolates (especially soy proteins) are being
used to fortify all type of pasta products such as macaroni, spaghetti, to improve the
nutritional value etc (Sipos, 2013). Protein isolates are important sources of protein
with high lysine content. They are used as proteinaceous ingredients in many food
products such as salad dressing, meat products and dessert. Whey proteins are mainly
used in beverage applications, due to their health benefits (Kudre, 2013). Different
protein products such as whey protein, soy protein isolates, wheat gluten, rice bran
protein, peanut protein, and cottonseed proteins were investigated for film
development (Rhim, 1998).
2.7.2.1 Whey Protein Isolates (WPI)
Whey is the liquid by-product of cheese which can further be processed into a
spray dried products like instance whey protein concentrates (WPC), whey protein
isolate (WPI) or whey protein hydrolysate (WPH) (Brucic et al., 2009). By definition
from the code of Federal Regulation, is the liquid substances obtained by separating
the coagulum from milk or cream in the cheese making. During cheese making, the
whey protein remains in the serum phase which represents about 20 percent of the
milk protein (Kimberlee, 2012). Whey proteins are widely used as ingredients in
40
Literature Review
different foods (dairy, meat and bakery products) due to their unique functional and
nutritional properties (Brucic et al., 2009).
2.7.2.2 Fish Protein Isolates (FPI)
The utilisation of unconventional raw material (dark muscle fish, fatty fish) and
also fish by-products (fish trims, fish frames etc.) a process was developed to
economically develop a functional protein isolates from these kind of raw materials.
Fish protein isolate is a protein concentrate which is prepared from fish muscle
without retaining the original shape of the muscle. It is not generally consumed
directly, but used as raw material for production of other value added products. Fish
protein isolate does not retain the original shape of muscle and is normally utilized as
ingredient for the production of value added products. The proteins of the muscle
tissue are first solubilised. The solubilisation can be accomplished by addition water
with alkali added to approximately pH 10.5 or higher, or with acid added to about pH
3.5 or lower. It is usually necessary to choose the pH at which the consistency of the
solution decreases to a value that allows the removal of undesirable material. The
mixture is then centrifuged, and due to density differences the oil rises to the top and
can then be removed. Other insoluble impurities such as bone or skin are also
sedimented at this stage. The muscle protein are then precipitated and collected by a
process such as centrifugation (Shaviklo, 2006).
2.7.2.3 Peanut Protein Isolates (PPI)
Peanut contains 26-29% protein with good nutritional quality. Peanut proteins
are used for their functional properties (emulsification, forming) or for their
nutritional properties in different food products. They are also used for human
nutrition in developing countries to supplement cereals, beverages and skim milk.
Peanut protein isolate can be prepared from the defatted peanut cake or powdered by
macerating with high salt phosphate buffer (20 mM Na2HPO4, 2 mM KH2PO4, 5.4 mM
KCl, 1M NaCl, pH 7.4), followed by centrifugation and supplementing the supernatant
with (NH4)2SO4 to 90% saturation. After centrifugation, the pellet can be dialysed
against distilled water overnight at 4C and then freeze-dried (Mouecoucou, 2004).
2.7.2.4 Soy Protein Isolates (SPI)
Soy protein isolate is a common isolate. It has high protein content of about
90%. It is made out of defatted soy meal by removing most of the fat and
carbohydrates (Seyam, 1983). Soybean is crushed into oil and defatted meal. The meal
is usually used as animal feed, while smaller amount is further processed into food
ingredients including soy flour, protein concentrate, protein isolates and textured
41
Literature Review
protein (Kinsella, 1976). Soy protein isolate is usually combined with other food
ingredients such as vitamins, minerals and flavour in preparation of soy protein shake
powder (Seyam, 1983). Advance in food technology resulted in the development of a
variety of soy product such as concentrates, isolate and extruded-expanded products,
this consequently leads to increased utilisation by technically developed regions of the
world (Young, 1979). The production of soy protein isolate involve solubilising the
protein and carbohydrate at neutral or alkaline pH and the recovery of the solubilised
protein, by separation and optionally washing and neutralization before drying (Moure
et al., 2006). Three steps involved in the processing of soy protein isolates (SPI) are the
soy flakes slurried with water under alkaline conditions (pH 6.8-10 at 27-66C using
sodium hydroxide and other alkaline substances approved for food use) so that the
protein and the oligosaccharides can dissolve into the solution. The protein solution is
then separated from the insoluble residue by centrifugation, in second step the
supernatant containing the protein and sugars was then acidified to isoelectric pH 4.5
(where the solubility of protein is minimal), using hydrochloric acid (HCl). This leads
to the precipitation of protein as curd, finally the solubility of the precipitated protein
is restored by neutralizing to alkaline pH of 6.5-7.0 after rediluting with fresh water or
spray dried in its acidic form and packed in multilayer paper bags (Lusas et al., 1995
and Anon, 2008).
2.7.2.5 Canola Protein Isolates (CPI)
According to USDA 2010, Canola meal has been the second largest feed meal
after soybean meal. It has a good amino acid profile with a well balanced amino acid
composition although it has found only marginal use in the food industry, due to the
presence of anti-nutritional factors. The vast majority of canola protein isolates are
prepared by alkaline extraction method followed by isoelectric precipitation. Although,
this extraction method generates high yield of nitrogen, but the isolate produced by
this method have been found to have poor solubility and digestivity, this is most
probably due to the nature of protein constituent of canola meal which consist of
alkaline-soluble fraction that can be easily denatured during the extraction process.
CPI thus possesses generally an unacceptable food-functional property including poor
water holding, gelling, oil binding, foaming and emulsification properties. Many studies
have been carried out to modify the properties of this isolate e.g. by succinylation,
acylation and enzymatic hydrolysis. However, enzymatic hydrolysis has been prepared
because of its less effect than chemical method (Alashi, 2011).
42
Literature Review
2.7.2.6 Chickpea Protein Isolate

Chick pea protein is the worlds third largest pulse crop in term of area, grown
mostly in West Asia and Mediterranean region. It is one of the major vegetable
proteins. Many functional properties of this protein isolate has been studied whereas,
information on gelation properties of chick pea protein isolate (CPI) was scarced. The
chick pea protein isolate dispersed with sodium and calcium salts showed different
rheological behaviour at different ionic strength and pH. Increasing the ionic strength
of dispersion could strengthen the gelation properties of CPI under acidic conditions,
however reduced the elastic parameters of CPI at pH of 7.0 (Zhang, 2007). It promoted
the culture of chickpea for the local consumption and fractionation of this agroresource for the production of local food ingredients as proteins (Ghribi et al., 2015).
2.7.2.7 Cashew Nut Protein Isolate
Cashew has considerable economic importance because its components have
numerous economic uses. The kernel has high food value with about 40-57% oil and
21% protein content. A cashew kernel meal contains about 42% crude protein, a low
crude fibre and 0.5% and 0.2% calcium and phosphorous, respectively, which is
comparable to that of peanut composition, which has been used for peanut protein
isolate and concentrate. Protein isolates and concentrates can be obtained from
defatted cashew nut powder by both alkaline extraction-isoelectric precipitation and
alkaline extraction methanol precipitation. Cashew nut protein isolates has water and
oil absorption capacities of 2.20 ml/g and 4.42 ml/g respectively, emulsifying stability
index (47%), foam capacity and stability (45% and 55%, respectively) and low
gelation capacity of 13.5% (Ogunwolu, 2009).
The wood apple seed meal and seed protein concentrate has been developed
by Rao et al (2011) and Sonawane et al (2016). But still wood apple seed protein
isolate based research was less evident and there is a need for the development of
wood apple seed protein isolate which could act as protein supplement in many food
formulations.
43
Materials and Methods
3. MATERIALS AND METHODS

The materials and methods pertaining to the study entitled Extractability and
Characteristics of Wood apple (Limonia acidissima) Seed Protein are discussed
under the following headings.
3.1 HYPOTHESES
3.2 PHASE I - CHARACTERISTICS OF WOOD APPLE AND SEED
3.2.1 Characteristics of wood apple

3.2.2 Characteristics of wood apple seed
3.3 PHASE II - ISOLATION OF PROTEIN FROM WOOD APPLE SEED
3.3.1 Preparation of wood apple seed protein concentrate

3.3.2 Methods of isolation of protein
3.3.3 Identification of efficient method of isolation
3.4 PHASE III - CHARACTERIZATION AND COMPARISON OF WOOD APPLE SEED FLOUR
(WASF), WOOD APPLE SEED PROTEIN CONCENTRATE (WASPC) AND
WOOD APPLE SEED PROTEIN ISOLATE (WASPI)
3.4.1 Physio-chemical and functional properties

3.4.2 Nutritional composition
3.4.3 Anti-nutritional properties
3.4.4 Phytochemical screening and quantification of total phenols
3.4.5 Antioxidant activity profile
3.4.6 In vitro protein digestibility
3.4.7 Thermal properties
3.4.8 Rheological properties
3.4.9 In vivo protein quality
3.5 STATISTICAL ANALYSIS
3.6 LIMITATIONS OF THE STUDY
3.1 HYPOTHESES
Based on the objectives of the study, the following hypotheses were set as a
guide for the investigation and analysis of the findings.
44
II
III
The method of isolation did not significantly influence the

quality of the wood apple seed protein isolate.
There was no significant difference between the characteristics

of WASF, WASPC and WASPI.
The protein quality of wood apple seed was not significantly

comparable with casein protein.
3.2.1 Characteristics of Wood Apple

The wood apple fruit were characterised by analyzing the agronomical
features and its components as follows.
3.2.1.1 Wood Apple
The wood apple (Limonia acidissima) is a globose with a woody brownish
pericarp, filled with a dark brown sub-acid pulp when ripe. The surface of fruit is very
rough and covered with white bloom. Seeds are numerous, oblong, embedded in the
edible pulp. Botanically, the fruit is hard-shelled, many seeded berry (Peter, 2008).
Ripe wood apple with hard shell, fairly large and globular shaped with soft, fleshy,
brownish edible pulp was selected for the study; purchased from the daily market of
Salem as a bulk as they are highly seasonal and available during the months of
September and March.
3.2.1.2 Agronomical Features
The agronomical features of wood apple fruit such as its popular name, origin
of the variety, fruit maturation, plant height, production and cultivation statistics were
collected from the Department of Medicinal and Aromatic crops, Directorate of
Horticulture and Plantation Crops, at Salem. The fruit was authenticated by the
Botanist in the Institute of Herbal Science, Plant Anatomy Research Centre, Chennai,
Tamil Nadu.
3.2.1.3 Wood Apple Fruit Components
The wood apple fruit components related parameters such as weight of whole
fruit, weight of shell, pulp, seed, fibre and seed pulp ratio were determined in ten
randomly selected fruits and averaged.
45
3.2.2 Characteristics of Wood Apple Seed

The wood apple seeds were collected and determined for its physical and
anatomical characteristics.
3.2.2.1 Extraction of Seeds from Wood Apple Pulp
The seeds are collected from the pulp in two different methods are shown in
flow chart (Flow chart 3.1).
Breaking the hard shell
Pulp with seed
Washing the pulp

under tap water
Collecting the seed using centrifugal juicer
Wood apple seed

Flow chart 3.1: Extraction of seeds from wood apple pulp
46
The outer hard shell of wood apple fruit was broken and the pulp was
separated from the shell by scooping. From the pulp, the seeds were manually
separated by washing under tap water in the first method. The seeds were collected
separately using the centrifugal juicer (Prestige PCJ 6.0) in the second method. The
seeds were then sun dried and packed in tight polythene bags for further analysis.
3.2.2.2 Physical Characteristics of Wood Apple Seed
The physical properties of seeds and grain is a wide knowledge that can be
useful in the farming, harvesting and storage or in processing such as drying, freezing
and other. This knowledge is important in the designing of machinery to harvest and
in preparation of processing chain from grain to food. Accurate design of machines
and processes in the food chain from harvest to table requires an understanding of
physical properties of raw material. The estimation of shape, size, volume, density,
specific gravity, surface area and other mechanical characteristics, which may be
considered as designing parameters for food production. The measurement
techniques allow computation of these parameters, which canthan provide
information about the effects of processing (Dobrzanski & Stepniewski, 2013). The
wood apple seed colour, mass, size and shape were determined by the following
methods.
A. Seed Colour
The colour of seeds was determined according to the method of Gomez et al
(1997). The colour was effectively observed by placing 20 wood apple seed samples
on a sheet of white paper. The difference in colour of the pericarp (outer coat of the
seed) was recorded.
B. Thousand Unit Mass of Seeds
One hundred units of wood apple seeds were weighed and recorded. 1000unit mass was calculated by multiplying the mass (g) of 100-unit of the seed by 10 to
have 1000 seeds weight (Nalladulai et al., 2002; Sirisomboon et al., 2007). The
average value was obtained and recorded as mean.
C. Dimensions of the Seed
A sample of 10 seeds was randomly picked and three major dimensions
namely minor diameter (thickness (T)), intermediate diameter (width (W)) and major
diameter (length (L)), were measured using an vernier calliper having least count of
0.01mm (Mohsenin, 1980).
47
D. Geometric Mean Diameter (Dg)

In order to calculate the geometric mean diameter (Dg) for each wood apple
seed, the three principal dimensions (Length (L), Width (W) and Thickness (T)) were
used. The geometric mean diameter was calculated based on the following equation
(Mohsenin, 1970).
Dg = (LWT) 1/3
E. Equivalent Diameter (De)
The equivalent diameter of wood apple seed was calculated from measured
Length (L), Width (W) and Thickness (T) of the seed and calculated by following
relationship equation (Mohsenin, 1970).
De = [L(W+T)2]
4
F. Arithmetic Mean Diameters (Da)
The arithmetic mean diameters were calculated using the following
relationship (Kiani et al., 2008; Mohsenin, 1986).
Da = (L+W+T)
3
G. Sphericity (S)
Sphericity (S) is defined as the ratio of the surface area of a sphere having the
same volume as the seed to the surface area of the seed. It is an important property
used in fluid flowand heat and mass transfer calculations. The sphericity was
determined using the following expression (Mohsenin, 1970).
= {(LWT)1/3/L} 100
H. Aspect Ratio (Ra)
In order to gather more information about the shape of the seed, aspect ratio
(Ra) of the seed was determined from the following relationship (Maduako &
Faborode, 1990).
Ra =
W
L
I. Projected Area (Ap) and Surface Area (As)

The projected and surface areas of wood apple seed were calculated using the
expression cited by Ozarslan (2002).
Ap = (
) L w
As = Dg2
48
Where Ap and As are the projected and surface areas (mm2), Dg = geometric mean
diameter (mm).
J. Volume (V) of the Seed
The volume of wood apple seeds was calculated based on Jain & Bal (1997)
through the following equation.
B2L2
V =
6(2L B)
Where B = (W/T)0.5
3.2.2.3 Anatomical Characteristics
The anatomical structure of wood apple seed was studied by Microscope and
the detailed procedure was carried out by the following mentioned procedure.
A. Preparation of Specimens
The healthy seed specimens of Limonia acidissima were fixed in FAA
(Formalin -5ml + Acetic acid -5ml +70% Ethyl alchohol-90 ml). After 24 hrs of fixing,
the specimens were dehydrated with graded series of tertiarybutyl alcohol as per the
schedule given by Sass (1940). Infiltration of the specimens was carried by gradual
addition of paraffin wax (melting point 58-60C) until TBA solution attained
saturation. The specimens were cast into paraffin blocks.
B. Sectioning
The paraffin embedded specimens were sectioned with the help of Rotary
Microtome. The thickness of the sections was 10-12 m. Dew axing of the sections
was by customary procedure (Johansen, 1940). The sections were stained with
Toluidine blue as per the method published by OBerien et al (1964). Since Toluidine
blue is a polychromatic stain, the staining results were remarkably good; and some
cytochemical reactions were also obtained. The dye rendered pink colour to the
cellulose or walls, dark green to suberin, violet to the mucilage, blue to the protein
bodies etc. Wherever necessary sections also stained with Safranin and Fastgreen
and IKI (for Starch).Powdered materials of different parts were cleared with NaOH
and mounted in glycerin medium after staining. Different cell component were
studied and measured.
C. Photomicrographs
Microscopic descriptions of tissues are supplemented wherever necessary.
Photographs of different magnifications are taken Nikon lab photo 2 microscopic Unit.
For normal observations bright field was used. For the study of crystals, starch grains
and lignified cells, polarized light was employed. Since these structures have
49
birefringent property, under polarized light they appear bright against dark
background. Magnifications of the figures are indicated by the scale-bars. Descriptive
terms of the anatomical features are as given in standard Anatomy books (Esau,
1964).
3.3 PHASE II - ISOLATION OF PROTEIN FROM WOOD APPLE SEED
Protein extraction methods can vary widely in reproducibility and in

representation of the total proteome, yet there are limited data comparing protein
isolation methods. The methodical comparison of protein isolation methods is the
first critical step for proteomic studies (Cilia et al., 2009).
3.3.1 Preparation of Wood Apple Seed Protein Concentrate (WASPC)
The protein rich meal, which is left behind after the oil has been removed from
the seed, is currently used as a protein source in livestock and aquaculture industries
(Uruakpa & Arntfield, 2005; Canola Council of Canada, 2009). The oil was extracted
from the flour of wood apple seeds according to the method described by El-Tinay et
al (1988). Sample was mixed with hexane (1:10 w/v) and stirred for 2 h at room
temperature and then the mixture was left overnight. In the next day, the mixture was
filtered and the residue was collected and dried for 12 h at room temperature. The
process was repeated two more times to extract the complete oil from the sample.
Later the fat free meal was air dried at room temperature and ground to pass through
a BSS 60 (240 m) mesh sieve. The final product was used as wood apple seed protein
concentrate (WASPC) and was packed in a polyethylene pouch to store at room
temperature for further investigations.
3.3.2 Methods of Isolation of Protein
Protein extraction from plant tissues is challenging due to relatively low
protein concentration and high levels of polysaccharides, polyphenols, pigments and
lipids (Carpentier et al., 2005). Interest in newer sources of protein has grown
due to protein shortage in developing countries. As part of the quest for newer
sources, some lesser-known oil seeds have been evaluated for their nutritional
qualities in India (Udayashekhara & Belavady, 1979; Udayashekhara, 1985, 1987,
1991, 1994; Rukmini & Udayashekhara, 1986; Vijayakumari et al., 1994). Protein
from wood apple seed protein concentrate was extracted by five methods (Table 3.1)
as follows.
3.3.2.1 Method I Trichloroacetic Acid (TCA)/Acetone Precipitation
The proteins were isolated from the WASPC by the method using Cilia et
al(2009) with some modifications made by Jyoti and Yadav (2013). The protocol used
50
TCA and acetone for extraction of the proteins. The WASPC (100mg) was
homogenized in 10% TCA containing 2% -Mercaptoethanol (-ME) using liquid
nitrogen. It was kept overnight for precipitation at -20C. Next day, the mixture was
centrifuged at 5000 g for 30 min at 4C. The supernatant was discarded and
precipitates were washed thrice with ice cold acetone. The precipitates were agitated
vigorously in between each wash. The precipitates were then air dried and were
dissolved in 1 ml of the modified lysis buffer (9 M urea, 2 M thiourea, 1% DTT and 4%
CHAPS). After gently stirring for 4 h at room temperature, samples were centrifuged
at 15,000g for 1 h. The supernatant solutions, contained the solubilized proteins, were
centrifuged again at 15,000g for20 min to further remove precipitate.
TABLE 3.1: METHODS OF ISOLATION OF PROTEIN FROM WOOD APPLE SEED
Methods
Reference
Trichloroacetic acid (TCA)/

acetone precipitation
Cilia et al (2009) with some modifications

made by Jyoti & Sudesh (2013).
Phenol extraction
Hurkman & Tanaka (1986)
Ammonium sulphate precipitation
Ee et al (2008)
TrisHCl buffer
Wang et al(2003) and Vidal et al(2008)
Alcoholic precipitation
Schwenke (2001)
3.3.2.2 Method II - Phenol Extraction

The protein was extracted based on method described by Hurkman & Tanaka
(1986) with slight modifications. The WASPC was extracted by grinding WASPC for 5
min in 1 ml of phenol (Tris pH 8.8 buffered) and extraction buffer (0.1 M TrisHCl, pH
8.8, 5 mM EDTA, 20 mM DTT, 30% sucrose). The sample was transferred into a
centrifuge tube, vortexed for 30 min and centrifuged at 25,000g for 10 min. The
upper, phenol layer was transferred into another tube. The lower, aqueous phase was
then re-extracted with 1 ml of phenol and extraction buffer, vortexed, centrifuged and
the phenol layer was combined with that collected earlier. The isolated protein was
dried at room temperature and used for further analysis.
3.3.2.3 Method III - Ammonium Sulphate Precipitation
The soluble matter was extracted from the WASPC with 10 volumes of
deionized water under constant stirring at room temperature for 1 h. After
centrifugation at 2147g for 10 min at 4C, the supernatant was filtered through
Whatman No. 1 filter paper and precipitated with ammonium sulphate until
51
saturation (>75% w/v) (Ee et al., 2008). The protein was recovered by centrifugation
at 2147g for 10 min, followed by dialysis against deionized water at 4C for 72 h prior
to lyophilization. This protein extraction was designated as ammonium sulphate
precipitate.
3.3.2.4 Method IV - TrisHCl Buffer Extraction
The WASPC was suspended in a 1:1 mixture of Tris-buffer, pH 8.0 and dense
SDS buffer (2%w/v SDS, 5%w/v sucrose, 0.1M Tris-HCl, pH 8.0, 5% v/v mercaptoethanol). The mixture was vortex mixed and the sample was obtained by
centrifugation at 12000 x g for 10 min according to Wang et al (2003) and Vidal et al
(2008). The precipitated protein extract were room dried and the isolated protein
was used for further analysis.
3.3.2.5 Method V - Alcoholic Precipitation
Alcoholic precipitation of proteins was carried out using the method of
Schwenke (2001). One volume of cold 95% ethanol and methanol was added to the
same volume (100 mg) of WASPC and left for at least 30 min. The mixture was kept
cold and stirred gently. The resulting precipitate was centrifuged for 20 min at 15000
rpm and the supernatant was carefully decanted while the residue was dried under
room temperature.
3.3.3 Identification of Efficient Method of Isolation
The use of proteomics is a powerful tool in Food Science in terms of process
optimization and monitoring quality, traceability, safety and nutritional assessment
(Pedreschi et al., 2010). A wide variety of extraction and fractionation tools for
proteins and peptides are available based on their physicochemical and structural
characteristics such as solubility, hydrophobicity, molecular weight, isoelectric point
(pI) and so on. The state-of-the-art of extraction and fractionation techniques for food
proteins and peptides are the first step prior to proteome studies. The most efficient
method of isolating protein from wood apple seed was identified based on the quality
(functional properties) and quantification of wood apple seed protein isolate (WASPI)
(protein yield, protein content and 1-D SDS PAGE) (Table 2).
3.3.3.1 Percent Recovery
The percent yield of WASPI extracted from five different methods was
calculated using a formula published by Onsaard et al (2010).
% Recovery=
Extracted sample (g) % Protein in extracted sample

Starting sample (g) % Protein in starting sample
x 100
52
3.3.3.2 Protein Content

The protein content of WASPI extracted from five different methods was
calculated by Kjeldhal method (Sadasivam & Manickam, 2005). The detailed
procedure is given in appendix IA.
3.3.3.3 Functional Properties
In order to evaluate if a protein is applicable and suitable in certain food
systems and food products, it is important to characterize the functionalities of the
protein (Kinsella, 1982; Vaclavik & Christian, 2003). For the proteins to be used in
foods they must possess or contribute characteristics that are appropriate in
interaction with other food components (e.g. water and lipids) or be suitable for
processing. The functional properties that are required from a protein vary with
different food applications and food systems. The three most important functional
properties of food proteins in general are solubility, emulsification and foaming
(Kinsella, 1982). It is important to remember that no single protein exhibits all the
functional properties (Vaclavik & Christian, 2003). According to Pomeranz (1981),
functional properties of proteins include: hydrophilic and hydrophobic properties,
surface properties and properties related to protein-protein interaction. Hence the
wood apple seed proteins isolated by five different methods were determined for its
functional properties in Table 3.2.
TABLE 3.2: FUNCTIONAL PROPERTIES AND CHARACTERIZATION OF WOOD APPLE SEED
PROTEIN ISOLATE
Parameters
References
A. Hydrophilic and Hydrophobic Properties

Water absorption capacity (g/g)
Rodriguez-Ambriz et al (2005)
Fat absorption capacity (g/g)
Lin & Zayas (1987)
Dispersibility(%)
Karuna et al (1991)
Solubility (%)
Klompong et al (2007)
Least gelation concentration (%)
Abbey & Ibeh (1988)
B. Surface Properties
Foaming property (ml)
Sze-Tao & Sathe (2000)
Emulsification capacity (EC) (ml)
Beuchatet al (1975)
C. Properties Related to Protein-Protein Interaction

Protein extractability (%)
Rao et al (2011)
Isoelectric precipitation of soluble protein (%)
Taher et al (1981)
D. Fractions of the Protein Isolate
Landry & Moureaux (1970)
E. Gel Electrophoresis (1-D SDS PAGE)
Laemmili (1970)
53
A. Hydrophilic and Hydrophobic Properties

Functional properties can be interpreted as a result of the thermodynamically
favourable protein-water interactions (wettability, swelling, water retention,
solubility) or unfavourable (foaming, emulsification) (Moure et al., 2006). The
interactions of protein with water are important in relation to dispersibility, water
absorption and binding, swelling, viscosity, gelation and surfactant properties as these
properties influence the important functions of proteins in meat, bakery and beverage
systems (Moure et al., 2006). Ease of dispersibility and wettability is important in
food formulations and is affected by surface polarity, topography, texture and area,
and by the size and microstructure of the protein particles (Kinsella, 1979).
a. Water Absorption Capacity (WAC)
Proteins have both hydrophilic and hydrophobic properties therefore can
interact with water and oil in foods (Butt & Batool, 2010). WAC was determined using
the method described by Rodriguez-Ambriz et al (2005), 100 mg of WASPI was mixed
with 1000l of distilled water using a stirrer. The protein suspension was then
centrifuged at 1800g for 20 min at 22C. The supernatant was decanted and the tube
was drained at 45angle for 10 min. Water absorption capacity was calculated by
dividing the volume of water absorbed by the weight of the protein sample.
WAC (%) =
Wf Wi
Ws
x 100
b. Oil Absorption Capacity (OAC)

OAC was determined using the method described by Lin and Zayas (1987).
One hundred milligrams of WASPI was vortex-mixed with 1000l of sunflower oil for
30 s. The emulsion was incubated at room temperature for 30 min and then
centrifuged at 3,600g for 10 min at 25C. The supernatant was decanted and drained
at 45 angle for 20 min. The volume of oil absorbed was divided with the weight of the
protein sample, to obtain the fat or oil absorption capacity of the sample.
OAC (%) =
Wf Wi
Ws
x 100
c. Least Gelation Concentration (LGC)

This is a qualitative parameter that expresses the minimum protein
concentration at which the gel does not slide along the test tube walls in inverted
position (Young and Scrimshaw, 1979). This was determined using the method
described by Abbey and Ibeh (1988); WASPI were mixed with 5 ml of distilled water
in a centrifuge tube to obtain 2-20% w/v concentrations. The centrifuge tube was
54
heated for 1h in a boiling water bath, cooled rapidly under running tap water and
further cooled for 2h in a refrigerator at 4C. The least gelation concentration was
regarded as the concentration at which the sample from the inverted tube did not fall
or slip.
d. Dispersibility
The dispersibilities of protein isolates were measured by the method of
Karuna et al (1991). One hundred mg of WASPI was placed in a stopperd measuring
cylinder and distilled water was added to reach a volume of 1ml. The mixture was
stirred vigorously and allowed to settle for 3 h; the volume of settled particles was
subtracted from 100 and the difference was reported as percentage dispersibility at
different pH 4.5, 7 and 10.
e. Solubility
This was determined by the method described by Klompong et al (2007). Two
hundred milligrams of WASPI was dispersed in 20 ml deionised water and the pH of
the solution was adjusted to 2, 4, 6, 8, 10 and 12 with 1 or 0.1 N HCl and 1 or 0.1 N
NaOH. The mixture was stirred at room temperature for 30 min, using magnetic
stirrer and then centrifuged at 7500g for 15 min. The protein content in the
supernatant was determined using the Kjeldhal method (Sadasivam a& Manickam,
2005). Protein solubility was then calculated as
Solubility % =
Protein content of supernatant

Total protein content of the sampl e
x 100
B. Surface Properties
Important properties of foods involve the interaction(s) of proteins and lipids,
e.g. emulsions, fat entrapment in meats, flavour absorption, lipoprotein complexes in
egg yolk, meats, milk, coffee whiteners, dough and cake batters. The surface
properties relates primarily to surface tension, bulk density, emulsification and
foaming characteristics of proteins (Kinsella, 1979). Emulsions and foams are two
phase systems commonly found in food systems, whose formation is significantly
affected by protein surface activity (Kinsella, 1979; Moure et al., 2006).
a. Foaming Properties
The foaming properties are used as an index of the whipping characteristics of
the protein isolate (Mwasaru, 1999). Foaming capacity and stability were determined
according to the method described by Sze-Tao & Sathe (2000). Two hundred and fifty
milligrams of WASPI were mixed with 2.5 ml of distilled water and the solution was
whipped for 3 min and whipped protein solution was then poured into a 100 ml
55
graduated cylinder. The total sample volume was taken at 0 min for foam capacity and
up to 30 min for foam stability. Foam capacity and foam stability were then calculated:
FC (%) =
Volume after whipping Volume before whipping

Volume before whipping
FS (%) =
Volume after standing Volume before whipping

x 100
x 100
b. Emulsification Capacity (EC)

Proteins are surface active agents that can form and stabilize the emulsion by
creating electrostatic repulsion on oil droplet surface (Makri et al., 2005).
Emulsification capacity (EC) was determined by the method of Beuchat et al (1975).
To one gram of WASPI, 25 ml of distilled water were added and mixed in a blender at
low speed. After complete dispersion, refined groundnut oil was added at a rate of 0.4
ml/s; blending was continued until phase separation was seen.
EC (ml) =
Height of emulsified layer

Heig ht of total contents of tube
x 100
C. Properties Related to Protein-Protein Interaction

a. Protein Extractability
The WASPI were extracted in water for 1 h at room temperature (28 2C).
The ratio of WASPI to water was studied in the range of 1:10 to 1:70 (w/v).
Subsequently, the extraction time was varied from 10 to 80 mins and the pH was
varied from 2 to 12. The required pH was adjusted using 0.5 M hydrochloric acid or
sodium hydroxide solutions. The unextracted material was separated by
centrifugation at 3000g for 30 mins. The supernatant was analysed for protein
content and the results were expressed as the protein/100 g (Rao et al., 2011).
b. Isoelectric Precipitation of Soluble Protein
Protein precipitability of WASPI was carried out (Taher et al., 1981) by
dispersing 7 g of sample in 350 mL distilled water and the pH of the suspension was
adjusted to 10. The mixture was stirred for 60 mins, and the suspension was
centrifuged at 3000g for 30 min. The supernatant liquid of 20 ml each was taken in a
number of graduated centrifuge tubes. The pH (26.5) was adjusted and the
suspensions were vortexed for 10 mins and centrifuged at 3000g for 30 mins. The
volume of supernatant was noted and protein contents were analysed. Protein
precipitability was calculated by the following equation in 100 g of soluble protein:
Protein precipitability (g/100 g soluble protein) =
V1P1V2P2
V1P1
x 100
56
where V1 and V2 are volumes of aliquots in mL before and after precipitation and P1,
P2 are mg of protein in 1 ml of V1and V2, respectively.
D. Fractions of the Protein Isolate
Seed storage proteins were initially classified according to their solubility
properties into albumins (water soluble), globulins (saline soluble), prolamins
(alcohol soluble) and glutelins (residue) (Osborne, 1924). Protein fractions were
extracted according to their solubilities in different solvents, as described by Landry
and Moureaux (1970). WASPI (3.5 g) was extracted twice with 50 ml distilled water
for 30 mins at room temperature. The extract was centrifuged at 3000g for 30 min
and the supernatant was used for the determination of a water-soluble protein
(albumin). The residue was then extracted successively in a similar manner with 1.0
M NaCl, 70% ethanol or 0.2% NaOH. The supernatant of each extract was collected
separately and used to estimate the salt (globulin), alcohol (prolamin) or alkali
(glutelin) soluble fraction. The residue remaining after successive extractions
represents the insoluble proteins.
E. Gel Electrophoresis (1D SDS-PAGE)
The electrophoresis of seed storage protein is a method to investigate genetic
variation and to classify plant varieties (Isemura et al., 2001). The technique of
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is
commonly used for separation of seed storage proteins (Ullah et al., 2010).
a. Step I: Protein Purification by Dialysis
Proteins can be purified from small molecules by dialysis through a
semipermeable membrane, such as a cellulose membrane with pores (Picture 3.2).
Molecules having dimensions significantly greater than the pore diameter are
retained inside the dialysis bag, whereas smaller molecules and ions traverse the
pores of such a membrane and emerge in the dialysate outside the bag. This technique
is useful for removing a salt or other small molecule, but it will not distinguish
between proteins effectively (Berg at al., 2002). The protein of WASPI was purified by
dialysis (Raghmuramalu et al., 2003). The main purpose of dialysis in biological
research is the removal of small molecular weight constituents from biological fluids.
This is achieved by dialysing the desired material in a cellulose tubing with a pore of
40-80A (4-8 nm) diameter, which allows the passage of compounds with molecular
weight less than 10,000 Da. The length of the dialysis tube was decided depending
upon the volume to be processed. The sides of the tube were opened for the flow of
57
glass distilled water and transferred to a boiling water bath. After this processing the
tubing should never be allowed to dry as this will cause distortion of pore size. The
tube was made into a bag by tying a knot on one end. The solution to be dialysed was
transferred into this bag. Another knot was made at the top of the bag. The bag was
then placed in a beaker containing the solution against which dialysis to be carried
out. In general one volume of desired solution is dialysed against 50 to 100 volumes of
buffer. The total period of dialysis could range from 8 24h. The efficiency of dialysis
could be increased by stirring and by changing the dialysate solution at frequent
intervals.
Picture: 3.1 Dialysis protein molecules (red) are retained within the dialysis
bag, whereas small molecules (blue) diffuse into the surrounding medium
b. Step 2: Protein Quantification and 1-D Electrophoresis
The proteins were quantified using Bradford reagent (Bradford, 1976) and
samples were analyzed in triplicates. SDS-PAGE (12%) was used for 1-D
electrophoresis analysis. The detailed procedure for determination of protein using
Bradford method is given in appendix IA.
c. Step 3: Gel Electrophoresis (SDS-PAGE) of WASPI
One dimensional electrophoresis of WASPI extracted by different methods
was done by following steps described as follows. SDS-PAGE analysis of seed storage
protein is reliable method because these proteins are not influenced by
environmental
conditions.
Sodium
dodecyl
sulphate
polyacrylamide
gel
electrophoresis (SDS-PAGE) was performed on extracted isolated protein on a 12%
58
polyacrylamide gel to determine molecular weights for proteins according to the

method of Laemmili (1970). SDS-PAGE, is a technique used in biochemistry, genetics
and molecular biology to separate proteins according to their molecular weight. The
electrophoretic mobility of proteins depends upon their size. The purpose of SDSPAGE is to separate proteins according
to their size. As proteins are amphoteric
compounds,
their
net
charge
can
therefore be determined by the pH of the

medium in which they are suspended.
Therefore, at a given pH and under nondenaturing
conditions,
the
electrophoretic separation of proteins is

determined by both size and charge of
molecules. As proteins are high molecular weight molecules, it needs porous gels to
get separated. Polyacrylamide gels are those which provide a means of separating
proteins by size as they are porous. Polyacrylamide is the best gel recommended to
provide such an environment. Polyacrylamide is a synthetic gel which is thermostable, transparent, strong and relatively chemically inert and can be prepared with a
wide range of average pore sizes. It can withstand high voltage gradients and is
feasible to various staining and destaining procedures and can be digested to extract
separated fractions or dried for autoradiography and permanent recording. A
polymer gel is formed of acrylamide monomers and the proteins are run through this
gel by electrophoresis, hence this entire process is called Polyacrylamide Gel
Electrophoresis (PAGE) (Laemmli, 1970).
i.
Preparation of Samples for 1D SDS-PAGE

Protein was extracted according to the method proposed by Sekhar &
Demason (1988) in 0.06 M Tris-HCl, 2.3% SDS buffer at pH 6.8 for 5h at 4C. The
suspension was centrifuged for 10 min at 3750g, and then the supernatant
precipitated by adding four volumes of cold acetone. A protein pellet was separated
by centrifuging at 18500g for 10 min after which the pellet was air dried.
ii.
Separation of Proteins by 1D SDS PAGE

All parts of the gel casting unit (glass plates, combs, spacers and assembled gel
cassette) were washed thoroughly with light detergent to remove any residues of
previous gels, before rinsing well with distilled water and drying on paper towel. The
59
gel casting tray was assembled by placing the glass plates together with a spacer
between them. A rubber strip was placed at the base of the casting unit and the glass
set on top of it. The screws on the casting unit were tightened to hold the glass plates
in place. The gap between the glass plates was filled with water to ensure there is no
leakage from the base or sides of the casting unit. After this, a comb was inserted
between the glass plates and a mark was made on the plate to ensure filling of the gel
to a sufficient height. The gel was cast by pouring the 12% separating gel buffer (1.5M
Tris-HCl buffer pH 8.7, acrylamide/bis-acrylamide 40% solution, 10% SDS buffer,
10% APS buffer, 20 l TEMED solution) between the glass plates up to the mark. nbutanol was spread over the surface of the gel to ensure a flat surface and left for an
hour to allow the gel to solidify (Flow chart 3.2).
Once the gel had polymerized, n-butanol was removed carefully with filter
paper before pouring the 4% stacking gel (0.5M Tris-HCl pH 6.8, acrylamide/bisacrylamide 40% solution, 10% SDS, 10% APS and 20 l TEMED solution) over the
separating gel and immediately inserting a comb into the stacking gel to form the
wells in the gel. The gels were allowed to polymerize for further one hour. The glass
plates were removed from the casting unit before clamping them onto the gel running
unit. The assembly was inserted into an electrophoresis tank and both the chambers
were filled with SDS-PAGE running buffer (25mM Tris-HCl (pH 8.3), 192 mM Glycine
and 0.1% SDS) before the combs were carefully removed. The air dried pellet of
protein (7 mg) obtained from the extraction step was dissolved in distilled water (1
ml). This protein solution (100 l) was added to an equal volume of SDS sample buffer
(0.5M Tris-HCl pH 6.8, SDS, glycerol, 2-mercaptoethanol and 0.1% bromophenol blue
solution). This was mixed well before 1020 l were loaded into the wells in the gel
using a pipette ensuring no dispersal in the running buffer in the tank. The tank was
covered with a lid and connected to the power pack.
The gel was run at constant voltage of 160 V for approximately one hour or
until the dye reached the bottom of the gel. After completion the gel was gently
removed from the glass plates and placed in a tray before staining with colloidal
Coomassie brilliant blue solution (5% (w/v) aluminium sulphate hydrate, 10% (v/v)
ethanol, 0.02% (w/v) Coomassie brilliant blue-G250, 2% (v/v) orthophosphric acid)
and left overnight on a shaker. The stain solution was removed and replaced with
destain solution (10% (v/v) ethanol and 2% (v/v) orthophosphoric acid) until the
background become clear and protein bands were visible. Gels were scanned and
60
analysed with Gel Analyzer 2010a software to estimate the molecular weight of
protein bands.
Relative molecular masses were compared using a wide range MW marker
(Sigma Chemicals Co., St. Louis, USA) consisting of myosin (205 kDa); galactosidase
(116 kDa); phosphorylase B (97 kDa); Bovine serum albumin (66 kDa); ovalbumin
(45 kDa); carbonic anhydrase (29 kDa); Cytochrome C (12 kDa) (Sigma Chemicals Co.)
was also mixed in the marker solution.
Flow Chart 3.2: Separation of proteins by 1-DE (SDS PAGE)

(WASF), WOOD APPLE SEED PROTEIN CONCENTRATE (WASPC) AND WOOD APPLE SEED
PROTEIN ISOLATE (WASPI)
The WASF, WASPC and WASPI (TCA/acetone precipitation method) was

compared by determining its characteristics (Table 3.3).
3.4.1 Physio-Chemical and Functional Properties
Functional properties are the fundamental physicochemical properties that
reflect the complex interaction between the composition, structure, molecular
conformation and physico-chemical properties of food components together with the
nature of environment in which these are associated and measured (Kinsella, 1976;
Kaur & Singh, 2006; Siddiq et al., 2009). Functional characteristics are required to
evaluate and possibly help to predict how new proteins, fat, fibre and carbohydrates
61
TABLE 3.3: CHARACTERISTICS OF WASF, WASPC AND WASPI
Parameters
References
Physio-Chemical and Functional Properties
Bulk density (g/ml)
Wang & Kinsella (1976)
Water absorption capacity (ml/g)
Beuchat (1977)
Oil absorption capacity (ml/g)
Lin et al (1974)
Foaming capacity and foaming stability (%) Lawhon et al (1972)
Emulsification capacity (ml)
Beuchat et al (1975)
Dispersibility (%)
Karuna et al (1991)
Wettability
Regenstein & Regenstein (1984)
Nutritional Composition
Moisture (%)
AOAC (2002)
Ash (%)
AOAC (2002)
Fat (%)
Sadasivam & Manickam (2005)
Protein (% )
Carbohydrate (%)
Onyeike et al (1995)
Crude fiber(%)
Iron, Potassium and Sodium (mg )
AOAC (2003)
Calcium and Phosphorous (mg )
AOAC (2003)
Amino acid profile by HPLC
Jarret et al (1986)
Anti-Nutritional Composition
Oxalate (mg)
Day & Underwood (1986)
Phytate (mg)
Reddy & Love (1999)
Birk et al (1963), Hudson & El-Difrawi
Saponin (mg)
(1979)
Tannin (mg)
Trease & Evans (1978)
Phytochemical Screening and
Swain & Hills (1959) as modified by
Quantification of Total Phenols
Thaipong et al (2006)
Antioxidant Activity Profile
Arnao et al (2001) with some
modifications as per Thaipong et al
ABTS
(2006)
Benzie & Strain (1996) with some
FRAP
(2006)
Williams et al (1995) with some
DPPH
(2006)
In Vitro Protein Digestibility
Malomo et al (2013)
Thermal Properties (DSC)
Gorinstein et al (1996)
Rheological Properties (RVA)
AACC (2000)
In Vivo protein quality
62
may behave in specific systems as well as demonstrate whether or not such protein
can be used to stimulate or replace conventional protein (Mattil, 1971; Kaur & Singh,
2006; Siddiq et al., 2009). The food property is characteristics of the structure, quality,
nutritional value and /or acceptability of a food product. A functional property of food
is determined by physical, chemical and/or organoleptic properties of a food. Example
of functional properties may include solubility, absorption, water retention, frothing
ability, elasticity and absorptive capacity for fat and foreign particulars. Typical
functional properties include emulsification, hydration (water binding), viscosity,
foaming, solubility, gelation, cohesion and adhesion (Chandra & Samsher, 2013).
3.4.1.1 Bulk Density
Bulk density is a function of particle size, particle size being inversely
proportional to bulk density. Particle size differences may be the cause of variations in
bulk density of the flours. The particle size also influences the package design and
could be used in determining the type of package material required (Appiah et al.,
2011). Higher bulk density is desirable since it offers greater packaging advantage as
greater quantity of flour can be packed within a constant volume (Leach et al., 1959).
The Bulk density was determined by the method of Wang & Kinsella (1976). About
three grams of WASF, WASPC and WASPI were placed in a 10 ml graduated cylinder
and gently packed by tapping the cylinder on the bench 10 times to a reasonable
height (approximately 5-8). The volume of the sample was recorded. Bulk density was
calculated as gram per millilitres of material.
Mass of the sample
of the sample
Bulk Density = Volume
(g/ml)
3.4.1.2 Water Absorption Capacity (WAC)

Water absorption capacity of flour is useful indicator of whether protein can
be incorporated with the aqueous food formulations, especially, those involving
dough handling (Osungbaro et al., 2010). Interactions of protein with water, is
important to properties such as hydration, swelling power, solubility and gelation
(Etudaiye et al., 2009). The WAC for WASF and WASPC was determined by the
method of Beuchat (1977). One gram of WASF and WASPC was weighed into a preweighed 15 ml centrifuge tubes (Wi). For each sample, 10 ml of distilled water was
added and mixed using a vortex at the highest speed for 2 mins. After the mixture was
thoroughly wetted, samples were allowed to stand at room temperature for 30 mins,
then centrifuged at 3000g for 25 min at 20C. The supernatant was decanted and the
63
centrifuge tube containing sediment was weighed (Wf). WAC was calculated using
following formula. The WAC of WASPI was determined by the procedure mentioned
3.3.3.3a.
WAC (%) =
Wf Wi
Ws
x 100
3.4.1.3 Oil Absorption Capacity (OAC)

According to Lahl & Braun (1994), lipid binding is dependent on the surface
availability of hydrophobic amino acids. Oil absorption capacity is important as oil
acts as flavor retainer and gives soft texture to food improving mouth-feel (Aremu et
al., 2006; Ubbor & Akobundu, 2009). OAC was determined for WASF and WASPC
according to the method of Lin et al (1974). WASF and WASPC (0.5 g) was mixed with
10 ml of refined oil in pre-weighed (Wi) centrifugal tube and vortexed for 10 mins.
The tubes were centrifuged for 25 mins at 3000g. The oil was drained off by inverting
for 10 min and centrifuge tubes were weighed (Wf).The OAC of WASPI was
determined by the procedure mentioned 3.3.3.3b. The OAC was calculated as
OAC (%) =
100
3.4.1.4 Foam Measurements

Foams are 2 phase systems composed of air bubbles surrounded by a
continuous liquid lamellar phase (Sanchez-Vioqueet al., 2001). Foams can be formed
and stabilized by either proteins or surfactants. FC is related to the readiness of
proteins to bind to the air-water interface to form foam particles, whereas FS is
related to the proteinprotein interactions that form strong interfacial membranes
that stabilized the foam particles (Kinsella, 1981). FC and FS were determined
according to the method of Lawhon et al (1972). The WASF, WASPC and WASPI (3g)
was dispersed in 100ml distilled water, adjusted to pH 7.0, transferred the contents to
a waring blender (Johnson, India) and whipped for 5 mins at 10,000 rpm. The
contents along with the foam were poured into a 250 ml measuring cylinder; the foam
volume was recorded after 30 s. FC is expressed as percentage increase in volume.
After 30 mins the volume of foam was measured and expressed as FS. Foaming
properties were expressed as
FC (%) =
FS (%) =

Volume before whipping
100
100
64
3.4.1.5 Emulsification Capacity (EC)

Proteins are an important group of emulsifying agents used in food. Proteins
reduce the oil-water interfacial tension and thus facilitate the formation of emulsions
as well as stabilize the oil droplets against coalescence (Kinsella, 1982). During the
process of emulsification, proteins with satisfactory emulsifying properties are able to
adsorb rapidly at the newly created oil-water inter-faces, followed by structural
change and rearrangement at the oil-water interface and subsequently the formation
of a cohesive film with viscoelastic properties due to intermolecular interactions
(Damodaran, 1989). Many physicochemical factors are involvedin this formation,
stability, and textural properties of emulsions (Khattab & Arntfield 2009).
Emulsification capacity (EC) was determined by the method of Beuchat et al (1975).
To the WASF, WASPC and WASPI (1 g), 25 ml of distilled water were added and mixed
in a blender at low speed. After complete dispersion, refined groundnut oil was
added; blending was continued until phase separation was seen. EC was expressed as
millilitres of oil emulsified per gram of material.
EC (ml) =
100
3.4.1.6 Dispersibility
The dispersibilities of WASF, WASPC and WASPI were measured by the
method of Karuna et al (1991). Ten grams of samples was placed in a stopperd
measuring cylinder and distilled water was added to reach a volume of 100 ml. The
mixture was stirred vigorously and allowed to settle for 3 h; the volume of settled
particles was subtracted from 100 and the difference was reported as percentage
dispersibility.
3.4.1.7 Wettability
The wettability was estimated for both samples according to the method of
Regenstein & Regenstein (1984). Two grams of WASF, WASPC and WASPI were
weighed and transferred to a beaker containing 80 ml distilled water and the behavior
of the powder was observed on the water surface immediately after adding the
sample. After 30 min observation, the material was stirred on the magnetic stirrer
sufficiently fast to form a vortex which reached the bottom of the beaker. The stirring
continued for one min and after which the grade describing wettability was recorded
as excellent, good and fair or poor according to the time and behavior of the
dispersion.
65
3.4.2 Nutritional Composition

Nutrition analysis refers to the process of determining the nutritional content
of foods and food products. The estimation of nutrient intake from food consumption
requires reliable data on food composition. These data are also the fundamentals of
food-based dietary guidelines for healthy nutrition, containing the necessary
information on food sources for different nutrients (Elmadfa & Meyer, 2010). The
moisture, ash, protein, fat, CHO and crude fibre content of WASF, WASPC and WASPI
were determined by the method described in Table 3.3. The detailed procedures for
these determinations are given in appendix IA. The energy content of WASF, WASPC
and WASPI was determined by multiplying the values obtained for protein (%), fat
(%) and CHO (%) by 4.00, 9.00 and 4.00 respectively and adding up the values
(Indrayan et al., 2005); expressed in Kcal/100g.
3.4.2.1 Mineral Extraction and Determination
Minerals are nutrients that exist in body and food in organic and inorganic
combinations. There are seventeen minerals essential for human nutrition, just only
5-3% of human body weight is mineral matter (Lin et al., 1974). Minerals of three
samples were extracted according to Chapman and Pratt (1982). Each sample was
burned in a muffle furnace at 550C. The dish which contained the samples was put in
a sand bath for 10 min after addition of 5 ml HCl. Then the solution was carefully
filtered in 100 ml volumetric flask, extracts were stored in bottles at 30C. The
extracted minerals calcium, phosphorous, iron, sodium and potassium contents were
measured with a Corning 405 flame photometer (AOAC, 1990). The detailed
procedures of mineral analysis are given in appendix IA.
3.4.2.2 Amino Acid Profile by HPLC
Amino acid composition and the ease with which digestive enzymes hydrolyze
a food protein are major determinants of food quality (Sze-Tao & Sathe, 2000). The
WASF, WASPC and WASPI were hydrolysed by incubation in 6 N HCl at 110C for 24 h.
The amino acid analysis was performed using an automated precolumn derivation
with O- phthaldiadehyde (OPA) using high performance liquid chromatography
(HPLC) analysis carried out in an Agilent 1100 (Agilent Technologies, Palo Alto, USA)
assembly system. The WASF, WASPC and WASPI were injected onto a Zorbax 80 A
C18 column (i.d., 4.6 180 mm, Agilent Technologies) at 40C with detection at 338
nm. The mobile phase A was 7.35 mm/l sodiumacetate/ triethylamine/
tetrahydrofuran (500:0.12:2.5, v/v/v), adjusted to pH 7.2 with acetic acid, while
66
mobile phase B (pH 7.2) was 7.35 mM/l sodium acetate/methanol/acetonitrile (1:2:2,
v/v/v). The amino acid composition was expressed as g of amino acid per 100 g of
protein (Tidjani et al., 2010). The detailed procedure of HPLC analysis is given in
appendix IA.
A. Chemical Score of Amino Acid
The nutritional parameter, amino acid score (%) of WASF, WASPC and WASPI
was calculated as follow according to FAO/WHO (2007).
AAS (%) =
mg of amino acid per g test protein sample

mg of amino acid per g WHO standard protein
x 100
The FAO/WHO standard for essential amino acids of child, adult and casein
protein (Abugoch et al., 2008) used for calculation of amino acid score is depicted in
Table 3.4.
TABLE 3.4: ESSENTIAL AMINO ACID CONTENT OF CHILD, ADULT
AND CASEIN REFERENCE PROTEIN (g/100g)
Amino Acid
Child*
Adult*
Casein**
Histidine
1.9
1.6
2.7
Threonine
3.4
0.9
3.7
Methionine
2.7
1.7
2.6
Leucine
6.6
1.9
8.4
Isoleucine
2.8
1.3
4.9
Phenylalanine
6.3
1.90
4.5
Lysine
5.8
1.6
7.1
Valine
3.5
1.7
6.0
Tryptophan
1.1
0.5
1.4
*FAO/WHO,
et al (2008)
2007; **Abugoch
B. Essential Amino Acid Index (EAAI)

The EAAI of WASF, WASPC and WASPI were determined on the basis of the
amino acid profiles. The Essential Amino Acid Index (EAAI) was calculated using the
method of Labuda et al (1982) according to the equation
EAAI = 9 [ ]/

where: [lysine, tryptophan, isoleucine, valine, threonine, leucine, phenylalanine,
histidine and methionine]a in test sample and [lysine, tryptophan, isoleucine, valine,
threonine, leucine, phenylalanine, histidine and the sum of methionine and cystine]b
content of the same amino acids in standard protein (%) (Casein) respectively.
67
C. Predicted Protein Efficiency Ratio (P-PER)

The P-PER was calculated for WASF, WASPC and WASPI as reported by
Alsmeyer et al (1979) using the formula as follows
P-PER = -0.468+0.454 (Leucine)-0.105 (Tyrosine)
3.4.3. Anti-Nutritional Composition
Recent epidemiological and controlled-case studies reported that many antinutrients that present in a low level give beneficial effects for prevention of diseases
like coronary diseases and cancers (Pandey & Rizvi, 2010). Due to this, they can be
considered as anti-nutritional factors with negative effects or non-nutritive
compounds with positive effects on health (Habtamu & Negusse, 2014). Phytate is
now considered to be endowed with anticancer activity (Singh & Agarwal, 2005;
Vucenik & Shamsuddin, 2006) and to possess certain health-beneficial properties,
notably antioxidative and anticalcification (prevention of kidney stone formation)
activities (Grases et al., 2000). Saponins have been associated with various health
beneficial and protective effects attributed to their antifungal and antibacterial
activity, cholesterol-lowering action and most of all, to their ability to inhibit
cancer cell growth (Jeon and Sung, 2000; Berhow et al., 2000). The antinutritional
properties in terms of tannins, oxalate, phytate and saponin content of WASF, WASPC
and WASPI were determined and detailed procedure for these determinations are
presented in appendix IB.
3.4.4. Phytochemical Screening and Quantification of Total Phenols
The preliminary screening tests may be useful in the detection of the bioactive
principles and subsequently may lead to the drug discovery and development
(Kavitha et al., 2013). The medicinal value of plants is attributed to the presence of
some chemical substances (bioactive non-nutrient plant compounds) which produce a
definite physiological action on human body (Edeoga et al., 2005). These chemical
substances are called phytochemicals (Sood et al., 2012). Some common examples of
phytochemicals are flavonoids, alkaloids, saponins, glycosides, terpenoids, tannins,
sterols and carbohydrates (Sood et al., 2012).
3.4.4.1 Preparation of the Seed Extracts
Preparation of WASF, WASPC and WASPI extracts using different solvents
(methanol, aqueous and ethanol) was done according to a combination of the
methods used by Lu & Foo (2001); Pizzale et al (2002). The WASF, WASPC and
WASPI were extracted with methanol, ethanol and aqueous extract for 1 minute using
68
an ultra turax mixer (13,000 rpm) and soaked overnight at room temperature. The
extracts were then filtered through what man No.1 paper in a Buchner funnel. The
filtered solution was evaporated under vacuum in a rotaevator at 40C to a constant
weight and then dissolved in respective solvents. The concentrated extracts were
stored in an airtight container in the refrigerator below 10C.
3.4.4.2 Qualitative Phytochemical Analysis
Qualitative phytochemical screening was done for evaluation of major
phytochemical constituents such as tannins, saponins, flavonoids, sterols, terpenoids,
carbohydrates, phenols, proteins and amino acids, alkaloids and glycosides using
standard procedure of analysis (Olayinka & Anthony, 2011; Kaur & Arora, 2011;
Sharma et al., 2011). The reactions in this analysis revealed the presence or absence
of these compounds in the WASF, WASPC and WASPI with different extractions
(methanol, ethanol and water). The detailed procedure is depicted in appendix II.
3.4.4.3 Quantification of Total Phenols
The total phenol content of WASF, WASPC and WASPI was determined in
methanol, ethanol and aqueous extracts. The detailed procedure is given in
appendix II.
3.4.5 Antioxidant Activity Profile
Primary sources of naturally occurring antioxidants are whole grains, fruits
and vegetables (Mishra et al., 2010). Antioxidants are applied in food industry as
additives limiting oxidation of food components, especially lipids. The oxidation leads
to losses in food quality and shelf life (St Angelo, 1996). The ABTS (2,2 -azinobis(3ethylbenzothiazoline-6-sulfonic acid) assay is a colorimetric assay in which the ABTS
radical decolorizes in the presence of antioxidants (carotenoids, phenolic compounds
and others). DPPH (2,2-diphenyl-1-picrylhydrazyl) is based on the premise that a
hydrogen donor is an antioxidant. This colorimetric assay uses the DPPH radical,
which changes from purple to yellow in the presence of antioxidants and is widely
used as a preliminary study (Moon & Shibamoto, 2009). FRAP method measures the
ability of antioxidants to reduce ferric iron. It is based on the reduction of the complex
of ferric iron and 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-dienechloride
(TPTZ) to the ferrous form at low pH. This reduction is monitored by measuring the
change in absorption at 593 nm, using a diode-array spectrophotometer (Alam et al.,
2013). The DPPH radical scavenging activity, ABTS radical cation decolorisation assay
and ferric reducing antioxidant power assay of WASF, WASPC and WASPI were
69
determined using standard procedures. The detailed procedures are given in

appendix III.
3.4.6 In Vitro Protein Digestibility (IVPD)
In vitro protein digestibility method is less expensive, less time consuming and
easier method for determining protein digestibility of different feed ingredients. This
method allows close observations of the dynamics of the breakdown of protein by
using only small amount of raw materials (Dimes & Haard, 1994).This was
determined by multi-enzyme method of Hsu et al (1977). To the WASF, WASPC and
WASPI weight equivalent to 6.25 mg protein/ml, 10 ml of distilled water was added. A
multi-enzyme (Sigma-Aldrich Inc, Germany) solution containing 1.6 mg trypsin, 3.1
mg chymotrypsin and 1.3 mg peptidase per ml of distilled water was equilibrated to
pH 8.0 and maintained in an ice bath. Samples were equilibrated to pH 8.0 and
maintained at 37C. A 1 ml aliquot of multi-enzyme solution was added to each
sample, stirred for exactly 10 min and pH of enzyme hydrolysate measured. In vitro
protein digestibility of sample was calculated using the following equation
% digestibility = 210.464 18.103x
where x represents the pH after 10 mins incubation.
3.4.7 Thermal Properties (DSC)
Differential scanning calorimetry (DSC) has been widely used in studying the
thermal stability of proteins and as one of the most sensitive technique for measuring
the thermodynamic parameters of thermal protein
unfolding (Chen & Oakley, 1995; Cordella et al.,
2003). Heating causes denaturation of protein as a
result from the disruption of bonds that are
involved in the formation and maintenance of the
protein structure (Stanley & Yada, 1994). The
temperature needed and the extent of these
changes was determined by the thermal stability of the protein, which can be studied
from the endothermic peaks of their differential scanning calorimetry (DSC) profiles.
DSC determines the calorimetric changes in proteins as a function of temperature. The
thermal denaturation of proteins is attributed to the rupture of intramolecular
hydrogen bonds. The denaturation temperatures are measures of the thermal stability
of proteins. Their determination under controlled conditions can provide direct
comparison of the thermal stability of the different proteins (Nurul & Azura, 2012).
70
The temperature midpoint of the transition for the enthalpy change (Tm)
occurs when the protein goes from native to denatured form. At the Tm, 50% of the
protein is in the native state, and 50% is in the denatured state, assuming a two-state
transition. Some proteins with different regions of activity or more than one
structural domain can have more than one Tm. Tm is an indicator of thermostability,
and in general, the higher the Tm, the more stable the protein. With a higher Tm, the
protein is less susceptible to unfolding and denaturation at a lower temperature as
well. By interrogating various conditions and additives, DSC can determine
formulations with the highest Tm that will correspond to the optimal formulation(s)
for stability (Schrier et al., 1993; Chan et al., 1996; Chen & Arakawa, 1996; Remmele et
al., 1998; Remmele & Gombotz, 2000; Mishra et al., 2010).
Differential Scanning Calorimetry (DSC 6220 SII model, Japan) equipped with
a thermal analysis station (Exstar Instruments) was used to determine thermal
characteristics of WASF, WASPC and WASPI (3mg) of sample was weighed onto the
aluminium DSC pan and pan was sealed and allowed to stand for 1 h at room
temperature. The scanning temperature range and heating rate were 30-140C and
10C/min, respectively, using an empty pan as reference. Onset, peak and conclusion
thermal degradation temperatures (C), enthalpy of degradation (H in J/g) were
noted to describe the thermal nature of samples (Gunaratne et al., 2011).
3.4.8 Rheological Properties (RVA)
The Rapid Visco Analyzer (RVA) is a measuring device used traditionally for
measuring starch pasting properties, but the
pasting properties of proteins can be measured
accurately. The RVA measures changing paste
viscosity during cooking or other food processing
operations (Whalen, 1995). RVA paste viscosity
measurements can reveal the extent of interaction
among a mixture of food components and the effect
of changes in physical processing conditions on
protein
functionality
(Meares
et
al.,
2004).
Elevating temperature in a mixed protein system

increases the collision of partially unfolded aggregated moleculesforming a network
(Richardson & Murphy, 1981).
71
The rheological properties of samples were determined in duplicate by a

Rapid Visco Analyzer (model RVA3D; Newport Scientific, Australia) according to AACC
standard method No. 61-02.01 (AACC, 2000). First, 25 ml of distilled water was added
directly to a metal RVA canister. Then 3.00 0.01 g of WASF, WASPC and WASPI were
weighed, added to the water and immediately measured by RVA. These measurements
were made using the standard Newport Scientific profile. An initial 10 s of high-speed
(960 rotations min-1) stirring was used to disperse the sample prior to the beginning of
the measuring phase at 160 rotations min-1. Temperature was held at 50 C for 1 min;
then raised to 95C in 3.5 mins; held for 2.5 min; cooled to 50C in 3.5 mins; and held for
2.5 mins. The RVA parameters such as peak viscosity (PV), the highest viscosity during
heating; trough (T), the lowest viscosity; break-down (BD), PV minus T; final viscosity
(FV), the viscosity at the completion of the cycle; and setback (SB), FV minus PV were
reported in cP (AACC, 2000).
3.4.9 In Vivo Protein Quality
Proteins are complex nitrogenous organic compounds occurring naturally in
all living matter and forming an essential part in animal food requirements (Alfaro et
al., 2004). Protein quality, also known as the nutritional or nutritive value of a food,
depends on its amino acid content and on the physiological utilization of specific
amino acids after digestion, absorption and minimal obligatory oxidation rates
(Friedman, 1996). Protein quality tests predict the nutritional quality of food proteins
and how available are these proteins for growth and cell maintenance. Protein quality
tests are designed to directly measure or estimate the dietary essential amino acid
content of the test protein or protein-containing food and how well the protein is
digested, absorbed and utilized in human growth and maintenance (Halimatul et al.,
2007).
The study protocol was approved by the Institutional animal ethical
committee of the Periyar University, Salem, Tamil Nadu, India (Appendix IV).
3.4.9.1 Animal Housing and Diet Preparation
The experimental diets were prepared according to Sadasivam & Manikcam
(2005) as shown in Table 3.5. Thirty male Wistar rats aged 22-31 days weighing 30-40 g
was obtained from Animal House of National school of Agro-Industrial Sciences. Animals
were divided into five groups with six animals each. The rats were placed in metabolic
cages (Picture 3.2).
72
Picture 3.2: Animal housing, feeding techniques and weight measurement of

rats
(a)
(b)
(d)
(c)
(e)
Picture 3.3: ( a) CD (Control), (b) PFD, (c) WASF diet, (d) WASPC diet and (e) WASPI diet
After an acclimatation period of 7 days during which the rats were feed standard diet,
each group of rats were fed on their experimental diets (Picture 3.3). The temperature of
laboratory was 274 C, while the experiment alternate 12 h periods of light and dark
73
(Badr et al., 2013). Rats received water and their experimental diets ad libitum for 28
days. Food intake and body weights were recorded daily and weekly respectively
using an electronic weighing balance during the experimental period (Picture 3.2).
TABLE 3.5: COMPOSITION OF THE DIETS USED IN THE EXPERIMENTS WITH
RATS (g/100g OF THE MIXTURE)
Control
Group
(g)
Exp.
Group I
(g)
Exp.
Group II
(g)
Exp.
Group III
(g)
PFD
(g)
Corn flour
70
70
70
70
80
Casein (Control)
10
WASF
10 (36)
WASPC
10 (13)
WASPI
10 (10.7)
Corn oil
10
10
10
10
10
Cellulose
Mineral salts
Vitamin mixture
Food Items
Values in parentheses indicates the quantity of WASF (g) (Exp. Group I), WASPC (g) (Exp.
Group II) and WASPI (g) (Exp. Group III)
3.4.9.2 Protein Quality Evaluation

After completion of the feeding experiment, the rats were deprived of food for
16 h, weighed and then anaesthetized by using diethyl ether. The liver, spleen,
kidneys, heart and stomach were rapidly excised and weighed in order to assess the
growth performance of the experimental rats. The difference between diet given and
refusals was taken as feed intake, which was further used to compute protein intake.
Feed intake, protein intake and bodyweight gain were used to compute the following
nutritional indices (Ijarotimi & Keshinro, 2013). Faeces and urines were collected
every day on the every week during treatment period and the nitrogen content was
determined using a Kjeldahl method. Based on the nitrogen content of the feeds,
faeces and urines, the protein efficiency ratio (PER), corrected PER, net protein ratio
(NPR), apparent digestibility (AD), true digestibility (TD), biological value (BV) and
net protein utilization (NPU) of five diets were calculated using the respective
formulas.
PER
C-PER =
Gain in body weight (g)

Protein Intake (g)
PER of Casein 2.5 PERof test protein
Exp . PER of Casein
74
Weight gain of test group + Weight loss of PFD

Protein Inatke (g)
NPR
AD
N in diet g N in feces (g)

N in diet (g)
TD
Ni (Nf 1Nf 2)
Ni
BV
NPU =
x 100
x 100
Ni Nf 1Nf 2 (NU 1NU 2)

Ni Nf 1Nf 2
x 100
BV TD
100
Ni = nitrogen of animal fed with test diet, NF1 = nitrogen execreted in faeces of animal
fed with test diet, NF2 = nitrogen excreted in faeces of animal fed with protein-free
diet. NU1 = nitrogen excreted in urine of animal fed with test diet NU2= nitrogen
excreted in urine of animal fed with protein free diet.
3.4.9.3 Clinical Observations
Each animal in the five groups was observed for the abnormalities, physical
appearance and mortality.
3.4.9.4 Biochemical Parameters and Histopathological Examination
Animals fasted overnight were euthanized on 30th day. Blood samples were
collected from each group by cardiac puncture and transferred to heparinised and
non heparinised centrifuge tubes. Non-heparinised blood samples of the animals in
each group were tested for biochemical parameters such as blood glucose, urea, BUN,
creatinine, uric acid, lipid profile (Cholesterol, triglycerides, HDL, LDL, VLDL), liver
function (Bilirubin (total), bilirubin (direct), bilirubin (Indirect)), total protein,
albumin, globulin, SGOT, SGPT, alkaline phosphatise, gamma GT, iron, TIBC, Ca, P, Mg,
Cu and amylase. Heparinised blood samples of the animals in each group were tested
for hematological parameters such as RBC, WBC, hemoglobin, hematocrit, differential
count (Neutrophils, lymphocytes, eosinophils, monocytes and basophils), platelet
count, MCV, MCH, MCHC, RDW-SD, RDW-CV, PDW, MPV,P-LCR and PCT. Hematological
and biochemical analysis was carried out using hematological and biological semi
automatic analyzer (3000 Evolution, Tulip Group, India). The detailed procedure
fordetermination of biochemical parameters are given in appendix IV.
A portion of the granulation tissue was subjected to histopathological studies.
The tissues were fixed in 10% neutral formalin solution for 24 h and dehydrated with
a sequence of ethanolxylene solution series (Mukherjee, 2000). The materials were
filtered and embedded with paraffin (40 60 C) and microtome sections of 5
75
thickness taken. The sections were again processed with ethanol-xylene solvent series
and stained with hemotoxylin-eosin dye. The histopathological changes observed
were photographed using a compound light microscope (Novex, USA).
3.5 STATISTICAL ANALYSIS
The average data (in triplicate) were tabulated, analyzed statistically and
interpreted. The statistical analysis like mean, standard deviation, one way analysis of
variance (ANOVA) with Post-Hoc comparison as LSD (Least Square Difference) test
were analyzed by the Statistical Package for the Social Sciences (SPSS) for windows,
Version 17.0.
3.6 LIMITATIONS OF THE STUDY
Further exploration of proteomic nature of wood apple seed protein isolate

through MALDI-TOF studies and Bio-informatics is limited due to time constraint.
76
Results and Discussion
4. RESULTS AND DISCUSSION

The
results
pertaining
to
the
study
entitled
Extractability
and
Characteristics of Wood Apple (Limonia acidissima) Seed Protein are discussed

under the following headings.
4.1.1 Characteristics of wood apple

4.1.2 Characteristics of wood apple seed
4.2 PHASE II - IDENTIFICATION OF EFFICIENT METHOD OF ISOLATION
(WASF), WOOD APPLE SEED PROTEIN CONCENTRATE (WASPC) AND
WOOD APPLE SEED PROTEIN ISOLATE (WASPI)
4.3.1 Physio-chemical and functional properties

4.3.2 Nutritional composition
4.3.3 Anti-nutritional composition
4.3.4 Phytochemical screening and quantification of total phenols
4.3.5 Antioxidant activity profile
4.3.6 In vitro protein digestibility
4.3.7 Thermal properties
4.3.8. Rheological properties
4.3.9 In vivo protein quality
PHASE I
4.1 CHARACTERISTICS OF WOOD APPLE AND SEED
4.1.1 Characteristics of Wood Apple

4.1.1.1 Agronomical Features
The botanical description of wood apple (Figs.4.1a and 4.1b) was from the
family Rutaceae, genus Limonia L. of species L. acidissima under synonyms F.
elephantum correa. The fruit authentication certificate was given as Fig.4.1c.
4.1.1.2 Wood Apple Fruit Components
The weight of fruit, shell, pulp, seed and fibre were analysed as components of
fruit and the results are shown in Table 4.1. In wood apple fruit, the shell comprises
more weight than pulp and seed. Whereas in mango fruits, the composition was
13.7% of shell, 10.1% of seed and 76.1% of pulp (Lemus et al., 2013). Mango seed
consists of a tenacious coat enclosing the kernel.
77
Fig.4.1a: Fruits and leaves of

wood apple
Fig.4.1b: Large woody fruit
Fig.4.1c: Authentication certificate of wood apple fruit

The seed content of different varieties of mangoes ranges from 9% to 23% of
the fruit weight (Palaniswamy et al., 1974) and the kernel content of the seed ranges
from 45.7% to 72.8% (Hemavathy et al., 1988). The pulp and seed accounting for 4%
to 12% of the total mass of the fruit was obtained from the processing of guava fruit
pulp (Uchoa-Thomaz et al., 2014).
78
TABLE 4.1: COMPONENTS OF WOOD APPLE FRUIT COMPARISON OF EXTRACTION

METHOD
Parameters
Under Tap Water
Juicer
Weight of fruit (g)
92.62.41 (100)
18718.64 (100)
Weight of shell (g)
46.93.92 (50.6)
91.614.19 (48.9)
Weight of pulp (g)
36.75.94 (39.6)
18.23.93 (9.7)
Weight of seed (g)
6.251.71 (6.7)
28.27.33 (15.0)
Weight of fibre (g)
12.92.7 (13.9)
35.410.9 (18.9)
Seed/Pulp ratio
0.160.03
1.590.45
Values are the average of ten determinants. Figure in parentheses indicates percentage.
The guava fruit consists of 20% pulp and 50% seed core (Bal et al., 2014). The
highest pulp:seed ratio was recorded in RCG-11 genotype guava (94.25) and lowest in
RCG-1 genotype guava (27.27) among eleven hybrid varieties of guava (Patel et al.,
2011). The wood apple seed was separated from whole pulp in two methods (under
tap water and juicer). The seed were collected more in juicer extraction method than
in running water process. The highest seed to pulp ratio was found in juicer
extraction method. The pulp to seed distribution was comparable to guava.
4.2.1 Characterization of Wood Apple Seed
The wood apple seed was determined for its physical and anatomical
characteristics and the results are interpreted as follows.
4.2.1.1 Physical Characteristics of Wood Apple Seed
The physical properties of seeds are essential for the design of equipment for
handling, processing, storing, sowing the seeds and are the most important factors in
determining the optimum vacuum pressure of the precision vacuum seeder (Karayel
et al., 2004). The colour of wood apple seeds was brown, similar to Achishiru
(Nigerian cowpea variety). The size and shape are important in designing of
separating, harvesting, sizing and grinding machines. The shape of the material is
important for an analytical predication of its drying behaviour (Cetin, 2007; Esref &
Halil, 2007). The study of size is essential for uniformity and packing in standard
cartons. Shape and physical dimensions, such as major, intermediate and minor
diameters, unit mass, volume and sphericity, are important in screening solids to
separate foreign materials and in sorting out various sizes of fruits and vegetables
(Stroshine, 2005). In recent years, physical properties have been studied for various
seeds such as locust bean seed (Ogunjimi et al., 2002); millet (Baryeh, 2002); quiona
seed (Vilche et al., 2003) and almond nut and kernel (Aydin, 2003), amaranth seeds
(Abalone et al., 2004), hemp seed (Sacilik et al., 2003), lentil seeds (Amin et al., 2004),
79
safflower seeds (Baumler et al., 2006), linseed (Selvi et al., 2006), coriander seeds
(Coskuner & Karababa, 2007), cucurbit seeds (Milani et al., 2007), wheat, barley,
chickpea and lentil seeds (Gursoy & Guzel, 2010), arigo seeds (Davies, 2010), Parkia
speciosa (Abdullah et al., 2011) and rice seed (Jouki & Khazaei, 2012).
The physical properties of wood apple seeds determined in this study are
shown in Table 4.2 and Fig.4.2. The longitudinal dimension (length) of the seed was
higher than the width and thickness. The dimensions of wood apple seeds were
higher than sesame seed (Tunde-Akintunde & Akintunde, 2004), coriander seeds
(Coskuner & Karababa, 2007), Tef seed (Eragrostis tef (Zucc.) Trotter) (Zewdu and
Solomon, 2007), chia seeds (Ixtaina et al., 2008) and mustard seeds (Sinapis alba L.)
(Damian, 2014) but lower than pumpkin seeds (Joshi et al., 1993), African yam beans
(Felix & Anthony, 2011), sunflower Seeds (Helianthus annuus L.) (Seifi & Alimardani,
2010; Ashwini & Vikas, 2014), rice seed (Oriza sativa) (Jouki & Khazaei, 2012) and
chickpea seeds (Ghamari et al., 2014). Dimensions of the seed are of paramount
importance in determining the aperture size of the machine to process the seed.
Apart from that, the dimensions could be useful in determining the shape of the seed.
Since the three semi-axes of the seed are unequal, the shape of wood apple seed was
considered as oblong ellipsoid.
The geometric mean diameter obtained can be used to determine the volume
and sphericity of the seed theoretically. The values were higher than tef seed
(Eragrostis tef (Zucc.) Trotter) (Zewdu & Solomon, 2007) and lower than watermelon
seed (Koocheki et al., 2007), Morama beans (Jideani et al., 2009) and Parkia speciosa
seeds (Abdullah et al., 2011). Similar result of aspect ratio and sphericity was found
in chia seeds (Ixtaina et al., 2008) and snake tomato seed (Salawu et al., 2014). The
low sphericity and aspect ratio of the seeds indicate that the wood apple seeds are
likely to slide on their flat surfaces rather than roll. This parameter is of utmost
importance in designing of hopper to handle the seeds. Similar seed volume was
found in coriander seeds (Coskuner & Karababa, 2007). The average projected area
and surface area was found to be 16.47 (0.23) and 35.46 (0.60) mm2 while the
mean volume determined for the seeds was 12.66 (0.45) mm3. This suggests that it
is advisable to store pumpkin seed at lower moisture content; since lower surface
area means less area for moisture absorption during storage (Abano & Amoah, 2011).
Similar results of size dimensions, geometric mean diameter, sphericity and
surface were found in fenugreek seed (Altuntas et al., 2005), flaxseed (Singh et al.,
2011) and Jatropha curcas seed (Salawu et al., 2013).
80
TABLE 4.2: PHYSICAL PROPERTIES OF WOOD APPLE SEED (WAS)
Physical Properties
WAS
Length (mm)
05.810.55
Width (mm)
03.610.30
Thickness (mm)
01.800.16
Dg (mm)
03.350.20
Da (mm)
03.740.67
De (mm)
06.530.50
Sphericity (%)
57.801.34
Aspect ratio (%)
62.130.15
1000 seed mass (g)
22.600.01
Projected area (mm2)
16.470.23
Surface area (mm2)
35.460.60
Volume
(mm3)
12.660.45
Dg - Geometric Mean Diameter, Da - Arithmetic Mean diameter, De - Equivalent Diameter.

Values are the average of ten determinants.
(a)
(b)
(c)
Fig. 4.2: Characteristic dimensions of wood apple seeds: L, length; W, width; T,

thickness; (a) Front view (b) Side view (c) Three-dimensional geometry
TABLE 4.3: CORRELATION OF WOOD APPLE SEED DIMENSIONS
Particulars
Ratio
Correlation Coefficient (r)
L/W
1.60
0.073
0.840NS
L/T
3.20
0.595
0.070NS
L/Dg
1.72
0.839
0.002*
W/T
1.99
-0.216
0.548NS
W/Dg
1.07
0.340
0.337NS
T/Dg
0.53
0.771
0.009*
* Correlation
p-Value
significant at the p<0.01, NS- Not Significant
81
The correlation of wood apple seed dimensions in terms of L/T, L/W, L/Dg,
W/T, W/Dg and T/Dg were determined and depicted in Table 4.3 and Fig.4.2 The
geometric mean diameter (3.35 mm) was lower than the length (5.81 mm) and width
(3.61 mm) and higher than thickness (1.8 mm). The L/T ratio exhibited the highest
value, while the L/Dg and L/W ratio were found similar. The coefficients of
correlation showed that L/Dg and T/Dg were found to be highly significant. This fact
indicates that the length and thickness of the seed was positively related to its
geometric mean diameter. Coskuner & Karababa (2007); Ixtaina et al (2008)
indicated that the length of the seed is positively related to its width, thickness and
geometric mean diameter.
The following general expression can be used to describe the relationship
among length, width and thickness of wood apple seed
L = 1.61W = 3.21T
4.2.1.2 Anatomical Characteristics
The wood apple (Limonia acidissima) seed were 5 or 6 2.55 mm in size. The
seeds are slimy and brown or dark brown in colour and the seeds are exalbuminous.
A. Seed Structure
The wood apple seed structure was viewed under polarized light microscope
for its cross sectional and longitudinal view to study its anatomical features. The
seeds were oblong elliptical. There were two cotyledons which were plano convex.
The flat sides of the cotyledons were juxtaposed and occur in close proximity within
the seed coat (Fig.4.3a). In median longitudinal section, the full embryo with
cotyledons and radicle were seen (Fig. 4.3b). The cotyledons were 20 mm long and 8
mm thick, while the radicle was thick and conical with 15 mm height and 10 mm
thickness (Fig.4.5b). Procambial strand was seen in the radicle and plumule was
minute and occurs in between cotyledons (Fig.4.3b). The cells of the cotyledons are
thin walled, polyhedral and are darkly strained. The cells contain many cell
inclusions, the starch grains being the major content.
B. Seed Coat
The wood apple seed coat (Testa) consists of three zones. The outer zone had
one or two layers brachyscelerids with lignified walls (Fig.4.4a). From the scelerotic
layer several conical bundles of hairs arose which were tightly held and conical in
shape. The surfaces of the tufts of hairs were irregularly bulged (Fig.4.4b). The
trichome bundles were up to 20 mm long. The middle part of the seed coat was thin
and includes three or four layers of tangentially elongated parenchyma cells and
82
discontinuous small clusters of lignified scelerids. The inner zone of the seed coat was
parenchymatous (sarcotesta) (Fig.4.5a). It includes six or seven layers of circular thin
walled compact cells with dense cells inclusions. The innermost layer of the
parenchymatous seed coat forms a thin layer of epidermis. The total thickness of seed
coat was 200-300 m. Along the boundary of the middle seed coat occurs a layer of
calcium oxalate crystals (Fig.4.5c). The crystals were prismatic type. The
crystalliferous layer consists of two or three rows of crystals and the layer was
continuous all along the seed coat.
Fig.4.3: (a) Vertical section of the seed, at right angles to the surface, (b) Cotyledons and
radicle in LS of the seed (Cot-Cotyledons; Pe-Procambium; Pl-Plumule; Ra-Radicle; SCSeed coat)
Fig.4.4: (a) Outer sclerotic seed coat with vertical bundles of epidermal trichomes (b)
Two trichome bundles enlarged (EPT-Epidermal trichome; MZ-Middle zone; SCTSclerotic tissue; Pa Sc- Parenchyma seed coat)
83
Fig.4.5: (a) Parenchymatous seed coat (b) Cotyledonary cells (c) Crystals in the seed
Coat (Cr-Crystals; Pa-Parenchyma; SC-Seed coat; Cot-Cotytedous; Cr-Crystals; EPEpidermis; IMS-Inner seed coat)
PHASE II
4.2 IDENTIFICATION OF EFFICIENT METHOD OF ISOLATION
The extraction of comprehensive protein populations from plants is

particularly challenging due to the metabolic and structural characteristics of plant
tissues, including the plant cell wall matrix (Giavalisco et al., 2003; Wang et al., 2003;
Rose et al., 2004; Saravanan & Rose, 2004; Carpentier et al., 2005). A number of
protein extraction protocols have been published for various plant tissues and a
particular protocol is generally optimized according to the aim of the study.
The most frequently used protocols for extraction of plant proteins involve a
precipitation step, which should separate proteins from interfering compounds.
Chemicals frequently used for this purpose are acetone, trichloroacetic acid
(TCA)/acetone and Tris-buffered phenol; this protein extraction is generally followed
by methanol/ammonium acetate precipitation (phenol) (Saravanan & Rose, 2004;
84
Pavokovic et al., 2007; Faurobert et al., 2007). Proteins from several plant species
such as banana, avocado, orange fruits, grapevine leaves and tomato pollen have been
successfully extracted using TCA/acetone or using phenol and therefore are the
methods of choice for recalcitrant plant tissues (Saravanan & Rose, 2004; Jellouli et
al., 2010). Phenol and trichloroacetic acid (TCA)-based extraction methods have been
found to give superior protein yield and good quality 2-DE gels for certain plant
tissues (Sheoran et al., 2009). The efficient extraction protocol was identified by
determining recovery and protein content of isolates, hydrophilic and hydrophobic
and surface properties, properties related to protein-protein interaction, protein
fractionation, quantitative protein estimation and protein profiling using 1D SDSPAGE electrophoresis of WASPI.
4.2.1 Recovery and Protein Content of Protein Isolates
Plants in general are recognized as recalcitrant subjects when used for
proteomic studies. Establishing a protein extraction protocol is an even bigger
challenge when proteomic analysis of fruit seeds are involved (Xie et al., 2007; Zheng
et al., 2007). The efficiency of protein extraction from any tissue depends strongly on
the efficiency of sample disruption. The wood apple seed were isolated using five
extraction methods to identify the most efficient protocol for extraction of proteins
from the seed and the results are presented in Table 4.4.
The amount of protein extracted by TCA/acetone as precipitating agent was
significantly (p<0.01) higher, which was followed by ammonium sulphate
precipitation, alcoholic precipitation, Tris-HCl extraction and phenol extraction
method in order. Rose et al (2004) revealed that homogenizing the sample in 10%
TCA dissolved in acetone was found to almost immediately precipitate proteins. It
also allowed interfering substances that dissolved in the acetone to be washed from
the precipitated proteins and provided a clean sample for IEF. Although this protocol
is effective for some plant tissues, it often results in the co-extraction of polymeric
contaminants. He & Wang (2008) reported that these contaminants precipitate with
proteins and cannot be removed by the final washing steps. Cilia et al (2009)
suggested that qualitatively the phenol extraction has been considered to give the
cleanest and most soluble pellet. Similar observation was noted in present study that
the purity of protein was relatively high (36.05%) in phenol precipitation method
though protein recovery percentage was very low (13.09%) in this method. However
the recovery percentage of protein and protein content of recovered wood apple seed
protein isolate using phenol as precipitating agent was significantly (p<0.01) lowest
85
among the extraction methods of the present study; the TCA/acetone method was
considered to be the efficient method for extraction of protein from WAS in terms of
both protein recovery and purity. In alcoholic precipitation, the purity was
moderately high (70.80%) but accordingly the protein recovery was low (49.02%)
like phenol extraction method. Although the protein recovery and purity was
moderately high (75.35% and 66.62% respectively) in ammonium sulphate
precipitation method, a considerable amount of the measured crude nitrogen could
be from non-protein sources as reported by Adewusi et al (2003); Agboola et al
(2012). The percentage of protein recovered was relatively high (36.7%) in Tris-HCl
buffer method when compared to phenol extraction method. According to Alsohaimy
et al (2007), acid precipitation in Tris-HCl buffer method might have negative effect
in the recovery of basic amino acids probably due to the chemical effects of the acidic
agent on the basic amino acids.
TABLE 4.4: PROTEIN RECOVERY AND PROTEIN CONTENT OF WASPI
Precipitating Agent
Protein Recovery (%)
Protein Content (%)
TCA/acetone
94.831.75a
86.860.32a
Phenol
13.090.44e
36.500.50e
Ammonium sulphate
75.350.45b
66.260.25c
TrisHCl buffer
36.700.61d
45.260.64d
Alcohol
49.020.43c
70.801.05b
Values are the average of three determinants. The alphabets following the values indicates
LSD between groups at p<0.01.
TCA/acetone is a very efficient precipitation method, which eliminates

proteolytic and other modifying enzymes (Wang et al., 2006). Similar results were
found in coniferous seeds with highest protein yield from TCA method than SDS and
phenol extraction (Zhen & Shi, 2011); in legume seeds, the highest protein yield from
TCA-acetone than phenol extraction (Bhardwaj & Yadav, 2013) and TCA/acetone
extraction method proved to be better than phenol extraction for Brassica seeds
(Devouge et al., 2007). The protein extraction yield and properties are influenced by
the type of extraction process and by different factors such as pH, salt concentration,
the ionic strength of the medium, net charge and electrostatic repulsions (Tan et al.,
2011).
4.2.2 Functional Properties
The functional quality of WASPI extracted by five different methods was
analyzed in terms of hydration property, surface properties, properties related to
86
protein-protein interactions, fractions of protein and SDS PAGE profile and results are
tabulated and presented as follows.
4.2.2.1 Hydrophilic and Hydrophobic Properties
The hydrophilic properties such as WAC, dispersibility, solubility and LGC;
hydrophobic property such as OAC were determined for WASPI extracted by five
different methods, compared and interpreted in Table 4.5.
A. Water Absorption Capacity
The WAC of WASPI extracted by TCA/acetone as precipitating agent was
significantly (p<0.01) high, however the least WAC was noted in ammonium sulphate
precipitation method (p<0.01) (Table 4.5). Water absorption is influenced by several
factors such as the number of hydration positions, physical environment, pH, solvent,
presence of lipids and carbohydrates (Kinsella, 1982). The low WAC (0.27 g/g) in
ammonium sulphate method could be due either to loss of solubility or protein
aggregation, thus decreasing the surface area exposed to the water phase as
mentioned by Ventatesh & Prakash (1993). The good WAC will enhance the uses of
isolate as binding agents in food processing and pharmaceutical industry (Ogundele
et al., 2013). Higher water absorption values of protein indicate the swelling ability
and property of dissociation for exposing additional binding sites (Rao et al., 2011).
WAC may be affected by conformation and environmental factors; conformational
changes in the protein molecules may expose previously enclosed amino acid side
chains, thereby making them available to interact with water (Paredes-Lopez et al.,
1991); Yusuf et al (2008) reported that the higher water absorption capacity of flour
may be due to the higher polar amino acid residues of proteins having an affinity for
water molecules. This might be the reason for higher WAC in WASPI by TCA/acetone
extracted method.
B. Oil Absorption Capacity
The OAC is an important functional trait in food industries to retention of
flavour and improvement of shelf-life and palatability. The mechanism of oil
absorption involves the physical entrapment of oil by food components and the
affinity of non-polar protein side chains for lipids (Sathe et al., 1982). The oil-holding
capacity of WASPI by TCA/acetone extraction method was significantly greater than
(<0.01) other extraction methods (Table 4.5). According to Olaofe et al (1998), WASPI
had both good water-holding and oil-holding capacity and could be utilised in food
industry, for ground meal formulation, meat substitutes and extenders, doughnuts,
baked goods and soups. The variation in OAC of WASPI extracted by different
87
methods might be due to the different property of non polar side chains of the amino
acids in the surface of the protein molecules as reported by Khalid & Elharadabou
(2013). Jitngarmkusol et al (2008) proclaimed that the major chemical component
affecting oil absorption capacity is protein, which is composed of both hydrophilic
and hydrophobic parts. Non-polar amino acid side chains can form hydrophobic
interactions with hydrocarbon chains of lipid. The high WAC and OAC of WASPI
extracted by TCA/acetone method could be explained by high amount of both polar
and non-polar amino acid residues when compared to other extraction methods.
TABLE 4.5: HYDROPHILIC AND HYDROPHOBIC PROPERTIES OF WASPI
Dispersibility (%)
Precipitating Agent
WAC (g/g)
OAC (g/g)
pH 4.5
pH 7
pH 10
TCA/acetone
1.260.05a
1.670.05a
53.830.50 ax
61.330.00 ay
68.830.11 az
Phenol
0.360.05d
0.280.03e
33.560.05 dx
37.160.05 dy
39.260.05 ez
Ammonium sulphate
0.270.05e
0.560.03c
42.060.05 bx
46.130.05 by
52.530.05 bz
TrisHCl buffer
0.530.05c
0.380.05d
24.100.10 ex
34.650.00 ey
41.400.00 dz
Alcohol
0.860.06b
0.730.07b
37.460.05 cx
40.560.05 cy
46.760.05 cz
Values are the average of three determinants. The alphabets (a,b,c,d,e) following the values
indicates LSD between groups in rows at p<0.01.The alphabets (x,y,z) following the values
indicates LSD between groups in columns at p<0.01.
C. Dispersibility
The dispersibility of a mixture in water indicates its reconstitutability
(Kullarni et al., 1991). The better temperature, ionic composition, pH and degree of
agitation of the solvent are major factors affecting dispersibility (Kinsella, 1976). The
results of dispersibility for the WASPI of different extractions, at different pH levels
(Table 4.5) exhibited that the percentage dispersibility was significantly (p<0.01)
high in TCA/acetone method irrespective of acid, neutral and alkaline pH. In acidic,
pH dispersibility of WASPI was significantly (p<0.01) lower than neutral, which was
significantly (p<0.01) lower than alkaline pH. Similarly the dispersibility of sesame
protein was significantly higher at neutral and alkaline pHs than acidic pHs (Khalida
et al., 2003). Sulieman (2007) reported that lentil protein isolate had a higher
dispersibility at pH 7.0. Similar results also found in fenugreek protein concentrate
(Nazar et al., 2007). The lowest dispersibility of 39.26% was obtained in phenol
extraction method at alkaline pH; 34.65% in Tris-HCl buffer method at neutral pH;
24.1% in Tris-HCl buffer method at acidic pH. Kinsella (1979) reported that high
88
dispersibility enhances other functional properties like emulsifying and foaming

properties during processing for the manufacture of cookies.
D. Solubility
Protein solubility of flours is helpful in forecasting the waterlipid
interactions (Kinsella, 1976). The protein solubility profile of WASPIs of different
extracts at different pH was shown in Table 4.6 and Fig. 4.6. The WASPIs extracted by
TCA/acetone and alcoholic precipitation method showed increase in solubility as pH
increases; significant at p<0.01. Similar results were shown by Erythrina variegata L.
seed proteins and L. fendleri seed protein showed high solubility at pH 10 and still
had substantial solubility at pH7, which implied that the protein may be useful in
highly alkaline, as well as, aqueous systems (Hojilla-Evangelista & Evangelista, 2009).
The presence of alkali generally improves protein solubility by causing dissociation
and disaggregation of proteins; however, high solubility at alkaline pH may also be
caused by extensive proteolysis (Kinsella, 1976). Similar results were also observed
in the proteins from different plants (Bora, 2002; Lawal et al., 2005), Ginkgo biloba
seed proteins (Deng et al., 2011) and sesame protein concentrates (Onsaard et al.,
2010). Generally, solubility decreases as the pH increases until it reaches the
isoelectric point. The loss of electrostatic repulsive forces provides beneficial
conditions for the formation of protein aggregates; high bulk density and large
diameter of the aggregates results in precipitation of protein (Singh et al., 2005); then
the solubility increases with further increase of pH. Electrostatic repulsive forces
between the positively charged proteins help to keep them apart and increase
protein-solvent interactions.
Similar observation were noted in WASPI extracted by TCA/acetone and
alcoholic precipitation method where the solubility decreases as the pH increases
until pH 4.0 (pI). Then the solubility increased with further increase in pH from 5.0.
This result was further confirmed in percentage of protein precipitation at various
pH. The pH independent solubility was also noted in canola 12S globulin by Gruener
& Ismond (1997); Lawal et al (2005) justified that electrostatic interactions which
involve ionisation of the interior non-polar groups by alkali and acid lead these
groups to disrupt the native structure of the proteins, thus shifting the equilibrium
towards the unfolded form and subsequently to expose the buried functional groups
in the protein molecules. This development causes intermolecular repulsion and
enhances protein solubilisation.
89
TABLE 4.6: PROTEIN SOLUBILITY OF WASPI
pH
TCA/acetone
Phenol
Ammonium
Sulphate
TrisHCl
Buffer
Alcohol
46.60.01
15.60.01
19.60.03
22.30.01
28.30.02
19.60.04
28.30.03
17.30.01
24.60.02
18.40.08
39.30.03
19.00.02
31.00.01
21.00.03
16.30.01
53.00.00
30.00.04
53.70.02
25.60.00
35.30.03
37.60.01
55.00.02
40.00.03
30.00.01
22.60.00
51.00.02
48.30.01
22.60.05
31.00.03
26.70.05
60.50.03
51.60.01
27.50.03
16.80.13
44.50.02
64.30.01
29.60.01
18.60.02
18.80.02
50.00.05
10
70.40.01
14.70.01
17.50.01
13.90.04
59.50.01
11
73.30.01
21.30.00
20.30.02
18.30.06
65.10.02
12
80.40.00
29.70.01
22.60.04
10.00.02
70.10.01
Protein Solubility (%)
Values are the average of three determinants.

90
80
70
60
50
40
30
20
10
0
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl buffer
Alcohol
7
pH
10
11
12
Fig.4.6: Protein solubility of WASPI

The maximum protein solubility of WASPI was noted at pH 12.0 in
TCA/acetone method and alcoholic precipitation method, pH 6.0 in phenol method,
pH 5.0 in ammonium sulphate method and pH 7.0 in Tris-HCl buffer method. Charles
& Guy (1999) reported that protein solubility in neutral salt solutions depend on
ionic solution and pH of the medium used for the extraction. Usman et al (2005)
stated that the effect of pH on the solubility of the protein that depends on whether
the desired protein is at its isolectric pH. Some protein fractions are at their minimum
solubility at their isolectric pH, while some are completely insoluble at this pH.
E. Least Gelation Concentration
Gelation is an important function in food formulation. The data in Table 4.7
shows that weak gel was formed at 8% (w/v) in TCA extracted WASPI, good gel was
90
formed at 12% even in alcoholic precipitated WASPI; the appearance of gel at 16%
and 20% was very strong; at 16% in WASPI precipitated by phenol, ammonium
sulphate and Tris-HCl buffer methods. No gel was formed at 4% in any of the
extraction methods (Table 4.7). The variation in LGC among the WASPI extracted by
different methods could be explained by gelation properties are interrelated to WAC;
gelation takes place more readily at higher protein concentration because of greater
intermolecular content during heating; high protein solubility as observed by Wiltoon
et al (1997). Similar results were reported by Ragab et al (2004) for cowpea protein
isolate. Gelation is not only an indication of protein quality but is related to type of
protein. Sathe & Salunkhe (1981) reported that various factors affect the formation of
gels, mainly non-protein components and protein solubility. Rapeseed flours,
concentrates and isolates were reported to possess poor gelation properties (Sosulski
et al., 1976). The lower LGC, the better is the gelation ability of the protein ingredient
(Akintayo et al., 1999).Variations in gelling properties may be ascribed to the ratios of
different constituents, such as proteins, carbohydrates and lipids. The least gelation
concentration was 7.5% for wheat protein isolate (Schmidt, 1981); 18% for lupin
seed protein concentrate (Sathe et al., 1982); 6% for defatted sesame seed (Inyang &
Nwadimkpa, 1992); 13% for cashew nut protein isolate (Ogunwolu et al., 2009); 18%
for bambara protein isolate (Eltayeb et al., 2011).
TABLE 4.7: LEAST GELATION CONCENTRATION OF WASPI
Precipitating Agent
Concentration
4%
8%
12%
16%
20%
TCA/acetone
++
+++
+++
Phenol
++
++
Ammonium sulphate
++
+++
TrisHCl buffer
++
++
Alcohol
++
++
++
Values are the average of three determinants. Symbols: -, no gel; +, weak gel; ++, strong gel;
+++, very strong gel.
According to Schmidt (1981), for a given type of protein, a critical

concentration is required for the formation of a gel and the type of gel varies with the
protein concentration. Considerably, higher protein concentration is usually required
for the gelation of globular proteins. Thus it implies that WASPI might comprise
more globular proteins, since LGC is at 12% and above. Adebowale & Lawal (2004)
suggested that improvement in gelation capacity following
the
increase
in
91
concentration of sample flours and protein isolate is because of decrease in

thermo-dynamic affinity of proteins for the aqueous solution, which increased the
interaction between proteins. Gelation is important in the confectionary products.
Thus, the WASPI could be used in confectionary products.
4.2.2.2 Surface Properties
The predominant surface properties such as foaming capacity, foaming
stability and emulsification capacity of WASPI was determined and the results are
interpreted as follows.
A. Foaming Properties
FC is related to the readiness of proteins to bind to the air-water interface to
form foam particles, whereas FS is related to the proteinprotein interactions that
form strong interfacial membranes that stabilized the foam particles (Kinsella, 1981).
The foaming capacity of WASPI extracted by TCA/acetone method was significantly
(p<0.01) greater than other extraction methods (Table 4.8). In alcoholic precipitation
method more than 50% FC was noted. The lowest FC of 24% was noted in ammonium
sulphate as precipitating agent. Similarly Moongngam et al (2014) noted that the
extraction of protein with pH adjustment had the greatest foaming ability whereas
cowpea protein concentrate obtain by salt precipitation (N(H4)2SO4) had lowest
ability. Foods made up by foams are e.g. whipped toppings, meringues, ice creams,
chiffon desserts and angel cakes (Kinsella, 1981; Yang and Baldwin, 1995). Hence
WASPI extracted by TCA/acetone method will act as good protein supplement in the
preparation of these food items.
The foaming properties seem to be correlated with the amount of
hydrophobic amino acids that are exposed at the surface of protein molecules
(Damodaran, 1990; Wang et al., 1999). Protein isolates with high FC does not
necessarily produce foam with high FS. The presence of polyphenols, according to
Sarker et al (1995), might be beneficial to foaming properties because polyphenols
are involved in the stabilization of proteinprotein complexes at the air-water
interface.
After 30 minutes of foam formation, 52% stability was noted in WASPI
extracted by TCA/acetone method (significant at p<0.01) (Table 4.8). Irrespective of
method of extraction, foaming capacity was directly correlated with the FS. The foam
was least stable (10.67%) in WASPI extracted by ammonium sulphate method. In
order to have foam stability, protein molecules should form continuous
intermolecular polymers enveloping the air bubbles, since intermolecular
92
cohesiveness and elasticity are important to produce stable foams (Kamara et al.,
2009). The WASPI extracted by TCA/acetone method had exhibited good
intermolecular cohesiveness and elasticity which reflected 50% foam stability after
30 minutes. Good foam stability of WASPI extracted by TCA/acetone method denotes
capacity to effectively reduce interfacial tension and favourable molecular orientation
at the oil and water interface.
B. Emulsification Capacity
The Emulsion capacity (EC) is the amount of oil that can be emulsified by a
standard amount of protein under specific conditions (Pearce & Kinsella, 1978). Many
physicochemical factors are involved in formation, stability and textural properties of
emulsions (Khattab & Arntfield, 2009). Kiosseoglou et al (1999) reported that the
emulsifying properties of seed proteins are related to the processing procedure and
to the protein composition. Several authors have reported that emulsion capacity
depends on the hydrophobic-lipophilic balance, which is affected by pH. Protein can
emulsify and stabilize the emulsion by decreasing the surface tension of the oil
droplet and providing electrostatic repulsion on the surface of the oil droplet
(Sikorski, 2002).
TABLE 4.8: FOAMING PROPERTIES AND EMULSIFICATION CAPACITY OF WASPI
Precipitating Agent
FC (%)
FS (%)
EC (ml)
TCA/acetone
66.700.50az
52.331.50 ax
24.340.56 ay
Phenol
39.331.05 cz
31.671.50 cy
10.660.50ex
Ammonium sulphate
23.661.15ez
10.671.15ex
18.330.56 cy
TrisHCl buffer
37.001.00 dz
29.331.15 dy
16.340.57 dx
Alcohol
54.660.50bz
39.331.15 by
21.001.00 bx
indicates LSD between groups in rows at p<0.01. The alphabets (x,y,z) following the values
The EC of WASPI extracted by TCA/acetone method was significantly greater

which was followed by alcoholic precipitation, ammonium sulphate precipitation,
Tris-HCl buffer extraction and the least EC in phenol extraction method (Table 4.8).
Khalid & Elharadallou (2013) suggested that addition of salt may increase the EC of
proteins by improving its solubility. Unfolding of proteins at oil and water interfaces
plays a significant role in formation and stability of emulsions. Other factors such as
adsorption kinetics, interfacial load, decrease of interfacial tension, rheology of the
interfacial film and its surface hydrophobicity also affect emulsion properties (Das &
Kinsella, 1990). Thus WASPI extracted by TCA/acetone method could be used as
93
emulsifying agent with better ability of protein to gel, solubility, surface

hydrophobicity and provide a structure for holding water, flavours, sugars and other
food ingredients useful in food application as reported by Nakai (1983).
4.2.2.3 Properties Related to Protein-Protein Interaction
Protein-protein interactions often occur as a result of food processing
designed to improve the functional properties of proteins for new product
formulations (Meyers, 2006). The properties related to protein-protein interaction
such as protein extractability and isoelectric precipitation of soluble protein of WASPI
was determined and the results are interpreted as follows.
A. Protein Extractability
The protein extractability of WASPI extracted by five different methods were
examined in terms of ratio of water to WASPI (1:70 w/v), extraction time (1 to 80
mins) and pH (2-12) and results are presented in Table 4.9 (a-c) and Figs.4.7 (a-c).
TABLE 4.9 (a): EFFECT OF TIME ON PROTEIN EXTRACTABILITY OF WASPI
Time
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl Buffer
Alcohol
10
2.10.03
1.00.01
1.30.03
1.40.01
1.20.02
20
3.80.01
1.20.00
1.40.01
2.00.01
1.50.03
30
5.50.04
1.60.01
2.00.06
2.40.00
2.20.02
40
6.20.02
2.20.01
2.10.02
2.00.03
2.70.04
50
7.30.03
3.60.05
3.40.01
3.80.01
3.90.00
60
7.80.09
4.60.00
4.00.03
4.30.02
4.70.01
70
8.20.01
5.70.03
5.30.01
5.40.00
5.20.01
80
9.00.01
6.00.05
6.20.02
7.00.01
6.50.01
TABLE 4.9 (b): EFFECT OF pH ON PROTEIN EXTRACTABILITY OF WASPI

pH
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl Buffer
Alcohol
34.60.03
22.50.01
12.90.01
24.10.06
21.90.01
35.80.01
28.70.00
20.10.02
28.90.01
24.50.02
46.80.02
29.00.03
26.30.01
33.40.00
28.90.00
56.70.08
32.00.04
31.60.01
35.90.02
33.70.01
58.90.02
35.00.01
39.40.05
42.70.04
38.10.05
64.20.02
37.00.02
40.30.00
44.20.01
42.90.01
70.60.01
42.00.05
55.40.01
48.90.02
50.70.07
72.80.00
58.00.03
58.90.02
53.50.01
51.90.01
10
77.90.01
62.80.01
60.00.00
57.10.02
65.20.02
11
82.50.03
66.00.07
62.60.01
63.20.01
67.00.01
12
93.70.01
68.00.00
64.40.05
71.00.01
72.10.01
94
TABLE 4.9 (c): EFFECT OF WATER TO WASPI RATIO ON PROTEIN EXTRACTABILITY OF

WASPI
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl Buffer
Alcohol
10
3.50.02
2.40.01
1.20.02
2.50.01
3.20.03
20
4.00.01
2.80.03
1.60.04
3.00.02
3.80.01
30
4.80.03
3.00.01
2.30.05
3.50.04
4.40.01
40
5.70.06
3.50.02
2.70.02
3.80.01
5.20.00
50
6.90.02
3.80.04
3.30.01
4.30.01
6.40.05
60
8.10.00
4.30.05
4.60.03
4.90.01
6.90.01
70
9.70.01
6.10.00
5.80.01
6.20.02
7.90.02
Protein Extracted (g/100g)
Volume
10
9
8
7
6
5
4
3
2
1
0
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl buffer
Alcohol
10
20
30
40
50
60
70
80
Extraction Time (min)
Fig.4.7 (a): Effect of time on protein extractability of WASPI

100
90
80
70
60
50
40
30
20
10
0
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl buffer
Alcohol
1
10 11
pH
Fig.4.7 (b): Effect of pH on protein extractability of WASPI
95
12
10
8
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl buffer
Alcohol
0
10
20
30
40
50
60
70
Water to WASPI ratio
Fig. 4.7 (c): Effect of water to WASPI ratio on protein extractability

TABLE 4.10 (a): REGRESSION STATISTICS OF TIME ON PROTEIN EXTRACTABILITY OF
WASPI
Regression
Equation
Adj.R2
p-Value of
Regression
Std
Error
p-Value of
Coefficient
TCA/acetone
y = 0.093x+2.04
0.934
0.000
6.66
0.034
Phenol
y = 0.080x - 0.4
0.952
0.000
0.34
0.287
Ammonium sulphate
y = 0.072x - 0.06
0.932
0.000
0.37
0.876
TrisHCl buffer
y = 0.075x+0.12
0.888
0.000
6.29
0.672
Alcohol
y = 0.076x+0.03
0.977
0.000
0.22
0.867
Method
TABLE 4.10 (b): REGRESSION STATISTICS OF pH ON PROTEIN EXTRACTABILITY OF

WASPI
Regression
Equation
Adj.R2
p-Value of
Regression
Std
Error
p-Value of
Coefficient
TCA/acetone
y = 5.631x + 23.71
0.976
0.000
2.13
0.000
Phenol
y = 4.882x + 9.548
0.926
0.000
3.32
0.018
Ammonium sulphate
y = 5.447x + 4.769
0.956
0.000
2.81
0.125
TrisHCl buffer
y = 4.401x + 14.90
0.987
0.000
1.18
0.000
Alcohol
y = 5.262x + 8.333
0.981
0.000
1.76
0.001
Method
TABLE 4.10 (c): REGRESSION STATISTICS OF EFFECT OF WATER TO WASPI RATIO ON

PROTEIN EXTRACTABILITY OF WASPI
Regression
Equation
Adj.R2
p-Value of
Regression
Std
Error
p-Value of
Coefficient
TCA/acetone
y = 0.103x + 1.971
0.968
0.000
0.33
0.002
Phenol
y = 0.053x + 1.571
0.838
0.002
0.419
0.013
Ammonium sulphate
y = 0.074x + 0.1
0.942
0.000
0.33
0.77
TrisHCl buffer
y = 0.056x + 1.785
0.939
0.000
0.25
0.001
Alcohol
y = 0.079x + 2.214
0.987
0.000
0.16
0.000
Method
96
Irrespective of effect of ratio of water, extraction time and pH, the

TCA/acetone method exhibited greater protein extractability from WASPI. A linear
relationship was noted in extractability of protein while increasing water ratio,
extraction time and pH in all methods of extraction. In order to predict optimum ratio
of water, extraction time and pH, regression equation (Tables 4.10 a-c) could be used.
B. Isoelectric Precipitation of Soluble Protein of WASPI
Proteins solubilized at pH 10 were precipitated at pH ranging from 2-6.5
(Table 4.11 and Fig. 4.8). The highest yield of precipitated water soluble wood apple
seed protein was obtained at pH 4.5 (TCA/acetone 84.6%; phenol 56%;
ammonuim sulphate 37.5%: Tris-HCl buffer 53.3% and alcohol precipitation
63.2%). (The percentage of protein precipitated was significantly (p<0.01) high in
TCA/acetone method and low in ammonium sulphate method. This further confirmed
by low soloubility of WASPI extracted by ammonium sulphate method.
TABLE 4.11: ISOELECTRIC PRECIPITATION OF SOLUBLE PROTEIN of WASPI
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl Buffer
Alcohol
27.60.02
22.00.01
08.00.02
12.00.00
28.30.01
2.5
19.60.01
18.80.03
08.70.01
20.00.01
18.30.03
14.10.03
19.30.01
08.90.03
18.80.01
21.90.01
3.5
32.10.04
21.10.05
10.00.07
23.40.05
23.70.02
73.20.05
46.00.01
25.90.01
29.30.01
50.60.04
4.5
84.60.01
58.00.06
37.30.09
53.30.06
63.20.01
70.30.00
56.90.07
30.00.01
49.20.01
52.80.00
5.5
37.60.05
45.00.01
21.00.01
25.60.00
29.40.01
29.60.01
53.00.08
18.60.01
13.40.01
35.10.01
6.5
44.30.01
51.60.01
22.00.01
17.20.02
46.30.00
% of Precipitation
pH
90
80
70
60
50
40
30
20
10
0
TCA/acetone
Phenol
Ammonium Sulphate
TrisHCl buffer
Alcohol
2.5
3.5
4.5
5.5
6.5
pH
Fig. 4.8: Isoelectric precipitation of soluble protein of WASPI at various pH

97
Similar observations were noted by Alsohaimy et al 2007) in chickpea seed

protein (81.4%), lupin seed protein (87.3%) and lentil seed protein (80%). Maximum
amount of extractable protein was noted at pH 5.5 (91 g/100 g) in wood apple seed
protein concentrate (Rao et al., 2011). Simth et al (1946) also observed maximum
precipitability of 79 g/100 g of soluble protein in linseed meal at pH 5.1.
4.2.2.4 Protein Fractionation
Fractionation of proteins is often employed to quality protein types within
food materials (Tounkara et al., 2013). Protein fractions (water-soluble/albumin,
salt-soluble/globulin, alkali-soluble/glutelin and alcohol-soluble/prolamin) were
extracted from WASPI obtained by different extraction methods (Table 4.12).
Albumins were the main fractions, contributing of 66.03 g% in TCA/acetone method,
52.66 g% in alcoholic precipitation, 46.76 g% in ammonium sulphate, 32.53 g% in
Tris-HCl buffer and 28 g% in phenol extraction. Similar observations were noted by
Morales-Arellano et al (2001) in San Juan yam bean seeds (52.1%). Globulins were
the second fraction, prolamins and glutelins were the lowest fraction in WASPI. The
albumins and globulins are mainly considered as storage proteins (Ramakrishna,
2007). Albumins are considered to have a better amino acid profile compared to
globulins and these largely account for the biologically active proteins in the seed
(Wang et al., 2003). A similar fractionation pattern was observed for amaranth
proteins (AOAC, 1980) and yam bean (San Juan and Costa-costa var.) seeds defatted
with hexane (Morales-Arellano et al., 2001).
The protein fractionation pattern of WASPI was different from those of other
legumes such as soybeans, peas and common beans in which globulins are the main
fractions (Utsumi et al., 1981; Gueguen & Barbot, 1988). The highest extraction of
albumins (66.03 g%), globulins (24.86 g%), prolamins (5.67 g%) and glutelins (6.13
g%) was noted in TCA method, Tris-HCl buffer method, Tris-HCl buffer method and
alcoholic precipitation method respectively. The high albumin extractability in
TCA/acetone method was highly correlated with protein solubility.
Hydrophobic interactions are the driving force for protein-protein
aggregation, leading to protein insolubilization (Li et al., 2014). A wide variety of
extraction and fractionation tools for proteins and peptides are available based on
their
physicochemical
and
structural
characteristics
such
as
solubility,
hydrophobicity, molecular weight, isoelectric point (pI) and so on. Generally, different
technologies focused on cell disruption, solubilization/precipitation, and enrichment
systems are needed to obtain the protein fraction of interest. Removal of interfering
98
compounds (mainly lipids, nucleic acids, phenolic compounds, carbohydrates,

proteolytic and oxidative enzymes and pigments) is crucial (Martinez-Maqueda et al.,
2013). The most common method used for the extraction of protein fractionation in
WASPI was TCA/acetone precipitation method as proposed by Damerval et al (1986);
Martinez-Maqueda et al (2013). The alcoholic extraction process after dehulling and
conventional deoiling has a high efficiency of protein recovery. Aqueous alcohols
(ethanol, isopropyl alcohol and butanol) are widely used on a commercial scale to
remove phenolics, oligosaccharides or inhibitors from defatted meals and seeds
(Moure et al., 2006). Accordingly prolamin and glutelin extraction from WASPI were
significantly (p<0.01) high in phenol and alcoholic precipitation method.
TABLE 4.12: PROTEIN FRACTIONS OF WASPI
Precipitating Agent
g of Protein Fraction/100g of Total Protein

Albumins
Globulins
Prolamins
Glutelins
TCA/acetone
66.030.15 az
20.560.05 by
0.730.05 ew
3.930.05 bx
Phenol
28.000.10 ez
16.030.05 dy
4.460.05 bx
0.330.06 ew
Ammonium sulphate
46.760.05 cz
19.330.05 cy
1.460.05 dw
2.200.00 cx
TrisHCl buffer
32.530.05 dz
24.860.06 ay
5.670.05 ax
0.860.05 dw
Alcoholic precipitation
52.660.05 bz
14.860.06 ey
1.830.05 cw
6.130.06 ax
indicates LSD between groups in rows at p<0.01.The alphabets (w,x,y,z) following the values
4.2.2.5 Quantitative Protein Estimation

Quantitative estimation of protein in WASPI by Bradford method
(Fig.4.9) revealed that TCA/acetone method (14.2 mg/ml) suggested the maximum
Protein concentration (mg/g)
amount of protein extracted and established as best extraction method.

16
14
12
10
8
6
4
2
0
TCA
Phenol
Tris-HCL
Amm. Sul.
Alcohol
Method of protein extraction
Fig.4.9: Protein estimation using Bradford assay
99
A very low amount of protein was estimated in ammonium sulphate method.

Similar level of protein estimation was exhibited in sunflower kernel of the hybrids
allium 10.04 mg/ml (Zilic et al., 2010) and 14.0 mg/ml in legumes (Vigna radiate and
Vigna mungo) extracted by Tris-HCl buffer method (Ranjan et al., 2012).
4.2.2.6 Protein Profiling using SDS-PAGE Electrophoresis
The profile of marker and WASPI extracted by five different methods were
depicted in Fig. 4.10. The relative MW of band was mainly ranged from 14.4 to 97.4
kDa. The band of WASPI extracted by TCA/acetone method consisted of 27 bands
with different intensities. A very high stained broad band was noticed between 45 to
55 kDa. The number of bands in WASPI extracted by ammonium sulphate method
revealed 16 weak stained bands. In Tris-HCl buffer extracted and alcoholic
precipitation method, only one band with strong intensity was noted between 43 kDa
and 66.2 kDa. In phenol extraction method nine bands with very weak stained were
identified.
Fig.4.10: Electrophorogram showing protein band of WASPI; Lane M: Standard

Protein Molecular Weight Marker; Lane S1: TCA/acetone precipitation; Lane S2:
Ammonium sulphate precipitation; Lane S3: Phenol precipitation; Lane S4:
Tris-HCl buffer precipitation; Lane S5: Alcoholic precipitation
According to Khoshroo et al (2013) the protein bands in WASPI ran as
separate groups with distinct molecular regions irrespective of method of extractions.
For convenient description, the whole group was classified into three groups
100
designated as (MW < 20 kDa), (20 50 kDa) and (50 120 kDa) according to the
MW of standard protein marker. A similar trend in SDS-PAGE separation was
observed between 45 and 97.4 kDa in wood apple seed meal and WASPC studied by
Rao et al (2011). Sunflower albumins are basic proteins with a MM 10-18 kDa range
(kortt & Caldwell, 1990). The SDS-PAGE profile of WASPI extracted by TCA/acetone
method showed a very similar number of proteins (Twenty-seven) when compared
to SDS-PAGE profile of sunflower seed and kernel proteins in the crude extracts for all
the genotypes in which the highest MW SDS-marker of protein observed was 94.5
kDa and the lowest was 11.5 kDa (Zilic et al., 2010). Jiang et al (1994) reported that
sunflower proteins showed bands at 21-57 kDa. As suggested by Zilic et al (2010) the
MW of proteins in WASPI ranged from 11.5-20.1 kDa could be due to 2S albumin
protein and 21-45 kDa could be due to 11s globumin named as Limonin according to
its genus name.
The result also indicated the subunits of WASPI (TCA/acetone method) with
MW of 11-15 kDa, 25-35 kDa, 40-45 kDa and 67-97 kDa was formed by disulfide bond
linked polypeptides. The major broad band with MW of 45-55 kDa was due to interchain disulfide bonds. Hence, it was evident that albumin, globulin, prolamin and
glutelin showed many bands concentrated at the MW ranged from 14.4-97.4 kDa with
major composition of MW between 11-15 kDa, 25-35 kDa, 40-55 kDa and 67-97 kDa
as reported by Mao et al (2014). As a kind of food protein, WASPI was complex with a
wide range of MW and multiple subunits. According to Mao et al (2014), the protein
separated from WAS like walnut could be considered as important functional protein
and added as food ingredient in cakes, ice-creams and desserts.
PHASE III
4.3 CHARACTERIZATION AND COMPARISON OF WOOD APPLE SEED FLOUR (WASF),
WOOD APPLE SEED PROTEIN CONCENTRATE (WASPC) AND WOOD APPLE SEED
PROTEIN ISOLATE (WASPI)
Seed proteins should possess the essential requisite functional properties

for successful utilization in various food products. These functional properties are
intrinsic physico-chemical characteristics which affect the behaviour of properties in
food systems during processing, manufacturing, storage and preparation. Critical
functional properties necessary in protein ingredients include gelation which is an
important function of proteins in food systems. The properties include emulsion
101
capacity, activities and stability. It also includes foam capacity and stability. Other
paramount functionalities are protein solubility, water and oil absorption capacity,
organoleptic properties and bulk density (Aremu et al., 2007).
4.3.1 Physio-Chemical and Functional Properties
Functional properties have been categorized as the non-nutritive characters
that food constituents play in a food system. Functionality of flour is important in the
preparation, processing, storage, quality and sensory attributes of foods. Knowledge
of functional property is critical for the development of new products and the
improvement of existing ones (Sonawane et al., 2016). The results of water and oil
absorption capacity, bulk density, foaming, emulsifying properties, dispersibility and
wettability of the wood apple seed flour, protein concentrate and protein isolate are
shown in Table 4.13.
4.3.1.1 Water Absorption Capacity (WAC)
The water-holding capacity is a critical property of proteins in viscous foods,
e.g. soups, dough, custards and baked products, because these are supposed to imbibe
water without dissolution of protein, thereby providing body thickening and viscosity
(Adeyeye et al., 1994; Seena & Sridhar, 2005).
The WAC of WASF, WASPC and WASPI was found to be 2.28 ml/g, 0.9 ml/g
and 1.15 ml/g respectively (Table 4.13). The WASF was more hydrophilic due to high
protein and carbohydrate content as reported by Voutsinas & Nakai (1983). It has
been reported that the protein concentrate exhibits poor water binding capacity
compared to that of the isolate. This is likely due to the fact that the isolates has great
ability to swell, dissociate and unfold, exposing additional binding sites, whereas the
carbohydrate and other components of the protein concentrate may impair it
(Kinsella, 1979).
Similar results were found in cowpea flour (2.27%), but were high when
compared to wheat flour (1.50%) (David et al., 2015). The water absorption
capacities of wood apple seed flour were found to be higher than 0.32 g/g for wheat,
1.68 g/g maize and 0.89 g/g cowpea reported by Owen (2001). Protein and
carbohydrate play an important role in binding water (Narayana & Rao, 1982). As
suggested by Chowdhury et al (2012) the proteins in WAS consist of more
hydrophilic subunit structure than wheat flour protein which can bind more water.
The high value of carbohydrate (may be presence of hydrocolloids, starch) could lead
to increased water absorption on higher supplementation (Narayana & Rao, 1982).
Water absorption capacity represents the ability of the products to associate with
102
water under conditions when water is limiting such as doughs and pastes. It suggests
that wood apple seed flour would be useful in foods such as bakery products which
require hydration to improve handling features. The WAC of WASPC could be
comparable with unfermented vacuum dried roasted peanut protein concentrate
(0.83 ml/g) (Yu et al., 2007); comparatively low in comparison with WAC of
fenugreek protein concentrate (1.68 ml/g) (El-Narsi & El-Tinay, 2007); seasame seed
protein concentrate (SPC-pH 9.0 1.98 g/g); SPC-pH 11.0- 3.53 and SPC-salt 2.32 g/g)
(Onsaard et al., 2010) and pumpkin defatted seed meal (2.94 g/g) (RodriguezMiranda et al., 2016).
The WAC of WASPI was low in comparison with cowpea protein isolate (1.68
ml/g) (Mwanjala et al., 1999); beach pea protein isolate (2.57-2.88 ml/g) (Chavan et
al., 2001); pigeon pea protein isolate (2.5 g/g) (Eltayeb et al., 2010); seasame seed
protein isolate (6.06 g/g) (Onsaard et al., 2010) and bambara protein isolate ( 2.21
ml/g) (Eltayeb et al., 2011).The difference in water-binding capacities of protein
isolates maybe due to protein concentration and possibly their conformational
characteristics (Chavan et al., 2001).
Water-binding capacity of proteins is a function of several parameters,
including size, shape, steric factors, conformational characteristics, hydrophilichydrophobic balance of amino acids in the protein molecules as well as lipids,
carbohydrates and tannins associated with proteins. Thermodynamic properties of
the system (interfacial tension, energy of bonding), physiochemical environment (pH,
ionic strength, vapour pressure, temperature, presence or absence of surfactants) and
solubility of protein molecules are among major factors responsible for the waterbinding capacity of protein isolates (Chou & Morr, 1979).
4.3.1.2 Oil Absorption Capacity (OAC)
Oil absorption capacity is attributed mainly to the physical entrapment of oils
(Kinsella, 1979). It is an indication of the rate at which the protein binds to fat in food
formulations (Singh et al., 2005). The OAC was observed to be significantly (p<0.01)
higher in protein isolate (1.46 ml/g) than the protein concentrate (1.06 ml/g) and
seed flour (0.9 ml/g) (Table 4.13). This could result from higher protein content of
the protein isolate leading to a greater extent of hydrophobic interaction between
protein and fat as suggested by Nassar (2008).The differences in fat absorption
capacity between the samples might be due to the presence of more non-polar amino
acids in WASPI and WASPC than in WASF, and also due to the partial denaturation of
proteins with exposition of hydrophobic amino acid groups during the process of
103
protein isolate production. The presence of several non-polar side chains may bind
the hydrocarbon chains of fats, thereby resulting in higher absorption of oil (Sathe et
al., 1982).
The OAC of WASF was observed low when compared to watermelon seed
flour (2.1 ml/g) (Akobundu et al., 1982); yam chick pea flour (2.60 g/g) (Valim &
Batistuti, 1998); prickly pear seed flour (3.6 g/g) (Nassar, 2008); potato flour (1.68
g/g), green gram flour (1.60 g/g), wheat flour (1.46g/g) and rice flour (1.24 g/g)
(Chandra and Samsher,2013); and bean seed flour (1.52 g/g) (Kisambira et al., 2015).
The water and oil binding capacity of food protein depend upon the intrinsic factors
like amino acid composition, protein conformation and surface polarity or
hydrophobicity. Chau & Cheung (1997) reported that surface area and
hydrophobicity improve oil absorption capacity. The ability of the proteins of these
flours to bind with oil makes it useful in food system where optimum oil absorption is
desired. This makes flour to have potential functional uses in foods such as sausage
production. The OAC also makes the flour suitable in facilitating enhancement in
flavor and mouth feel when used in food preparation. Due to these properties, the
protein probably could be used as functional ingredient in foods such as whipped
toppings, sausages, chiffon dessert, angel and sponge cakes etc. The lower oil
absorption capacity of WASF could be due to low hydrophobic proteins which show
superior binding of lipids (Adeleke & Odedeji, 2010).
The OAC of WASPC was higher when compared to unfermented vacuum dried
peanut protein concentrate (0.9 ml/g) (Yu et al., 2007) and fluted pumpkin seed
concentrate (0.65 g/g) (Wani et al., 2011); but was low in comparison with cowpea
protein concentrate (1.60-2.2 ml/g) (Lin & Zayas, 1987); fenugreek protein
concentrate (1.56 ml/g) (El-Nasri and El-Tinay, 2007); unfermented roasted peanut
protein concentrate (1.67 ml/g) (Yu et al., 2007); cashew nut concentrate (3.32 ml/g)
(Ogunwolu et al., 2009); walnut protein concentrate (2.50 g/g) (Mao & Hua, 2012);
and winged bean concentrate (1.5 ml/g) (Dwiani et al., 2014). Variation in oil
absorption capacity might be due to the different proportion of non polar side chains
of the amino acids on the surface of the protein molecules. Several authors have
reported the oil absorption capacity reflected the non polar side chains of the protein
as well as to different conformational features of the protein (Khalid & Elharadallou,
2013).
OAC of WASPI was higher in comparison with cowpea protein isolate (1.11
g/g) (Ragab et al., 2004); mungbean protein isolates (1.13 g/g) and pea protein
104
isolates (1.40 g/g) (Butt and Butool, 2010); pigeon pea isolate (1.30 g/g) (Eltayeb et
al., 2010); and melon protein isolates (1.12 g/g) (Wani et al., 2011). But similar in
comparison with sesame protein isolate (1.5 ml/g) (Khalida et al., 2003); cowpea
protein isolates (1.45 g/g) and was lower than that of chickpea protein isolate (1.7
ml/g) (Lopez et al., 1991); pigeon protein isolates (1.68 g/g); carinata protein isolate
(2.17 ml/g) (Pedroche et al., 2004); walnut protein isolate (3.11 g/g) (Mao & Hua,
2012) and almond protein isolate (2.92 g/g) (Sze-Tao & Sathe, 2000). The relatively
high oil absorption capacity of WASPI suggests that it could be useful in food
formulation where oil holding capacity is needed such as sausage and bakery
products (Ahamed et al., 2007).
TABLE 4.13: PHYSIO-CHEMICAL AND FUNCTIONAL PROPERTIES OF WASF, WASPC AND
WASPI
Properties
WASF
WASPC
WASPI
Water absorption capacity (ml/g)
2.28+0.01cz
0.9+0.01ax
1.15+0.00by
Oil absorption capacity (ml/g)
0.92+0.00ax
1.06+0.00by
1.46+0.00az
Foaming capacity (ml)
85.00+1.00ax
138.00+0.34by
174.00+0.05cz
Foaming stability (ml) (after 30

mins)
72.00+0.00ax
(84.7%)
132.00+0.05by
(95.7%)
169.33+0.01cz
(97.3%)
Emulsifying capacity (ml)
53.66+0.50cz
30.33+0.00ax
36.67+0.01by
Bulk density (g/ml)
0.37+0.00ax
0.55+00by
0.75+0.00cz
Dispersibility (%)
41.70+0.01ax
65.90+0.02by
74.23+0.00cz
indicates LSD between groups in rows at p<0.01. The alphabets (x,y,z) following the values
indicates LSD between groups in columns at p<0.01. Figure in parentheses indicates
percentage.
4.3.1.3 Foaming Properties

The foaming capacity (FC) of a protein refers to the amount of interfacial area
that can be created by the protein and foam stability (FS) refers to the ability of
protein to stabilize against gravitational and mechanical stresses (Fennema, 1996).
Foam formation and foam stability are a function of the type of protein, pH,
processing methods, viscosity and surface tension (Yasumatsu et al., 1972; Fekria et
al., 2012). Akubor & Chukwu (1999) reported that foams are used to improve the
texture, consistency and appearance of foods. Foam stability is important since the
usefulness of whipping agents depend on their ability to maintain the whip as long as
possible (Lin et al., 1974). The WASPI, because of its higher protein content,
possessed good amount of foaming capacity (174 ml) with greater stability (169 ml
105
after 30 mins) when compared with WASPC and WASF which showed only 138 ml
and 85 ml foam volume and the stability was also poor (6 ml and 13 ml after 30 mins)
(Table 4.13). As suggested by Mao & Hua (2012), the WASPI had a more flexible
protein structure in aqueous solutions and interacted strongly at the airwater
interface to form more stable foams when compared to the WASPC and WASF.
The foaming capacity and stability of WASF was greater than watermelon
seed flour (18.7 ml and 93.5% after 30 mins) (Lakshmi & Kaul, 2011); walnut flour
(24.35 % and 10.23 % after 30 mins) (Mao & Hua, 2012); wheat flour (12.9 % and
1.94 %); rice flour (3.52 % and 0.98 %); green gram flour (24.2% and 14.07%); yam
bean seed flour (42 % and 28 % after 60 min) (Kisambira et al., 2015).
The FC and FS of WASPC was greater than prickly pear protein concentrate
(50% and 30% after 30 mins) at pH 6.0 (Nassar, 2008); cashew nut protein
concentrate (40 % and 40 % after 60 min) (Ogunwolu et al., 2009); wood apple seed
protein concentrate with 58 ml of foaming capacity and 50 ml of foaming stability
after 75 mins (Rao et al., 2011); walnut protein concentrate (38.78% and 28.18%
after 30 mins) (Mao & Hua, 2012). When compared to result noted by Rao et al
(2011) in WASPC, the present study revealed greater FC while observing similar FS.
WASPC revealed greater FC and FS than other studied protein concentrates.
It was also evident that WASPI showed greater foaming capacity and stability
than freeze dried field pea protein isolates (143%) (Sumner et al., 1980); cowpea
protein isolates (35.30 % and 79.40%) and pigeon pea protein isolates (34.0% and
77.80%) (Mwasaru et al., 2000); foaming capacity of cowpea protein isolates (65%)
(Ragab et al., 2004); cashew nut protein isolates (45% and 55% after 30 mins)
(Ogunwolu et al., 2009);pigeon pea protein isolate (68% and 71% after 60 mins);
cowpea protein isolate (68% and 65% after 60 mins); mungbean protein isolate (110
% and 58 % after 60 mins); pea protein isolate (78% and 79% after 60 mins) (Butt
and Batool, 2010); watermelon seed protein isolate (28 ml and 87.4% after 30 mins)
(Lakshmi & Kaul, 2011) and walnut protein isolate (46.34% and 30.56% after 30
mins) (Mao & Hua, 2012). Foam capacity requires rapid adsorption of protein at an
air-water interface during whipping or bubbling, ability to undergo rapid
conformational change and rearrangement at the interface. Conversely, foam stability
requires a thick, elastic, cohesive, continuous, air permeable protein film around each
gas bubble (Were et al., 1997). The foam capacity and stability were enhanced by high
protein concentration and high protein concentration increases the viscosity and
facilitates the formation of a multilayer, cohesive protein film at the interface
106
(Damodaran, 1997). Thus it was opined that WASF, WASPC and WASPI with greater
FC and FS might act as excellent protein supplement in ice-creams, cakes etc.
4.3.1.4 Emulsion Capacity (EC)
The ability of proteins to form emulsions is important as it permits
development of foods in which proteins and lipids are held together. Proteins being
composed of charged, non-charged polar and nonpolar amino acids can serve as
emulsifiers, able to interact with both water and oil in food systems (Amadou et al.,
2010). The emulsion capacity of WASF (53.66%) was significantly (p<0.01) higher
than the protein concentrate (30.3%) and protein isolate (36.6%) (Table 4.13).
Several other authors have reported that different factors may affect emulsifying
properties, such as net charge, pH, inter-facial tension and protein conformation
(Hung & Zayas, 1991; Lawal et al., 2004). The capacity of proteins to enhance the
formation of emulsion is important for many applications in cakes, coffee whiteners
and frozen desserts (Elkhalifa & Bernhardt, 2010). WASPI showed a higher activity
than WASPC, which could be attributed to the concentration of the protein. Similar
result was found in watermelon seed defatted flour and isolate (Lakshmi & Kaul,
2011). The capacity of a protein to stabilize an emulsion might be expected to be
related to the interfacial area that can be coated by the available protein (Ivey et al.,
1970).
The EC of WASF was higher when compared to yam bean seed flour 35.7%
(Kisambira et al., 2015), defatted C. Lanatus and L. acidissima flour (47.58% and
40.60%) (Sonawane et al., 2016); similar to walnut flour (53.28%) (Mao & Hua,
2012). The difference in emulsifying activity of WASF, WASPC and WASPI, probably
be due to the difference in protein concentration. At low protein concentration,
protein adsorption at the oilwater interface is diffusion controlled, since it will
spread over the surface before it can be adsorbed. At high protein concentration, the
activation energy barrier does not allow protein migration to take place in a
diffusion-dependent manner (Sze-Tao & Sathe, 2000). Therefore, emulsifying activity
was decreased with increased protein concentration. Similar result was found in
walnut flour, walnut protein concentrate and walnut protein isolate (Mao & Hua,
2012).
The EC of WASPC was lower than fenugreek protein concentrate (135 ml/g at
pH 12) (El-Nasri & El-Tinay, 2007); prickly pear seed protein concentrate (50% at pH
6.0) (Nassar, 2008); walnut protein concentrate (52.98%) (Mao & Hua, 2012); black
cowpea protein concentrate (80.25%) (Moongngarm et al., 2014).
107
The EC of WASPI was lower than pigeon pea (49%), cowpea (47.50 %), peas
(45.50%) and mung bean (41.10%) protein isolates (Butt & Batool, 2010); walnut
protein isolate (50.01%) (Mao & Hua, 2012).Several natural and processed foods,
such as milk, egg yolk, coconut milk, soy milk, butter, margarine, mayonnaise,
spreads, salad dressings, frozen desserts, frankfurter, sausage and cakes, are
emulsion-type products where proteins play an important role as emulsifiers. Plant
proteins normally have poor surface activity compared to animal protein such as
ovalbumin and bovine serum albumin which are widely utilized for emulsification
role in food systems. The difference in the surface activity between the plant and
animal protein is related to the difference in protein conformation and the presence
of other macronutrients such as carbohydrates which may not necessarily have good
emulsification properties (Damodaran, 1996).
4.3.1.5 Bulk Density
The bulk density of the flours could be used to determine their handling
requirement, because it is the function of mass and volume (Owen, 2001). Bulk
density is also important in infant feeding where less bulk is desirable. The bulk
density of WASPC was significantly (p<0.01) higher (0.75 g/ml) than the WASPC
(0.55g/ml) and WASF (0.37 g/ml) (Table 4.13). The flour was the least dense it would
occupy greater space and therefore would require more packaging material per unit
weight and so could have high packaging cost (Shittu & Tanimu, 2012), however,
flour would be easier to transport as it was lighter.
The bulk density of WASF was lower in comparison with cowpea flour (0.64
g/cm3) (Akubor et al., 2003); chickpea flours (0.54 0.57 g/ml) (Kaur & Singh, 2005);
field pea flour (0.5410.562 g/ml) and pigeon pea flour (0.4710.467 g/ml) (Kaur et
al., 2006); tigernut (Cyoperus esculentus) seed flour (0.55-0.62 g/cm3) (Oladele &
Aina, 2007);pea seed flour and pigeon seed flour (0.55 g/cm3 and 0.46 g/cm3) (Butt &
Batool, 2010); and yam bean seed flour (0.59 g/cm3) (Kisambira et al., 2015). The low
bulk density of wood apple seed flour would be an advantage in the use of the flour
for preparation of complementary foods (David et al., 2015).It was reported that the
low values of bulk densities make the flour suitable for high nutrient density
formulation of foods (Shad et al., 2011).
The bulk density of WASPC was higher than cashew nut protein concentrate
(0.31 g/ml) (Ogunwolu et al., 2009) and lower than fenugreek protein concentrates
(0.66 g/ml) (El-Nasri & El-Tinay, 2007).
108
The bulk density of WASPI was higher than that of sesame protein isolate and
soy protein isolate (0.16 and 0.21 g/ml) respectively (Kanu et al., 2007); cashew nut
protein isolate (0.25 g/ml) (Ogunwolu et al., 2009); cow pea protein isolate (0.71
g/cm3); pea protein isolate (0.68 g/cm3); mungbean protein isolate (0.55 g/cm3) and
pigeon pea protein isolate (0.53 g/cm3) (Butt & Batool, 2010). The higher bulk
density is desirable for it great ease of dispersibility and reduction of paste thickness
which is an important factor in convalescent child feeding (Padmashree et al., 1987).
4.3.1.6 Dispersibility
The dispersibility of a mixture in water indicates its reconstitutability
(Kullarni et al., 1991). The result on dispersibility of WASF, WASPC and WASPI (Table
4.13) revealed a significantly (p<0.01) highest dispersibility in WASPI (74.2%), which
was followed by WASPC (65.9%). The lowest dispersibility of 41.7% was noted for
WASF. High dispersibility enhances other functional properties like emulsifying and
foaming properties during processing for the manufacture of cookies (Kinsella, 1979).
Accordingly high dispersibility of WASPI reflected high foaming capacity of WASPI
than WASPC and WASF.
The dispersibility of seasame protein isolate was 85% at neutral pH (Khalid et
al., 2003); 65.35% for fenugreek protein concentrates at neutral pH (El-Nasri & ElTinay, 2007).The better temperature, ionic composition, pH and degree of agitation of
the solvent are major factors affecting dispersibility (Kinsella, 1976).
4.3.1.7 Wettability
Wettability is an important property when protein powders are dispersed to
produce aqueous beverages and batters (Zayas, 1997). Wettability of proteins is
affected by surface polarity, topography, texture, area and by the size and
microstructure of the protein particles (Hagerdal & Lofqvist, 1978). Wettability grade
of WASPI was excellent since it wet slightly when it comes in contact with water and
after 30 mins the wetted sample and the powder sunk to the bottom. Similar result
was found in pigeon pea protein isolate as it took 27 mins for complete wetness
(Eltayeb et al., 2010). This time of wetting was less than that of cotton seed protein
isolate reported by Elkhatim (1994); watermelon protein isolate reported by Hayat et
al (1999).
The WASF and WASPC had good wettability property as 80% of sample got
wet in 30mins. Similar result was found in watermelon protein isolate (Hassan,
1994); chickpea flour (Osman, 2004); and groundnut cultivars (Fekria et al., 2012).
109
4.3.2 Nutritional Composition

4.3.2.1 Centesimal Composition
The centesimal composition of seed flour, protein concentrate and protein
isolate of L. acidissima seed is represented in Table 4.14. It could be observed that
there were significant (p< 0.01) differences among the studied wood apple seed flour,
protein concentrate and protein isolate in their contents of crude protein, crude
lipids, crude fiber, ash, moisture and carbohydrate.
A. Moisture
The WASF had significantly (p<0.01) highest moisture content than WASPC
and WASPI (Table 4.14). The moisture content of WASF, WASPC and WASPI were
within the acceptable limit of not more than 10% for long term storage of flour (Singh
et al., 2005). Moisture content of foods is influenced by type, variety and storage
condition (Eshun, 2012). The low moisture content of WASF, WASPC and WASPI
would enhance the storage stability by avoiding mould growth and other biochemical
reactions (Singh et al., 2005).
The moisture content of WASF (4.06%) was lower when compared to
pumpkin seed flour (5.02%) (Aisegbu, 1987); baobab seed flour (4.3%) (Osman,
2004); gourd seed flour (4.85%) (Ogungbenle, 2006); papaya seed flour (7.6-8.1%)
(Adesuyi & Ipinmoroti, 2011); watermelon seed flour (4.86%) (Lakshmi & Kaul,
2011); A. africana seed (7.4 5%) (Adebayo & Ojo, 2013); African yam bean seed flour
(9.23%) (Ndidi et al., 2014); brebra seed flour (4.24%) (Andualem & Gessesse, 2014);
avocado seed flour (8.6%) (Ifesan et al., 2015) and raw groundnut flour (5.08%)
(Mustapha et al., 2015); but higher in comparison with toasted and sundried
groundnut flour (3.02% and 3.05%) (Mustapha et al., 2015).
The moisture content of WASPC (1.7%) was lower in comparison of almond
seed protein concentrate (4.25%) (Yusuf, 2003); watermelon defatted flour (3.80%)
(Lakshmi & Kaul, 2011) and walnut protein concentrate (4.48%) (Mao & Hua, 2012).
The moisture content of WASPI (0.08%) was lower in comparison of hemp
protein isolate (2.79%), soy protein isolate (2.89%) (Wang et al., 2008); watermelon
protein isolate (5.00%) (Lakshmi & Kaul, 2011); walnut protein isolate (4.50%) (Mao
and Hua, 2012); Mucuna and Canavalia beans protein isolate (8.9% and 9.9%)
(Metsagang et al., 2013). As suggested by Mustapha et al (2015), WASF, WASPC and
WASPI was suitable and adaptable for further use in food formulation of infant
feeding due to their low moisture content.
110
B. Ash
The ash content corresponds with quantity of minerals and was found
significantly (p<0.01) higher in WASPC (4.5%) than WASF (3.8%) and WASPI (0.4%)
(Table 4.14). The ash content in WASF (3.83%) was higher when compared
toamaranthus cruentus seed flour (3.35%) (Escudero et al., 2004); snake gourd seed
flour (2.93%) (Yusuf et al., 2007); Arachis hypogaea seed flour (1.38-1.48%) (Ayoola
& Adeyeye, 2010); watermelon seed flour (2.87%) (Lakshmi & Kaul, 2011); A.
Africana seed flour (1.18%) (Adebayo & Ojo, 2013); brebra seed flour (3.24%)
(Andualem & Gessesse, 2014); African yam bean seed flour (2.61%) (Ndidiet al.,
2014); breadnuts seeds (3.43%) and yam bean seed flour (3.36%) (Adeleke &
Abiodun, 2010); groundnut seed flours (1.51-2.33%) (Mustapha et al., 2015) and
avocado seed flour (2.4%) (Ifesan et al., 2015); similar to baobab seed flour (3.8%)
(Osman, 2004); but lower when compared to pumpkin seed flour (5.08%) (Fagbemi,
2007) and papaya seed flour (9.9-11.5%) (Adesuyi & Ipinmoroti, 2011).
The ash content of WASPC (4.56%) was higher in comparison to almond seed
protein concentrate (2.15%) (Yusuf, 2003); amaranthus cruentus seed protein
concentrate (3.70%) (Escudero et al., 2004); defatted cashew nut flour (4.4%)
(Vincent et al., 2009) and walnut protein concentrate (Mao & Hua, 2012); but lower
in comparison with watermelon defatted seed flour (5.21%) (Lakshmi & Kaul, 2011).
The ash content of WASPI (0.40%) was lower in comparison of hemp protein
isolate (2.38%) (Wang et al., 2008); soy protein isolate (2.63%) (Wang et al., 2008);
watermelon seed protein isolate (1.28%) (Lakshmi & Kaul, 2011); walnut protein
isolate (2.27%) (Mao and Hua, 2012); Mucuna and Canavalia beans protein isolate
(3.12% and 3.72%) (Metsagang et al., 2013). Pomeranz & Clifto (1981) reported that
ash contents of seeds should be in the range 1.5-3.5%, suitable for consumption. The
ash contents of WASF and WASPI fall within this range thus it can be recommended
for human consumption.
C. Protein
The WASPI (92.93%) had significantly (p<0.01) higher value of protein
content compared to WASPC (76.46%) and WASF (27.56%) (Table 4.14). This could
be due to the removal of fat content. The WASF (27.56%) is highly comparable to
protein rich foods such as soybean, cowpea, pigeon pea, melon, pumpkin and snake
gourd seed flours ranging between 23.1-33.0% (Olaofe & Sanni, 1988). The WASF
contain higher content of protein when compared to amaranthus cruentus seed flour
(16.60%) (Escudero et al., 2004); baobab seed flour (18.4%) (Osman, 2004); benni
111
seed flour (16.418.1%) (Yusuf et al., 2008); breadnut seed flour (4.8%) (Adeleke &
Abiodun, 2010); A. Africana seed flour (18.75%) (Adebayo & Ojo, 2013); African yam
bean seed flour (21.78%) (Ndidi et al., 2014); avocado seed flour (23%) (Ifesan et al.,
2015); groundnut flour (24.48-26.37%) (Musthapa et al., 2015); similar to
watermelon seed flour (27.5%) (Lakshmi & Kaul, 2011); but lower in comparison
with soybean seed flour (35.1%) and melon seed flour (33.3%) (Olaofe & Sanni,
1988); pumpkin seed flour (30.42%) (Fagbemi, 2007); snake gourd seed flour
(30.18%) (Yusuf et al., 2007); papaya seed flour (29.1-36.3%) (Adesuyi & Ipinmoroti,
2011); Feronia Limonia L. seed flour (33.79%) (Rao et al., 2011); brebra seed flour
(29.7%) (Andualem & Gessesse, 2014); L. Acidissima seed flour (33.7%) (Sonawane et
al., 2016). Therefore, the seeds can be used as alternative sources of protein in diets.
The protein content of WASPC (76.50%) was lower in comparison to almond
seed protein concentrate (89.95%) (Yusuf, 2003); bambara protein concentrate
(85.97%) (Eltayeb et al., 2011); but higher in comparison to amaranthus cruentus
seed protein concentrate (52.56%) (Escudero et al., 2004); watermelon seed defatted
flour (61.29%) (Lakshmi & Kaul, 2011); similar to L. acidissima seed protein
concentrate (77%) Sonawane et al., 2016).
The protein content of WASPI (92.9%) was higher in comparison of hemp
protein isolate (90.5%) (Wang et al., 2008); watermelon seed protein isolate (87.9%)
(Lakshmi & Kaul, 2011); Mucuna and Canavalia beans protein isolate (85.2% and
63.8%) (Metsagang et al., 2013); similar to soy protein isolate (92.7%) (Wang et al.,
2008).
This result showed that the WASF, WASPC and WASPI can supply the
recommended daily intake of protein for children (23.0 -36.0g/100g) (Ferrao et al.,
1987). These results indicate that wood apple seeds can be included in food
formulations as a source of protein.
D. Fat
The WASF (33.43%) had significantly (p<0.01) higher value of fat content
than WASPC (1%) and WASPI (0.01%) (Table 4.14).The crude fat of WASF was low
when compared to pumpkin seed (47.01%) (Fagbemi, 2007); groundnut flour (40.142.13%) (Musthapa et al., 2015); but higher in comparison with baobab seed flour
(12.2%) (Osman, 2004); amaranthus cruentus seed flour (8.77%) (Escuderoet al.,
2004); breadnut seeds (3.4%) (Adeleke & Abiodun, 2010); papaya seed flour (29.431.6%) (Adesuyi & Ipinmoroti, 2011); A. Africana seed flour (16%) (Adebayo and Ojo,
2013); African yam bean seed flour (2.84%) (Ndidi et al., 2014); brebra seed flour
112
(48.5%) (Andualem & Gessesse, 2014); avocado seed flour (14%) (Ifesan et al.,
2015); and similar to Corchorous olitoriusseed flour (32.95%) (Oloye et al., 2014). Fat
is an important food constituent because it promotes fat soluble vitamins absorption
(Ranhotra et al., 1984).
The fat content of WASPC (1%) was lower when compared to amaranthus
cruentusseed protein concentrate (5.90%) (Escudero et al., 2004) and Watermelon
seed defatted flour (0.95%) (Lakshmi & Kaul, 2011).
The fat content of WASPI (0.01%) was lower in comparison of hemp protein
isolate (0.36%) and soy protein isolate (0.42%) (Wanget al., 2008); watermelon seed
protein isolate (0.75%) (Lakshmi & Kaul, 2011); M. pruriens protein isolate (2.01 %)
and C. Ensiformis protein isolate (3.01%) (Metsagang et al., 2013); lupine seed isolate
(0.09%) (Lopez, 2015).During protein isolation, the fats are lost probably due to the
non solubilisation in the TCA/acetone solution of extraction (Metsagang et al., 2013).
E. Carbohydrate
The wood apple seed flour had significantly (p<0.01) higher carbohydrate
value (23%) than protein concentrate (11.59%) and protein isolate (0.5%) (Table
4.14). The carbohydrate content of WASF was higher when compared to soyabeans
(20%); fluted pumpkin seed flour (11.4%) (Fagbemi, 2007); snake gourd flour
(7.59%) (Yusuf et al., 2007); groundnut flour (4.2 9.1%) (Musthapa et al., 2015); but
lower than groundnut flour (26%) (Rao et al., 1989); baobab seed flour (45.1%)
(Osman, 2004); defatted cashew flour (25.3%) (Vincent et al., 2009); breadnut seed
flour (26.1%) (Adeleke & Abiodun, 2010); papaya seed flour (8.4-12.1%) (Adesuyi &
Ipinmoroti, 2011); A. Africana seed flour (55.59%) (Adebayo & Ojo, 2013);Corchorous
olitoriusseed flour (27%) (Oloye et al., 2014); African yam bean seed flour (63.51%)
(Ndidi et al., 2014); brebra seed flour (14.3%) (Andualem & Gessesse, 2014) and
avocado seed flour (44.7%) (Ifesan et al., 2015).
The carbohydrate content of WASPC (11.59%) was higher in comparison to
almond seed protein concentrate (3.15%) (Yusuf, 2003); lower in comparison with
winged bean protein concentrate (13%) (Dwiani et al., 2014); asparagus bean flour
protein concentrate (24.27%) (Alagbaoso et al., 2015).
The carbohydrate content of WASPI (0.50%) was lower in comparison of
asparagus lupine seed isolate (3.3%) (Lopez, 2015); bean flour protein isolate
(2.08%) (Alagbaoso et al., 2015); Parkia biglobosa seeds protein isolate (8.8%)
(Ogunyinka et al., 2016).
113
TABLE 4.14: PROXIMATE COMPOSITION OF WASF, WASPC AND WASPI
Parameters
WASF
WASPC
WASPI
Moisture (g%)
4.06+0.05c
1.7+0.05b
0.08+0.00a
Ash (g%)
3.83+0.05b
4.56+0.05c
0.40+0.00a
Protein (g%)
27.56+0.05a
76.46+0.34b
92.93+0.11c
Fat (g%)
33.43+0.05c
1.00+0.00b
0.01+0.00a
Carbohydrates (g%)
23.10+0.00c
11.59+0.00b
0.50+0.00a
Fibre (g%)
08.20+0.08c
4.69+0.05b
0.10+0.00a
Energy (kcal/100g)
503.270.00
361.360.01
373.720.01
F. Fibre
The fibre content was significantly (p<0.01) higher in WASF (8.2 g/100g)
than WASPC (4.6g/100g) and WASPI (0.1g/100g) (Table 4.14). The crude fibre in
WASF was higher than soybean seed flour (4.28%) (Temple et al., 1991); amaranthus
cruentus seed flour (4.29%) (Escudero et al., 2004); gourd seed flour (2.8%)
(Ogungbenle, 2006); breadnut seed flour (1.2%) (Adeleke & Abiodun, 2010); papaya,
watermelon, orange, apricot seed flours (5.19%, 3.47%, 5.5% and 3.43%)
respectively (Samia et al., 2012); A. Africana seed flour (0.13%) (Adebayo & Ojo,
2013); Corchorous olitorius seed flour (6.60%) (Oloye et al., 2014); brebra seed flour
(2.41%) (Andualem & Gessesse, 2014); avocado seed flour (7.1%) (Ifesan et al.,
2015); comparable to that of apple seed flour (8.3%) and snake gourd (8%) (Yusuf et
al., 2007); African yam bean seed flour (8.38%) (Ndidi et al., 2014); yam bean seeds
(8%) (Kisambira et al., 2015) and lower when compared with baobab seed flour
(16.2%) (Osman, 2004); papaya seed flour (8.9-9.4%) (Adesuyi & Ipinmoroti, 2011);
groundnut flour (17.3-22.7%) (Musthapa et al., 2015). It is good to know the result is
close to the recommendation of Achinewhu (1982) for legumes with mean values
ranging between 5-6%. Crude fibre help in the maintenance of normal peristaltic
movement of the intestinal trait hence diet containing low fibre could cause
constipation and could lead to colon diseases, piles, cancer and appendicitis (Nieman
et al., 1992).
The fibre content of WASPC (4.69%) was lower when compared to
amaranthus cruentus seed protein concentrate (12.90%) (Escudero et al., 2004);
watermelon seed defatted flour (9.50%) (Lakshmi & Kaul, 2011) and higher in
114
comparison to Mucuna and Canavalia beans protein isolate (1.7% and 1.3%)
(Metsagang et al., 2013).
The fibre content of WASPI (0.1%) was lower than watermelon seed protein
isolate (3.0%) (Lakshmi & Kaul, 2011) and Parkia biglobosa seeds protein isolate
(1.1%) (Ogunyinka et al., 2016).
In certain cardiovascular diseases such as cancer, atherosclerosis and aging,
excess consumption of fat has been implicated (Antia et al., 2006). The WASF and
WASPC had higher fat content compared to the protein isolate. The low fat content of
the WASPI was desirable, because it could be consumed instead of animal protein
that contained high fat content (Adeyeye, 2013). Aside from low fat content, the
WASPI also contain low fiber content, which could be attributed to the processing
method in which other nutrients have been removed. Poor fiber content in protein
isolates has previously been reported (Ojukwu et al., 2012). On the other hand, the
WASPI contains low carbohydrate content. This could also be attributed to the
processing method in which the isoelectric precipitate removed soluble
carbohydrate. The low carbohydrate and fat levels could be beneficial to diabetic
individuals with increased serum lipid levels (Ogunyinka et al., 2016).
G. Energy
The nutritive value was high in WASF (503 kcal/100g) than WASPC (361
kcal/100g) and WASPI (373 kcal/100g), as the seed flour contains carbohydrates,
protein and fat in adequate amount (Table 4.14). This could be due to the high level of
crude fat and protein in the studied sample. Similar result of nutritional composition
was found in Hempseed, which contains 20-25% protein, 20-30% carbohydrates, 2535% oil and 10-15% insoluble fibre and a rich array of minerals (Callaway, 2004 &
Dimic et al., 2009).
4.3.2.2 Mineral Composition
The mineral content of the wood apple seed components increased on
defatting. The high amount of potassium was observed in the WASPC; similar results
were found in plant foods from Nigeria soil (Achinewhu, 1982). The WASPC had
significantly (p<0.01) more Ca, P, Na and K when compared to WASF and WASPI
(Table 4.15). High amount of Ca, K and Mg in diets have been reported to reduce
blood pressure. Calcium is responsible for teeth and bone formation in conjuction
with P, Mg, Mn, vitamin A, C, D, chlorine and protein. Phosphorus is always found with
calcium in the blood (Oloye et al., 2014). Similar results were found in wood apple
(Feronia limonia L.) seed and Limonia acidissima respectively (Rao et al., 2011;
115
Sonawane et al., 2016). Based on these results and on the latest recommendations
from ICMR (RDA, 2010/ http://icmr.nic.in/final/rda-2010.pdf/2013), wood apple
seed possess high levels of most essential minerals, hence these could be used in the
formulation of different food products in order to meet the requirements of growing
population. The P content of WASF and WASPC were higher in comparison with the P
value of pumpkin seeds (47.68mg/100g) (Elinge et al., 2012); Juglans regia seeds
(0.87mg/100g) (Ogungbenle & Anisuiowo, 2014) and melon seeds (5.77 mg/100g)
(Jacob et al., 2015).
The WASF mineral components such as P, Na, K, Ca and Fe was lower when
compared to conophor nut flour P (465 mg/100 g), Fe (1.55 mg/100g) and calcium
(42.06 mg/100g) (Enujiugha, 2003); breadnut seed flour Ca (185 mg/100g), K (325
mg/100g), Na (248 mg/100g), P (363 mg/100g), (Adeleke & Abiodun, 2010); A.
africana seed flour iron (2.48mg/100g), calcium (340mg/100g) (Adebayo & Ojo,
2013); brebra seed flour P (1062 mg/100 g), K (281 mg/100 g), Na (93.26 mg/100g),
Fe (27.81 mg/100g) and Ca (61.55 mg/100g) (Andualem & Gessesse, 2014);
groundnut flour Ca (90-122.33 mg/100g), Na (62.23-66.13 mg/100g), Fe (4.53-5.10
mg/100g), K (501-535 mg/100g) (Musthapa et al., 2015); but higher when compared
to egusi melon seed flour Na (13 mg/100g), K (96.1 mg/100g), Ca (28.2 mg/100g), P
(125.3 mg/100g) (Ojieh et al., 2008); cashew nut flour P (14 mg/100 g), K (27.5
mg/100 g), Na (8.2 mg/100g), Fe (0.6 mg/100g) and calcium (21.5 mg/100g)
(Akinhanmi et al., 2008); papaya seed flour Na (16.2 - 33.6 mg/100g), K (17-43.6
mg/100g), Ca (2.5-4.1 mg/100g), P (28.5-58.6 mg/100g) (Adesuyi & Ipinmoroti,
2011); avocado seed flour Ca (0.8 mg/100g), K (4.16 mg/100g), Na (1.4 mg/100g), P
(0.09 mg/100g) (Ifesan et al., 2015).
Na/K plays a very important role in diet as it controls high blood pressure in
the body. Studies had showed that lower sodium and higher potassium intake helps
to reduce high blood pressure in hypertensive patients. The recommended Na/K ratio
should be less than one (Jacob et al., 2015). The Na/K value of WASF, WASPC and
WASPI was 0.09, 0.11 and 0.46 respectively. The Na/K ratios obtained in raw, toasted
and sundried groundnut flour are 0.12, 0.13 and 0.12 respectively (Musthapa et al.,
2015). As suggested by NRC (1989) and Yusuf et al (2007), the WASF would probably
be a good source for electrolyte balance and controls high pressure in the body. Food
is considered good if the ratio Ca/P > 1 and poor if < 0.05, while recommended
Na/K is 0.60 (Nieman et al., 1992). The Ca/P ratio of WASF, WASPC and WASPI was
medium since it is <1 and >0.05; Na/K ratio was <0.6.
116
TABLE 4.15: MINERAL COMPOSITION OF WASF, WASPC AND WASPI
Parameter
WASF
WASPC
WASPI
Calcium (mg%)
34.000.05b
46.900.02a
9.30.02c
Phosphorus (mg%)
141.160.01b
189.210.05a
28.120.12c
Sodium (mg%)
36.000.15b
43.200.02a
16.060.05c
Iron (mg%)
0.080.02a
0.10.15b
Trace
Potassium (mg%)
396.000.10b
4010.05a
34.780.01c
Na/K ratio
0.09
0.11
0.46
Ca/P ratio
0.24
0.25
0.33
Bioaccessibility of a nutrient is the fraction of ingested nutrient that is

available for utilization in normal physiological functions and for storage. Factors that
influence its availability include chemical state of the nutrient, its release from food
matrix, its interactions with other food components, presence of suppressor and
other cofactors, formation of stable compounds that are slowly metabolized and so on
(Parada & Aguilera, 2007). The bio-accessibility of minerals in the oilseeds is a less
researched area, but the information is of high significance as oilseeds form major
protein sources and is extensively used in complementary foods, supplementary
foods and bakery products. Though the quantity of total iron was higher in WASF,
percent availability was lower in WASPC as the absorption inhibitory components
also enhanced along with other nutrients on defatting (Lakshmi & Kaul, 2011).
4.3.2.3 Amino Acid Analysis
Nutritional qualities of proteins are based on their amino acid composition.
The quality of protein is very much determined by the amino acid composition, as
amino acids are fundamental building blocks of protein. Table 4.16 and Fig.4.11 (a-c)
shows the amino acid content of WASF, WASPC and WASPI respectively. As a
reference, the FAO/WHO/UNU (2007) recommended mode of the essential amino
acid for (25 years old) child, adult and EAA content of casein were included for
comparison and the calculated percentage of EAA score of WASF, WASPC and WASPI
was given in Table 4.17 and 4.18. The essential amino acids (histidine, threonine,
valine, leucine, isoleucine and lysine) of WASF, WASPC and WASPI were in higher
percentage than the FAO/WHO/UNU reference pattern for child and adult while it
was lower when compared to casein protein. High arginine content in the seeds is
indicative of possession of therapeutic value (Lakshmi & Kaul, 2011). The arginine
content of WASF and WASPI was comparatively greater than arginine content of
117
soyabean meal (7.3 g/100g), canola-rapeseed meal (5.8 g/100g) and pennycress seed
(7.85-7.87 g/100g) (Hojilla-Evangelista et al., 2013). Arginine is an essential amino
acid for childrens growth (NRC, 1989; Olaofe et al., 2008). Thus wood apple seed
showed good sources of essential and non essential amino acids and can be used to
supplement food substances for proper growth of children and good health of adults.
TABLE 4.16: AMINO ACID PATTERNS OF WASF, WASPC AND WASPI (g/100g Protein)
Amino Acid
WASF
WASPC
WASPI
Histidine
2.30.02
2.40.02
2.50.01
Threonine
2.80.02
3.30.02
3.40.00
Methionine
2.00.09
2.10.07
2.50.03
Leucine
6.30.04
6.50.01
7.10.04
Isoleucine
2.40.03
3.80.06
4.60.02
Phenylalanine
3.80.01
4.00.05
4.30.00
Lysine
5.80.05
5.90.01
6.90.02
Valine
3.20.01
4.10.03
5.60.01
Tryptophan
0.50.01
0.490.04
0.60.01
Aspartic acid
4.30.02
3.70.11
1.80.20
Glutamic acid
5.00.00
4.40.32
2.30.19
Serine
2.20.01
1.90.18
1.60.06
Glycine
1.20.05
1.50.16
1.50.13
Alanine
1.50.04
3.00.22
6.20.06
Arginine
8.50.03
3.20.07
9.60.09
Tyrosine
1.00.02
1.20.02
1.70.26
Essential Amino Acid
Non Essential Amino Acid
Percentage of Amino Acid with Different Characteristics**

Basic
16.60.04
11.50.06
19.00.02
Acidic
9.30.05
8.10.01
4.10.04
Hydrophobic
19.20.01
23.50.03
30.30.00
Uncharged polar
7.20.02
7.90.02
8.20.02
EAAI
0.96
0.97
0.98
P-PER
2.2
2.3
2.5
*FAO/WHO/UNU:
Values are the average of two determinants.

Daily requirements for human
child and adult (FAO, 2007), **Basic, acidic, hydrophobic, uncharged polar: (Lysine, arginine,
histidine), (Aspartic acid, glutamic acid), (Alanine, isoleucine, leucine, methionine,
phenylalanine, valine), (Glycine, serine, threonine, tyrosine), respectively.
118
According to EAA content of casein protein (Abugoch et al., 2008), valine,

isoleucine, leucine and lysine were the limiting amino acids in ascending order in
WASF; valine, leucine, lysine and isoleucine were the limiting amino acids in
ascending order in WASPC and leucine, tryptophan, valine and isoleucine were the
limiting amino acids in ascending order in WASPI. As per the percentage of amino
acid with different functional group characteristics in WASF, WASPC and WASPI
hydrophobic (alanine, isoleucine, leucine, methionine, phenylalanine, valine) and
basic amino acid (lysine, arginine, histidine) were found to be in highest percentage
followed by acidic (aspartic acid, glutamic acid) and uncharged polar (glycine, serine,
threonine, tyrosine) amino acid groups.
Siddeeg et al (2014) reported that the hydrophobic, acidic and uncharged
polar groups were found in highest percentage in bread fortified with seinat seed
protein concentrate and isolate which was followed by total aromatic and basic
amino acid groups. Azhari et al (2014a) also reported that seinat seeds continued
high amount of hydrophobic amino acids. As per data in Table 4.17 the essential
amino acid in WASPI such as histidine, leucine, isoleucine, lysine, valine was at higher
concentration than reference protein pattern of FAO/WHO/UNU (>100); theronine
was equal to 100 and methionine, phenylalanine and tryptophan was <100 which
reflected the tryptophan was the first limiting amino acid, phenylalanine was the
second limiting amino acid and methionine was the third limiting amino acid. The
wood apple seeds contained high amount of hydrophobic amino acids.
TABLE 4.17: ESSENTIAL AMINO ACID SCORES (%) OF WASF, WASPC AND WASPI
COMPARED TO THE FAO/WHO PATTERN FOR (25 YEARS OLD) CHILD
Amino Acid
WASF
WASPC
WASPI
Histidine
121.00.00
126.60.01
131.50.00
Threonine
82.350.03
97.050.00
100.00.01
Methionine
74.070.01
77.770.02
92.590.02
Leucine
95.450.02
98.480.00
107.570.02
Isoleucine
85.710.00
135.710.01
164.280.00
Phenylalanine
60.310.01
63.490.03
68.250.01
Lysine
100.00.00
101.720.00
118.90.00
Valine
91.420.02
117.140.01
160.00.00
Tryptophan
45.450.00
44.540.02
54.550.02
119
Fig.4.11 (a): HPLC chromatogram indicating amino acids in WASF
Fig.4.11 (b): HPLC chromatogram indicating amino acids in WASPC
Fig.4.11 (c): HPLC chromatogram indicating amino acids in WASPI

Nutritionally, a protein-based food material is said to be of good nutritional
quality when its protein efficiency ratio is 2.7, biological values is >70%, essential
amino acid index (EAAI) is >0.70 (Oser, 1959).
120
TABLE 4.18: ESSENTIAL AMINO ACID SCORES (%) OF WASF, WASPC AND WASPI
COMPARED TO THE CASEIN REFERENCE PROTEIN
Amino Acid
WASF
WASPC
WASPI
Histidine
85.180.01
88.880.01
92.590.00
Threonine
75.670.03
89.180.00
91.890.01
Methionine
76.920.09
80.760.01
96.150.01
Leucine
75.00.04
77.380.00
84.520.02
Isoleucine
48.970.03
77.550.00
93.870.03
Phenylalanine
84.440.01
88.890.02
95.560.01
Lysine
81.690.00
83.090.01
97.180.01
Valine
53.340.01
68.330.01
93.340.00
Tryptophan
35.710.01
35.000.02
42.850.02
The EAAI of WASF, WASPC and WASPI was 0.96, 0.97 and 0.98 respectively.
The P-PER of WASF, WASPC and WASPI was 2.2, 2.3 and 2.5, respectively. The P-PER
of wood apple seed was higher than some legumes and concentrates like lathyrus
sativus (1.03) (Aremu et al., 2007); Luffa cylindrical (1.49) (Olaofe et al., 2008);
pumpkin seed (1.80) (Atuonwu & Akkobundu, 2010); Bucholzia coriacea seed (1.952.12) (Ijarotimi et al., 2015); but lower than Citrullus colocynthis and Citrullus vulgaris
seeds (2.71 and 2.58) (Ogundele et al., 2012) and comparable with Prosopis africana
(2.3) (Aremu et al., 2006). The nutritional outcome of WAS in terms of essential
amino acid index and predicted protein efficiency ratio showed that the seeds contain
good amount of protein and can be used in complementing with other protein-based
food materials.
4.3.3 Anti-Nutritional Composition
The results of anti-nutritional composition of WASF, WASPC and WASPI are
shown in Table 4.19. Antinutritional components (tannin, phytate, oxalate and
saponin) were significantly (p<0.01) reduced on extraction and purification of wood
apple seed protein as isolate. As revealed in the present study, the total phenolics and
tannins contents of Mucuna and Canavalia were reduced to about 50% during the
isolating process (Metsagang et al., 2013). The tannin content in WASF, WASPC and
WASPI (0.89, 0.65 and 0.30 mg/100g) respectively were lower compared to soaked
tamarind seed (STS); STS had higher tannin content with 4.84% (Akajiaku et al.,
2014). The lower content of tannin in WASPI can be attributed to the fact that tannin
are water soluble compounds which makes them easy to be eliminated by washing
the seed on collection as suggested by Siddhuraju et al (1995). Tannins are astringent
121
in taste and help in healing of wounds and inflamed mucous membrane (Njoku &
Akumefula, 2007). Tannins are potential metal ion chelator, proton precipitating
agents and biological antioxidant (Okonkwo, 2009). The values were also lesser than
melon seeds (39 mg/100g) (Jacob et al., 2015); greater than B.eurycoma (0.039%)
and P.guineense seeds (0.026%) (Bolanle et al., 2014); canavalia beans protein isolate
(0.02%) (Metsagang et al., 2013).
TABLE 4.19: ANTI-NUTRITIONAL COMPOSITION OF WASF, WASPC AND WASPI
(mg/100g)
Anti-Nutrients
WASF
WASPC
WASPI
Tannin
0.89+0.01a
0.65+0.02b
0.300.04c
Phytate
0.60+0.00a
0.38+0.00b
0.120.08c
Oxalate
0.33+0.05a
0.03+0.00b
0.020.11c
Saponin
0.45+0.00a
0.21+0.00b
0.080.05c
The phytate content of WASF, WASPC and WASPI were very low when
compared with those reported for some commonly used legumes, cowpea (2-2.9%),
pigeon pea (2-2.4%) and African yam beans (2.4%) (Oboh, 2006); mucuna varieties
(white-0.9%, black-0.86% (Siddhuraju and Becker, 2001) while similar range of
values (0.48 to 1.092) in canavalia flour (Sridhar & Seena, 2008); dry beans (0.53.0%) (Deshpande & Cheryan, 1984) and soyabeans (0.6-1.5%) (Ologhobo & Fetuga,
1984). During proteins isolation, there was an increase in phytate level with values in
Mucuna and Canavalia isolates of 1.87% and 1.35% respectively, probably resulted
from its ability to complex with proteins which co-precipitate at the isoelectrical
point (Metsagang et al., 2013). Contradictory result was noted in WAS where phytate
content was significantly (p<0.01) reduced during isolation of protein. Phytic acid
binds calcium, iron, zinc and other minerals, thereby reducing their availability in the
body (FAO, 1990). It also inhibits protein digestion by forming complexes with them.
However, the phytate content can further be lowered by processing (Esenwah &
Ikenebomeh, 2008) and has been considered as an anti-nutritional component in
cereals, seeds and beans. However, resent research have shown that phytic acid has
many health benefits such as antioxidant, anticancer, hypocholesterolemic and
hypolipidemic effects. In fruits and vegetables, phytic acid helps to prevent oxidative
browning by inhibiting polyphenol oxidase. It may be used as a safe preservative and
antioxidant in food products by suppressing oxidative reactions catalyzed by iron
122
(Fasidi & Olorunmaiye, 1994). The decrease in phytic acid content by soaking,
cooking of pre-soaked bean or germination may be due to leaching out of this
compound in water (Osman, 2007).
Oxalate level was comparatively low in wood apple seed flour (0.33mg/100g)
than (3.06%) present in M. scandenis, and it is known to cause great risk of renal
absorption. Heat treatment (cooking) has been found to be an effective measure in
reducing the oxalate levels in leafy vegetable, thus, making the food prepared from
these accessions safe for human consumption (Lumu & Katongole, 2011).
The saponin in wood apple seed flour and wood apple seed protein
concentrate was 0.45 mg and 0.21 mg/100g respectively. According to Harborne
(1984), saponins have anti-hyper cholesterol, anti- inflammatory, anti-mutagenic,
cardiac depressant property and also appear to kill or inhibit cancer cells without
killing the normal cells in the process (Okwu & Josiah, 2006; Nafiu et al., 2011), while
some others have reported the expectorant action (Ayoola & Adeyeye, 2010). Plants
containing saponins are used to heal wounds (Okwu & Josiah, 2006) because
saponins have the ability to precipitate and coagulate red blood cells (Sood et al.,
2012). The present study results were lower than Cassipourea congoensis seeds,
which were rich in oxalate (10.21+1.11), tannin (2.84+0.12) and saponin (7.17+0.18)
(Nkafamiya et al., 2007) and leguminous seeds (Mucuna Ghana, Mucuna preta and
Mucuna Veracruz mottle) had higher content of oxalate (8.31+0.03), phytic acid
(85.47+0.62) and tannin (10.30+1.15) (Amoo et al., 2009).
Some of these anti-nutrients can be reduced by processing and cooking, for
example, boiling can reduce the soluble oxalate content of a food, if the water used for
boiling is discarded (Odugbemi, 2006). The toxic and anti-nutrient effects of
compounds in the plants could be removed by several processing methods such
as soaking, germination, boiling, autoclaving, fermentation, genetic manipulation
and other processing methods (Enechi & Odonwodu, 2003; Soetan, 2008). The
effect of processing such as soaking, soaking and boiling, had significant difference in
the reduction of the anti-nutrients concentrations and toxicants present in Mucuna
pruriens (Velvet Beans) seeds (Nwaoguikpe et al., 2011).
Phenolic compounds, notably tannins are known to have ability to decrease
digestibility by complexing with dietary proteins and to lower the activity of several
digestive enzymes (e.g. -amylase, trypsin, chymotrypsin, lipase) (Liener, 1994). The
loss of phenolic and tannins content might be due to leaching of phenols into
extraction water during precipitation of proteins at acidic pH. Phytic acid in legumes
123
has been reported to lower the nutritional value due to limiting the bioavailability of
dietary minerals, essential trace elements and also proteins (Brune et al., 1989;
Ryden & Selvendran, 1993). Phytic acid, 3,4-dihydroxyphenylalanine (L-DOPA), as
well as condensed tannins and polyphenols are known to interact with protein and
form complexes. These interactions could decrease the solubility of proteins and
increase the degree of cross-linking which resulted in impairment of protease access
to peptide bonds (Genovese & Lajolo, 1996).
4.3.4 Preliminary Phytochemical Screening and Total Phenol Content
Phytochemical constituents such as tannins, flavonoids, alkaloids and several
other aromatic compounds or secondary metabolites of plants serve as defense
mechanism and curative properties against predation by many microorganism,
insects and herbivores (Britto & Sebastiana, 2011). The analysis of phytochemical
screening of WASF, WASPC and WASPI of different extractions (Table 4.20)
(methanol, aqueous and ethanol) revealed the presence and absence of phyto
constituents of the wood apple seed. The major compounds were present in aqueous
extract of WASF, WASPC and WASPI rather than in methanol and ethanol extracts.
The protein and amino acid were highly present in WASPI and WASPC when
compared to WASF. This might be due to defatting of wood apple seed flour. Phenolic
compounds could be a major determinant of antioxidant potentials of food plants and
have been associated with the health benefits derived from consuming high levels of
fruits and vegetables. Similar results were found in Solanum indicum Linn., which has
high preservation capacity and nutritional values, because total phenolic compounds
prevent from damage of nutrients contain double bonds such fatty acids, flavor
compounds even proteins and amino acids and other compounds (Kahkonen et al.,
1999). But maximum phenolic content was found in methanolic extract than in
aqueous extract in Aegle marmelos seeds (Sharma et al., 2011). Among the screened
phytochemicals flavonoids, phenolics, carbohydrate, proteins and amino acids were
present in high amount in aqueous extract of WASF; flavonoids, amino acids and
protein in aqueous extract of WASPC; flavonoids, phenolics and protein in aqueous
extract of WASPI. The intensity of glycosides and saponins were reduced on
extraction and isolation of protein from WASF. Steroids and sterols were not
identified in aqueous and ethanol extract of WASF, WASPC and WASPI though it was
observed in very low intensity in methanol extract of WASF and WASPC. The changes
in intensity of saponin and tannin reflecting the content in WASF, WASPC and WASPI
was highly correlated with quantitative profile of these compounds.
124
TABLE 4.20: PHYTOCHEMICAL SCREENING OF WASF, WASPC AND WASPI
WASF
WASPC
WASPI
Ethanol
Extract
Aqueous
Extract
Methanol
Extract
Ethanol
Extract
Aqueous
Extract
Methanol
Extract
Ethanol
Extract
Aqueous
Extract
Methanol
Extract
Alkaloids
++
++
++
++
Flavonoids
++
+++
++
+++
+++
++
Amino acids
+++
+++
++
Glycosides
Saponins
++
Phenolics
++
+++
++
++
+++
++
Tannin
++
Carbohydrate
+++
Protein
++
++
+++
++
+++
+++
Phytochemicals
Steroids and Sterols

+
+++ = Present in higher amounts, + = Present, ++ = Moderately present, - = absent
125
Mahour et al (2008) reported similar result on preliminary qualitative

chemical test of fruit pulp of Feronia elephantum corr. that flavanoids, triterpenoids,
tannin, phyto sterol, amino acid and carbohydrate were the important constituents
while alkaloids and cardiac glycosides were found to be absent. It was also suggested
that flavonoids and triterpenoids are the active constituents in fruit pulp of Feronia
elephantum corr. and are responsible for its pharmacological activities. Flavonoids
found in the extracts are potent water soluble antioxidants (Borhade, 2012). Sharma
et al (2011) reported the presence of alkaloids, carbohydrates, proteins, glycosides
and phenolics in both the aqueous and methanolic extracts of seeds of Aegle
marmelos.
The various phytochemical compounds detected are known to have
beneficial importance in medicinal sciences. Alkaloids have been associated with
medicinal uses for centuries and one of their common biological properties is their
cytotoxicity (Nobori et al., 1994). Several authors have reported the analgesic
(Antherden, 1969; Harborne, 1973) antispasmodic and antibacterial properties of
alkaloids (Stray, 1998; Okwu & Okwu, 2004). Glycosides are known to lower the
blood pressure according to many reports (Nyarko & Addy, 1990) and were present
in ethanol extracts of quince seeds, musk melon seeds and bottle gourd seeds
indicating the benefits in reducing inflammation, protecting against endotoxemia,
used in cardiac treatment of congestive heart failure (Jisika et al., 1992; Matsumori et
al., 1997; Balch & Balch, 2000; Aiyelaagbe & Osamudiamen, 2009; Sood et al., 2012)
Similar phtyochemical screening results were
also found in flaxseeds (Amin &
Thakur, 2014), that glycosides and steroids were absent in all three extracts. Thus,
the results showed that the wood apple seeds may be used for treatment of
congestive heart failure, cancer, lowering blood cholesterol in blood, healing of
wounds, endotoxemia and reduction of inflammation.
4.3.4.1 Total Polyphenol Content (TPC)
Phenolic compounds are considered to be secondary metabolites that are
synthesized by plants during normal development, in response to stress conditions
and the compounds occur ubiquitously in plants as a diversified group of
phytochemicals derived from phenylalanine and tyrosine. In food, phenolics may
contribute to the bitterness, astringency, color, flavor, odor and oxidative stability of
the products. In addition, they have numerous beneficial effects, such as free radical
scavenging oxygen species, modulate the activity of some specific enzymes, inhibit
cell proliferation and have antimicrobial, anti-inflammatory and anti-allergic
126
potential (Manach et al., 2004). The TPC as GAE of WASF, WASPC and WASPI are
given in Table 4.21. Among the ethanol, methanol and aqueous extracts of WASF,
WASPC and WASPI, methanol showed the highest polyphenol content (significant at
p<0.01) than aqueous and ethanol extracts. Similarly maximum phenol content was
found in methanolic extract of Aegle marmelos seeds (Sharma et al., 2011). WASPI
had significantly (p<0.01) high polyphenol than WASPC and WASF. Similar results
were found by Thaipong et al (2006) in guava genotypes of Allahabad Safeda (344.9
g GAE/g) and Fan Retief (300.8 g GAE/g).
TABLE 4.21: TOTAL PHENOL CONTENT OF WASF, WASPC AND WASPI (g GAE/g)
Samples
Ethanol Extract
Aqueous Extract
Methanol Extract
WASF
120.300.12c
220.080.02b
248.210.16a
WASPC
216.910.07c
212.670.05b
328.330.09a
WASPI
304.150.22c
346.230.13b
400.120.05a
4.3.5 Antioxidant Activity Profile

The antioxidant activity of extracts depends on the type and polarity of the
extracting solvent, the isolation procedures and the purity of the active compounds,
as well as the assay techniques and substrate used (Meyer et al., 1998).
ABTS and DPPH assay is based on the antioxidant ability to react with ABTS
and DPPH radical action generated in the assay system. In contrast, the Ferric
Reducing Antioxidant Power (FRAP) assay measures the reduction of ferric iron
(Fe3+) to ferrous iron (Fe2+) in the presence of antioxidants, which are reductants
with half-reaction reduction potentials above Fe3+/Fe2+. These methods are widely
used to evaluate antioxidant activity in foods and biological systems (Sonawane &
Arya, 2013). The presence of electron-donating and electron-with drawing
substituents in the ring structure of phenolics as well as the number and arrangement
of the hydroxyl groups determines their antioxidant potential (Zhang et al., 2003). A
huge number of phenolic compounds show differences in possible bio-chemical
modification (glycosylation, acetylation, manolynation, esterification to organic acids
etc.) (Balasundram et al., 2006; Dai & Mumper, 2010). The antioxidant activity of
WASF, WASPC and WASPI in three extracts (methanol, ethanol and water) was
determined by different in vitro methods such as, DPPH free radical scavenging, ABTS
and reducing power assay.
127
4.3.5.1 DPPH Radical Scavenging Assay

The DPPH radical has been widely used to test the ability of compounds as
free-radical scavengers or hydrogen donors and to evaluate the antioxidative activity
of plant extracts and foods (Soare et al., 1997; Porto et al., 2000). The DPPH
scavenging activity was found to be dose dependent. This radical scavenging activity
of plant extracts could be related to the nature of phenolics, thus contributing to their
electron transfer/hydrogen donating ability (Shanmugapriya et al., 2011).
The concentration dependent radical scavenging activities of WASF, WASPC
and WASPI in different extracts (methanol, ethanol and water) are depicted in Table
4.22 and Fig. 4.12 (a-c). The DPPH radical scavenging activity of WASF, WASPC and
WASPI was considered to be dose dependent irrespective of different extracts.
Among the extracts, methanol (WASF: 94.6 % at 1.0 mg/ml; WASPC: 96.9 % at 1.0
mg/ml: WASPI: 97.3% at 1.0 mg/ml) revealed significantly (p<0.05) a high DPPH
scavenging activity followed by aqueous extract (WASF: 90.5% at 1.0 mg/ml; WASPC:
92.4% at 1.0 mg/ml: WASPI: 93.5% at 1.0 mg/ml) and then by ethanol extract (WASF:
33.8% at 1.0 mg/ml; WASPC: 40.2% at 1.0 mg/ml: WASPI: 66.4% at 1.0 mg/ml). The
DPPH scavenging activity was significantly (p<0.05) low in ethanol extracts. Similar
observation in the scavenging effect of L.acidissima fruit extract on DPPH radical
followed the extract based order of methanol> aqueous>ethylacetate>choloroform.
This could reveal that methanol would be the best extraction medium to extract the
polyphenols in fruits followed by water as suggested by Darsini et al (2013).
Turkmen et al (2006) reported that solvent with different polarity had significant
effect on phenolic compound and antioxidant activity. Musa et al (2011) found that
the increase in polarity of a solvent (upto 50% water) enhances the solubility of
antioxidant compounds. Generally polar solvents provide slightly more active
extracts than mixtures with less polar solvents. This factor may also have affected the
results when using the methanol/water solvent, providing greater extraction of
components (Benites et al., 2015).
WASPI had evidenced the greater DPPH activity (methanol: 97.3%at 1.0
mg/ml; ethanol: 66.4% at 1.0 mg/ml: water: 93.5% at 1.0 mg/ml) than in WASPC
(methanol: 96.9%at 1.0 mg/ml; ethanol: 40.2% at 1.0 mg/ml: water: 92.4% at 1.0
mg/ml) and WASF (methanol: 94.9%at 1.0 mg/ml; ethanol: 33.8% at 1.0 mg/ml:
water: 90.5% at 1.0 mg/ml).
The IC50 data in Table 4.23 showed that WASPI scavenged 50% DPPH radical
at the lowest sample concentration followed by WASPC and WASF irrespective of
128
extract. According to Norshazila et al (2010), the IC50 of methanol extract of WASF

(0.467 mg/ml), WASPC (0.363 mg/ml) and WASPI (0.293 mg/ml) was greater than
the positive control BHA (0.13 mg/ml). Sonawane & Arya (2013) revealed that the
antioxidant potential of commonly consumed fruits has been rated in the order of
Jambhul>wood apple>ambadi>ambat chukka by DPPH assay. The regression
equation according to concentration versus percentage inhibition could be used to
predict the percentage inhibition at unknown concentration of extract (Table 4.23).
The Adj.R2 value in Table 4.23 reflected the percentage influence of various
concentration of extracts on percentage inhibition of DPPH radical which was found
to be greater than 85% and was significant (p<0.05) irrespective of solvent, flour,
concentrate and isolate variation. The DPPH scavenging activity of WASF was lower
than that of pomegranate seed flour (6.4-13.1 mg/ml) (Jing et al., 2012), Marylandgrown soyabeans (7-10 mg/ml) (Slavin et al., 2009).
4.3.5.2 ABTS Radical Scavenging Assay
ABTS radical, a protonated radical has characteristic absorbance maxima at
734nm which decreases with the scavenging of the proton radicals (Mathew &
Abraham, 2006).
The effect of methanol, ethanol and aqueous extracts of WASF, WASPC and
WASPI on ABTS radical scavenging activity was presented in Table 4.24 and Fig. 4.13
(a-c). The methanol extract (WASF: 94.6 % at 1.0 mg/ml; WASPC: 96.9 % at 1.0
mg/ml: WASPI: 97.3% at 1.0 mg/ml) revealed significantly (p<0.05) a high ABTS
scavenging activity followed by aqueous extract (WASF: 90.5% at 1.0 mg/ml; WASPC:
92.4% at 1.0 mg/ml: WASPI: 93.5% at 1.0 mg/ml) and then by ethanol extract (WASF:
33.8% at 1.0 mg/ml; WASPC: 40.2% at 1.0 mg/ml: WASPI: 66.4% at 1.0 mg/ml). As
reported by Yu et al (2002), the methanolic extracts were fast and effective
scavengers of the ABTS radicals.
According to IC50 value (Table 4.25) WASPI showed the scavenging activity at
the lowest concentration followed by WASPC and WASF irrespective of extract. The
ABTS radical scavenging activity of WASF, WASPC and WASPI was considered to be
dose dependent irrespective of different extracts. Similar concentration in ABTS
scavenging activity was noted in Artocarpus heterophyllus seed extracts i.e. 50.36% at
400 g/ml for ethyl acetate matched with ABTS radical scavenging activity of
aqueous extract of WASPI (392.14 g/ml) (Shanmugapriya et al., 2011); 52.04% at
500 g/ml for aqueous fraction matched with ABTS radical scavenging activity of
129
ethanolic extract of WASPI (518.69 g/ml); and also noted in Manikara zapota seed
extract i.e. 50.67% at 400 g/ml for ethyl acetate matched with ABTS radical
scavenging activity of aqueous extract of WASPI (392.14 g/ml); 51.75% at 500
g/ml for aqueous fraction matched with ABTS radical scavenging activity of
ethanolic extract of WASPI (518.69 g/ml) (Shanmugapriya et al., 2011).
WASPI had evidenced the greater ABTS activity (methanol: 97.3%at 1.0
mg/ml; ethanol: 66.4% at 1.0 mg/ml: water: 93.5% at 1.0 mg/ml) than in WASPC
(methanol: 96.9%at 1.0 mg/ml; ethanol: 40.2% at 1.0 mg/ml: water: 92.4% at 1.0
mg/ml) and WASF (methanol: 94.9%at 1.0 mg/ml; ethanol: 33.8% at 1.0 mg/ml:
water: 90.5% at 1.0 mg/ml). The high molecular weight phenolics have more ability
to quench free radicals and their effectiveness depends on the molecular weight, the
number of aromatic rings and nature of hydroxyl group substitution than the specific
functional groups (Hagerman et al., 1998).
The regression equation derived from concentration dependent radical
scavenging activity could predict the % inhibition at unknown concentration of
extracts. The concentration of extracts influenced the % inhibition of ABTS radical at
minimum of 83% and maximum of 99% which was significant at p<0.01. The ABTS
scavenging activity of WASF was greater than pomegranate seed flour (7.4 12.9
mol TE/g) (Jing et al., 2012); brown, green and yellow soyabeans (around 10 mol
TE/g) (Salvin et al., 2009) and black pearl grape seeds (78.8-110.2 mol TE/g) (Xu et
al., 2011).
4.3.5.3 Ferric Reducing Antioxidant Power (FRAP) Assay
The Ferric Reducing Antioxidant Power (FRAP) assay measures the reduction
of ferric iron (Fe3+) to ferrous iron (Fe2+) in the presence of antioxidants, which are
reductants with half-reaction reduction potentials above Fe3+/Fe2+ (Sonawane &
Arya, 2013). The reducing activity of seed extract was determined by ferric reducing
assay. The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Meir et al., 1995). The reducing power assay is often
used to evaluate the ability of an antioxidant to donate an electron (Yildirim et al.,
2000). The higher the absorbance of the reaction mixture, the higher would be the
reducing power. Methanolic extract of pulp and seed showed some degree of electron
donation. Reducing power of different extracts increased with the concentration of
the extract. Methanolic extracts of fruit pulp and seed may act as electron donors and
could react with free radicals to convert them into more stable products and then
130
terminate the free radical chain reactions (Irshad et al., 2012). Difference found
between extracts, are probably due to the difference in their phenolic contents
(polyphenolic and anthocyanins extracts), and/or their electron-donating activity
(Boulekbache-Makhlouf et al., 2013).
TABLE 4.23: DPPH RADICAL SCAVENGING ACTIVITY- IC50 VALUE, REGRESSION
EQUATION AND Adj.R2 VALUE
Sample
WASF
WASPC
WASPI
Extract
IC50 value
Regression Equation
Adj.R2
p-value
Methanol
467.340.01
y = 0.083x + 11.21
0.976
0.00*
Water
492.500.02
y=0.084x+8.632
0.975
0.00*
Ethanol
1950.630.01 y = 0.022x + 7.086
0.857
0.00*
Methanol
363.020.01
y = 0.086x+18.78
0.938
0.00*
Water
423.840.00
y=0.078x+16.94
0.962
0.00*
Ethanol
1317.300.03 y = 0.026x + 15.75
0.945
0.00*
Methanol
293.900.02
y = 0.082x + 25.90
0.933
0.00*
Water
325.130.01
y=0.072x+26.59
0.953
0.00*
Ethanol
8100.01
y = 0.049x + 10.29
0.933
0.00*
TABLE 4.25: ABTS RADICAL SCAVENGING ACTIVITY- IC50 VALUE, REGRESSION

EQUATION AND Adj.R2 VALUE
Sample
WASF
WASPC
WASPI
Extract
IC50 value
Regression Equation
Adj.R2
p-value
Methanol
674.810.01
y = 0.079x - 3.318
0.990
0.00*
Water
704.810.10
y = 0.075x - 2.861
0.991
0.00*
Ethanol
923.910.02
y = 0.069x - 13.75
0.826
0.00*
Methanol
432.950.23
y = 0.088x + 11.90
0.959
0.00*
Water
453.130.12
y = 0.088x + 10.12
0.959
0.00*
Ethanol
566.830.04
y = 0.083x + 2.953
0.979
0.00*
Methanol
365.180.01
y = 0.083x + 19.69
0.973
0.00*
Water
392.140.20
y = 0.084x + 17.06
0.969
0.00*
Ethanol
518.690.05
y = 0.079x + 9.028
0.983
0.00*
As per IC50 value (Table 4.27) aqueous and methanol extract of WASPI
showed FRAP at 861.29 g/ml and 771.81 g/ml respectively, while WASPC and
WASF reduced 50% of ferric ion at >1 g/ml. This revealed that WASPI was more
effective in reducing ferric ion to ferrous ion at lowest concentration than WASPC and
WASF. The FRAP of WASF, WASPC and WASPI was considered to be dose dependent
irrespective of different extracts (Table 4.26 and Fig. 4.14 a-c). The regression
equation derived from the concentration dependent FRAP could predict the %
reduction of ferric to ferrous ion at unknown concentration of extracts.
131
TABLE 4.27: FRAP ASSAY - IC50 VALUE, REGRESSION EQUATION AND Adj.R2 VALUE
Sample
WASF
WASPC
WASPI
Extract
Methanol
IC50 value
22650.05
Water
Regression Equation
y = 0.014x + 18.29
Adj.R2
0.956
p-value
0.00
2863.630.03 y = 0.011x + 18.50
0.916
0.00
Ethanol
28100.02
y = 0.016x + 05.04
0.937
0.00
Methanol
39520.02
y = 0.005x + 30.24
0.975
0.00
Water
1736.50.01
y = 0.020x + 15.27
0.955
0.00
Ethanol
3275.530.05 y = 0.013x + 7.418
0.991
0.00
Methanol
771.810.01
y = 0.022x + 33.02
0.945
0.00*
Water
861.290.02
y = 0.031x + 23.30
0.879
0.00*
Ethanol
2237.20.01
y = 0.02x + 5.256
0.944
0.00
The concentration of extracts influenced the % reduction of ferric ions at

minimum of 88% and maximum at 99% which was significant at p<0.01 (Table 4.27).
Sudha et al (2011) reported that the ferric reducing power of pepino fruit
extracts was increased with increasing concentration as shown in WASF, WASPC and
WASPI and also suggested that the ethyl acetate extract of pepino fruit extract
exhibited good hydrogen donating ability or reductant. Whereas methanolic extract
of WASPI exhibited good hydrogen donating ability to reduce ferric to ferrous ions.
The reducing power of WASF was greater than FRAP values of pomegranate
seedflour (15.04 mol TE/g) (Pande & Akoh, 2009; Jing et al., 2012).
TABLE 4.28: CORRELATION MATRIX BETWEEN TPC AND DPPH, ABTS AND FRAP ASSAY
VALUES
TPC
TPC
DPPH
ABTS
FRAP
DPPH
-.738**
ABTS
-.849**
.750**
FRAP
-.389*
.452*
.314
132
TABLE 4.22: DPPH RADICAL SCAVENGING ACTIVITY OF WASF, WASPC AND WASPI
AT VARIOUS CONCENTRATION IN DIFFERENT EXTRACTS
Methanol
Ethanol
Water
Conc.
(g/ml)
WASF
WASPC
200
25.60.12
30.60.06
34.90.19 13.50.10 18.20.04 22.30.02 23.90.03 34.20.04 34.60.12
300
34.90.03
40.90.02
49.40.29 15.30.13 24.70.19 25.40.11 30.20.12 37.50.19 48.10.01
400
46.30.67
53.80.11
60.70.20 15.90.14 26.40.28 29.50.44 43.90.09 44.60.02 55.40.12
500
52.80.14
64.70.01
68.90.14 16.70.32 29.60.70 33.60.03 51.20.17 54.50.18 67.50.09
600
68.90.23
80.30.25
84.30.33 19.30.27 34.20.05 39.40.01 66.50.60 70.40.01 75.20.02
700
70.10.09
85.10.03
88.60.09 20.10.18 35.10.19 44.10.19 69.40.02 77.80.24 80.30.30
800
73.40.01
90.60.19
90.30.17 23.80.20 36.80.01 47.80.05 72.50.21 82.40.35 84.50.22
900
86.10.50
94.70.21
93.50.23 25.90.44 38.90.67 50.90.23 84.90.02 84.70.05 90.40.06
1000
94.90.02
96.90.07
97.30.47 33.80.28 40.20.02 66.40.28 90.50.19 92.40.12 93.50.21
WASPI
WASF
WASPC
WASPI
WASF
WASPC
WASPI
133
TABLE 4.24: ABTS RADICAL SCAVENGING ACTIVITY OF WASF, WASPC AND WASPI
200
Methanol
WASF
WASPC
WASPI
15.90.23 23.80.05 31.90.04
300
18.10.10 35.10.08 42.40.16 11.40.05 24.90.00 32.80.03 17.90.10 33.50.28 40.50.01
400
29.70.09 53.80.14 59.20.09 13.70.28 37.20.01 43.80.00 28.60.02 52.80.09 57.30.05
500
34.60.40 59.70.02 62.70.03 15.90.02 44.90.05 48.50.14 33.90.03 57.30.05 60.80.11
600
43.20.26 63.50.50 68.30.01 18.40.02 59.20.22 60.90.01 40.90.02 62.10.31 65.70.04
700
52.90.44 79.50.12 82.10.10 22.70.01 63.80.50 64.70.02 48.60.19 77.90.00 79.40.02
800
60.30.09 82.90.06 85.30.07 39.70.40 69.10.14 72.30.11 58.00.44 80.90.08 84.20.22
900
66.90.21 92.70.01 94.90.12 54.80.01 77.40.03 79.40.09 65.30.02 89.40.13 93.90.06
1000
79.80.25 94.20.31 99.30.07 66.40.05 82.40.20 85.70.16 74.90.01 93.30.09 96.50.01
Conc.
g/ml
WASF
9.20.01
Ethanol
Water
WASPC
WASPI
WASF
WASPC
WASPI
17.50.02 20.30.08 15.20.00 21.70.14 28.70.02
134
TABLE 4.26: FRAP ASSAY OF WASF, WASPC AND WASPI

Methanol
Ethanol
Water
Conc.
g/ml
WASF
WASPC
WASPI
WASF
WASPC
WASPI
WASF
WASPC
WASPI
200
20.10.02
31.10.00
35.10.01
8.10.05
9.20.10
10.10.03
19.20.06
20.10.01
24.00.03
300
23.20.04
32.20.20
40.10.02
10.00.03
10.30.01
13.20.12
21.40.02
21.30.03
31.10.14
400
24.40.00
32.80.34
42.20.04
12.30.02
13.30.21
14.40.04
23.20.04
24.20.02
39.30.03
500
26.20.3
33.20.03
43.30.23
13.20.03
14.10.02
14.90.05
24.30.05
25.70.01
42.10.05
600
26.10.20
33.50.04
47.20.03
13.70.01
15.20.03
16.40.09
24.80.06
26.20.04
45.60.02
700
27.40.02
33.80.09
50.30.04
15.10.09
17.40.02
18.00.03
25.10.02
28.10.01
49.10.09
800
28.20.09
34.10.05
51.10.04
16.30.02
20.60.05
21.60.04
27.20.04
30.30.02
50.10.01
900
30.50.01
37.20.02
51.30.09
20.20.03
21.20.02
23.20.07
29.10.02
34.10.01
50.30.04
1000
30.20.10
40.10.01
52.00.30
22.10.01
24.40.01
25.60.02
29.80.01
39.20.03
50.50.06
135
120
% Inhibition
100
80
60
WASF
40
WASPC
20
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
% Inhibition
Fig. 4.12a DPPH radical scavening activity in methanolic extract of

WASF, WASPC and WASPI
100
90
80
70
60
50
40
30
20
10
0
WASF
WASPC
WASPI
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.12b DPPH radical scavening activity in aqueous extract of

70
% Inhibition
60
50
40
30
WASF
20
WASPC
10
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.12c DPPH radical scavening activity in ethanolic extract of

136
120
% Inhibition
100
80
60
WASF
40
WASPC
20
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.13a ABTS radical scavening activity in methanolic extract of

120
% Inhibition
100
80
60
WASF
40
WASPC
20
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
% Inhibition
Fig. 4.13b ABTS radical scavening activity in aqueous extract of

90
80
70
60
50
40
30
20
10
0
WASF
WASPC
WASPI
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.13c ABTS radical scavening activity in ethanolic extract of

137
60
% Reduction
50
40
30
WASF
20
WASPC
10
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.14a Ferric reducing activity in methanolic extract of

60
% Reduction
50
40
30
WASF
20
WASPC
10
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.14b Ferric reducing activity in aqueous extract of

30
% Reduction
25
20
15
WASF
10
WASPC
WASPI
0
200
400
600
800
1000
Conc. (g/ml)
Fig. 4.14c Ferric reducing activity in ethanolic extract of

138
4.3.5.4 Correlation analysis between TPC and DPPH, ABTS and FRAP Assay
Pearson correlation matrix between TPC and DPPH, ABTS and FRAP assay
(Table 4.28) revealed a high degree significant (p<0.01) negative correlation noted
between TPC (as GAE) and DPPH; TPC (as GAE) and ABTS. A low degree significant
(p<0.05) negative correlation was noted between TPC (as GAE) and FRAP assay.
Darsini et al (2013) reported a contradictory result that a high degree positive
correlation between TPC and DPPH, FRAP assay. Thus it proved that free radical
scavenging activity roughly connected to phenolic composition and strongly
dependent on structure of phenolic compounds (Duh et al., 1999; Kwee & Niemeyer,
2011). Furthermore, a weak correlation between FRAP and TFC (R2= 0.267) was
observed in L. acidissima fruit extract. Similar significant negative correlation was
noted between TFC and EC50 of DPPH in China-grown pomegranate seeds (Jing et al.,
2012); between polyphenol and DPPH (r = -0.76), linoleic acid inhibition (r = -0.82) in
bitter orange respectively (Moulehi et al., 2012); between gallic acid and DPPH
radical scavenging activity (r = -0.037), TRAP (r = -0.0728) in lupin seeds (Siger et al.,
2012); between TEAC and TPC (R2= -0.8676) of 62 fruits (Fu et al., 2011).
4.3.6 In Vitro Protein Digestibility
Digestibility of protein is an index for the nutritional value of proteins present
in seed flour. Where, in vitro protein digestibility could be attributed to good and high
concentration of essential amino acids (Atta & El-Shenawi, 2013). The highest
(significant at p<0.01) in vitro protein digestibility value was obtained for WASPI
(91.560.05%) than WASPC (84.200.01%) and WASF (77.460.02%). Similar result
was noted by Ogunbusola et al (2012) in white melon (Cucumeropsis mannii) seed
flour in which protein isolates showed highest digestibility compared with defatted
and full fat flours. This might be due to the presence of trypsin and chymotrypsin
inhibitors and the globular structure of protein reported the lower digestibility of
proteins by the enzymes. Richardson (1991) suggested that the removal of protease
inhibitors during extraction increases the in-vitro protein digestibility observed in
protein isolates. In addition, seed proteins are denatured during isolation, rendering
the protein isolates more accessible to digestive enzymes and improve the hydrolysis
(Lynch et al., 1977). Thus the improvement in digestibility of WASPI might be
attributed to denaturation of protein, destruction of the trypsin inhibitor or reduction
of tannins and phytic acid (Mubarak, 2005). The significant reduction of tannin and
phytic acid in WASPI further confirmed these concepts. The TCA-soluble nitrogen
release during pepsin and trypsin digestion can reflect the changes in the digestibility
139
and the differences in digestion pattern (especially the trypsin digestion) (Wang et al.,
2008).
The in-vitro protein digestibility of WASF was similar to seeds of papaya
(80%), apple (77%), prickly pear (76%) and paprika (78%) (Samia et al., 2012);
lower when compared to seeds of guava (92%), watermelon (91%), apricot (88%)
and orange (86%) (Samia et al., 2012), Cucumeropsis mannii flour (84%) (Ogunbusola
et al., 2012). But higher in comparison to bambara bean flour (70-74%) (Mune et al.,
2011); bitter orange seed flour (75%) (Atta & El-Shenawi, 2013); Mucuna and
Canavalia beans flour (38.6% and 30.6%) (Metsagang et al., 2013); similar to
cucurbita pepo seed flour (77%) (Atuonwu & Akobundu, 2010).
The in-vitro protein digestibility of WASPC was lower when compared to
bambara bean protein concentrate (91-94%) (Mune et al., 2011); defatted
Cucumeropsis mannii flour (85%) (Ogunbusola et al., 2012); cowpea protein
concentrate (86.81-88.74 %) (Mune et al., 2014); but higher in comparison with baru
nuts protein concentrate (65%) (Guimaraes et al., 2012) and bean seed protein
concentrate (76.7%) (Segura-Campos et al., 2014).
The in-vitro protein digestibility of WASPI was similar to cowpea seed protein
isolate (91%) (El-Jasser, 2011); lower when compared to chickpea protein isolate
(95.6- 96.1%) (SanchezVioque et al., 1999); lupin protein isolates (86.3- 93.9%)
(Lqari et al., 2002); Cucumeropsis mannii protein isolates (96.7%) (Ogunbusola et al.,
2012); but higher when compared to Mucuna and Canavalia beans protein isolate
(43.2% and 35.4%) (Metsagang et al., 2013). The high digestibility of WASPI was also
attributable to the low concentration of anti-nutritional factors as well as secondary
metabolites in the seeds (Soetan & Oyewole, 2009).
4.3.7 Thermal Properties
The DSC thermograms of the WASF, WASPC and WASPI are presented in
Fig.4.12 and the values of the melting temperature (Tm), peak of denaturation
temperature (Td) and the transition enthalpy (H) are given in Table 4.29 and Fig.
4.15. The temperature midpoint of the transition for the enthalpy change (Tm) occurs
when the protein goes from native to denatured form. At the Tm, 50% of the protein is
in the native state, and 50% is in the denatured state, assuming a two-state transition.
Some proteins with different regions of activity or more than one structural domain
can have more than one Tm. Tm is an indicator of thermostability, and in general, the
higher the Tm, the more stable the protein. With a higher Tm the protein is less
susceptible to unfolding and denaturation at a lower temperature as well. During a
140
chemical process heat is either released (exothermic) or absorbed (endothermic).

The transition from native to denatured protein is generally endothermic
(www.microcalorimetry.com/2012). Three endothermic peaks were observed in the
thermograms of WASF and WASPC; two endothermic peaks were observed in WASPI;
the first peak was smaller intensity than the second peak. In the DSC pro-files, the
endothermic events are usually associated with the rupture of hydrogen bonds, or the
unfolding of globular proteins during thermal denaturation (Arntfield & Murray,
1981; Privalov, 1982; Meng & Ma, 2001; Fitzsimons et al., 2006).The Tm and Td are
measures of the degree of protein denaturation and are influenced by the heating rate
and the protein concentration. A decrease in the enthalpy value of DSC transitions
reflects the degree of denaturation due to treatments of the isolates and/or greater
tendency of aggregation during the DSC scan. The aggregation can be ascribed to an
increase in the surface hydrophobicity of the proteins as a result of structural
modifications (Adebowale et al., 2008). High Td peaks of 105.4, 104.8, and 102.5C
and of 2.20, 4.47 and 2.08 W/g for globulin II, glutelin and TG fractions, respectively,
and peaks of 76.4 and 70.5C and of 0.37 and 0.97 W/g for globulin I and albumin
fractions, respectively, were observed in Ditaxis hetarantha proteins by EspinoSevilla et al (2013). Martinez & Anon (1996) reported peaks of Td in amaranth at 64C
for albumins and a peak of 96C for glutelin and one of 94C for 11S globulins. Condes
et al (2012) reported two characteristic endotherms: one at 70.7C, which can be
attributed to the denaturation of albumins and a minor globulin fraction (7S
globulin), and a second endotherm at 98.6C that corresponds to denaturation of the
protein fractions of globulin 11S and glutelins for the amaranth isolate. This behavior
was similar to that of D. Heterantha proteins. The higher Td shown by D. Heterantha
proteins indicates a thermostable character, which is commonly related to a higher
number of hydrophobic interactions (Myers, 1990). According to Espino-Sevilla et al
(2013) the first peak between 55C and 75C in WASF (Td-73.6C), WASPC (Td59.1C) and WASPI (Td-56.9C) reflected the presence of albumin and globulin I (7S
globulin) which were of less thermostable; the denaturation between 125C and
130C in WASF, WASPC and WASPI suggested the presence of glutelin, globulin II or
11S globulin in WAS protein, which were more thermostable than albumin and
globulin I (7S globulin). The presence of albumin, globulin I (7S globulin) and globulin
II (11S globulin) was consistent with the SDS-PAGE result.
Enthalpy changes may be associated with molecular changes as a result of
protein unfolding. The enthalpy value is correlated with the net content of the
141
ordered secondary structure of a protein. The changes are due to a combination of

endothermic reactions, such as disruption of hydrogen bonds and exothermic
reactions, such as disruption of hydrophobic interactions (Li et al., 2008). Changes in
protein enthalpy could be used to predict the extent of protein denaturation
(Biliaderis, 1983). The H reflects the proportion of undenatured protein in a sample
or extent of ordered structure (Arntfield & Murray, 1981). The amount of protein
denaturated in WASF below 80C was less than WASPC which was followed by
WASPI. A stable and correctly folded protein is an absolute requirement for a
successful biotherapeutic. All biological processes depend on proteins being stable
and in the appropriate folded conformation. The use of enzyme in industrial
processes may require reaction to be conducted at high temperature in order to
improve productivity and the results obtained suggested that WAS protein could be
considered as potential protein component for various industrial applications (Nurul
& Azura, 2012). A very less intense endothermic peak between (69C - 77C) WASF
could be due to the interference of starch granules which compresses the protein and
induces the protein to lose moisture as suggested by Kim & Lee (1987).
4.3.8. Rheological Properties (RVA)
The pasting temperature is an indication of the minimum temperature
required to cook or gelatinize the flour (Kaur & Singh, 2005). The pasting properties
of WASF, WASPC and WASPI are presented in Fig. 4.16 and the values are presented
in Table 4.30. The peak viscosity was significantly (p<0.01) high in WASPC than
WASF and WASPI. The trough viscosity, final viscosity and setback viscosity of
WASPC was significantly (p<0.01) greater than WASPI and WASF. WASPI had
exhibited significantly (p<0.01) greater breakdown viscosity than WASPC and WASF.
The differences in the starch and protein composition in the flours could affect
pasting viscosity and properties (Morris et al., 1997; Batey & Curtin, 2000). Peak
viscosity is the maximum viscosity developed during or soon after the heating
portion. It is an index of the ability of starch-based foods to swell freely before their
physical breakdown (Adebowale et al., 2008). Similarly increase in peak viscosity was
noted in low amylase content of flour or starch (cassava) sample by the addition of
soy protein concentrate (Zaidul et al., 2007; Chinma et al., 2013). Proteins contain
many hydrophilic groups (such asCOOH,NH2, OH, andSH) all of which are
capable of forming cross links with starch and these cross links may be responsible
for their higher paste viscosity as compared to cassava starch paste (Goel et al.,
1999).
142
TABLE 4.29: DSC THERMOGRAMS OF WASF, WASPC AND WASPI

Sample
Peak I
Peak II
Peak III
Tm (C)
Td (C)
H Enthalpy (J/mg)
Tm (C)
Td (C)
H Enthalpy (J/mg)
Tm (C)
Td (C)
H Enthalpy (J/mg)
WASF
69.30.04
76.90.03
0.010.05
113.80.02
115.90.30
0.900.07
116.20.05
129.60.06
34.70.02
WASPC
45.10.60
59.10.06
4.080.04
101.30.46
103.60.01
0.940.03
115.30.09
130.30.02
25.80.07
WASPI
56.20.51
56.90.08
0.750.70
105.60.05
125.30.32
27.00.02
Values are average of two determinations.
Fig. 4.15: DSC thermogram of WASF, WASPC and WASPI
143
Trough viscosity measures the ability of the paste to withstand breakdown

during cooling (Iwe et al., 2016). The breakdown viscosity is an index of the stability
of the starch and a measure of the ease with which the swollen granules can be
disintegrated (Kaur et al., 2007). The higher the setback viscosity, the lower the
retrogradation of the flour paste during cooling and the lower the staling rate of the
products made from the flour (Adeyemi & Idowu, 1990). Setback viscosity has been
correlated with the texture of various end products. The breakdown and setback
viscosity of WASF and WASPI were significantly (p<0.01) lower than WASPC
respectively. The setback viscosity was found to be completely nullified in WASPI.
Similarly a decrease in setback viscosity was noted in pea starch while increasing the
proportion of peanut protein isolate in pea starch blends (Sun & Xiong, 2014). This
confirmed the higher potential of retrogradation (Oduro et al., 2000) and less
cohesiveness (Kiin-Kabari et al., 2015) of WASPI paste prepared by heating at 95C
and cooling down at 50C. Onwulata et al (2014) mentioned that a typical RVA profile
was obtained for whey protein isolate at 20% concentrate, but as concentrate
increased to 40%, profile showed an inflated peak during the cooling phase. Similar
inflated peak profile was noted in WASPC and WASPI when compared to WASF which
was further reflected through nullified setback viscosity in WASPI. When compared
to pasting profile of Brachystegia eurycoma seed flour (Ikegwu et al., 2010); tomato
seed meal (Ekthamasut, 2006); and bambara groundnut protein concentrate (KiinKabari et al., 2015), the pasting property of WASF, WASPC and WASPI was very low
and indicates the low viscous load likely to be encountered during mixing as reported
by Moure et al (2006). Final viscosity is commonly used to define the quality of a
particular starch-based flour since it indicates the ability of the flour to form a viscous
paste after cooking and cooling. It also gives a measure of the resistance of the paste
to shear force during stirring (Adebowale et al., 2005, 2008). Peak time is a measure
of the cooking time and it ranged between 2.07-4.47 mins for WAS.
TABLE 4.30: PASTING PROPERTIES OF WASF, WASPC AND WASPI
Samples
Peak
Viscosity
(cP)
Trough
Viscosity
(cP)
Breakdown
Viscosity
(cP)
Final
Viscosity
(cP)
Setback
Viscosity
(cP)
Peak
Time
(mins)
WASF
25.00.10a
21.00.30a
4.000.10a
38.341.45b
13.11.98b
4.47c
WASPC
36.00.60c
31.51.09c
5.001.67b
50.320.99c
14.001.17c
3.20b
WASPI
34.00.12b 23.30.46b 11.000.04c
34.1126.3a
0.000.00a
2.07a
Values are average of two determinations. The alphabets following the values indicates LSD
between groups at p<0.01.
144
Fig. 4.16: Pasting properties of WASF, WASPC and WASPI

4.3.9. In Vivo Protein Quality
4.3.9.1 Physical Appearance
The physical appearance of rats in all the group rats was normal in all the
groups throughout the eight weeks of the experiment. The eyes, mouth and hair of the
animals used in their respective groups appeared to be normal throughout the period
of the study.
4.3.9.2 Growth Performance
Gain in body weight as a measure of the nutritive value of dietary proteins has
been most popular (Kalyani et al., 2012). The casein diet (CD) feed was consumed
maximally, followed by WASPI, WASPC and WASF feed, while the protein free diet
(PFD) was consumed the least (Table 4.31 and Fig. 4.17) . The food intake of rats fed
with CD and WASPI were significantly higher (p<0.05) compared to WASF and
WASPC. The average protein intake of the rats was significantly (p<0.05) higher in CD
and WASPI than WASF and WASPC (Table 4.31 and Fig. 4.18). These findings indicate
that the total protein consumed by the different groups paralleled the total food
intake of the rats. There was steady increment in the body weight of the rats during
the experimental period. The control (Casein diet) was found to have the highest
weight gain followed by the WASPI when compared to WASPC and WASF. There were
significant differences (p<0.05) in body weights among groups, except for rats fed
with protein-free diet (PFD) (Table 4.31 and Fig. 4.19). The PFD had loss of body
weight due to less food intake. As suggested by Pugalenthi et al (2007), the lower
food intake was probably due to the difference between the diets in protein quality
and effects of anti-nutritional compounds in the WASF. The reduction in weight gain
145
of PFD could be not only due to lower feed intake but also due to lower protein
utilization (Rahman et al., 2014).
Digestibility of protein is related to the quality of protein in the feed. The
higher ratio of total essential amino acid to total amino acid contents in samples
resulted in rapid growth of rats. The high ratio of essential amino acids to total amino
acids in meat products explain their superiority in protein quality when used as new
protein sources for rats (Hoffman & Falvo, 2004).
4.3.9.3 Protein Quality Indices
Protein efficiency ratio (PER), C-PER, net protein utilization (NPU) and net
protein ratio (NPR) of casein, WASF, WASPC and WASPI are shown in Table 4.32. The
highest (significant at p<0.01) PER value was found for CD and WASPI, followed by
WASPC and WASF. As suggested by Friedman (1996) and depending on PER of CD
and WASPI, both were considered to be equally good quality protein and can be used
in infant formulation; while PER of WASF and WASPC considered to be intermediate
quality as it was ranged between 1.5-2.0. The PER of WASF was similar to PER value
of boiled roselle seed (1.71) (Halimatul et al., 2007).
TABLE 4.31: BODY WEIGHT, FEED INTAKE, % PROTEIN CONSUMED OF RATS BY PFD,
CD, WASF, WASPC AND WASPI DIETS
Body weight (g)
Total feed intake

(g/rat/28days)
(g)
Protein consumed
(g/rat/28days)
(g)
PFD
-2.60.05a
190.470.01 a
CD
8.010.02 e
269.920.03 e
31.70.01 d
WASF
2.900.01 b
237.510.01 b
21.30.05 a
WASPC
3.410.02c
254.660.01 c
26.40.02 b
WASPI
7.900.05 d
264.110.01 d
31.10.03 c
Diet
Values are average of six rats in each group. The alphabets following the values indicatesLSD
TABLE 4.32: PER, C-PER, NPR AND NPU OF CD, WASF, WASPC AND WASPI DIETS
Diet
PER
C-PER
NPR
NPU
CD
2.130.10d
2.500.01d
1.450.03d
83.590.09 d
WASF
1.720.01a
2.010.03 a
1.010.17a
61.700.08 a
WASPC
2.010.02b
2.300.01b
1.120.01b
64.550.03 b
WASPI
2.110.05c
2.480.02c
1.380.11c
82.720.09 d
Values are average of six rats in each group. The alphabets following the values indicates LSD
146
14
Food Intake (g)
12
10
8
PF
CD
WASF
WASPC
WASPI
0
1st wk
2nd wk
3rd wk
4th wk
Weeks
Fig. 4.17: Changes in food intake of rats of different dietary groups during
the growth study. Values are expressed as average of six animals (n=6)
Protein consumed (g)
12
10
8
CD
WASF
WASPC
WASPI
2
0
1st wk
2nd wk
3rd wk
4th wk
Weeks
Fig.4.18: Changes in protein intake of rats of different dietary groups during

40
Body weight (g)
35
PF
30
CD
25
WASF
20
WASPC
15
WASPI
10
Initial
1st wk
2nd wk
3rd wk
4th wk
Weeks
Fig. 4.19: Changes in body weight of rats of different dietary groups during
147
TABLE 4.33: TD, BV AND AD OF CD, WASF, WASPC AND WASPI DIETS
Diet
TD (%)
BV (%)
AD (%)
CD
85.93 0.41d
97.28 0.33d
83.32 0.01d
WASF
65.18 0.26a
94.670.10a
64.54 0.06a
WASPC
67.10 0.10b
96.21 0.06b
66.78 0.08b
WASPI
84.890.18c
97.45 0.04 c
83.230.50 c
The PER of WASPC was higher in comparison with tomato seed protein
concentrate (1.45) (Sogi et al., 2005). The close PER values for test diets to casein
indicates adequate combination of essential amino acids in test diets. Plant proteins
are categorized into three groups of PER, high PER (<2.3 to >1.6), medium PER (<1.6
to >1.0) and low PER (<1.0). The present study revealed that all the test and standard
protein diets had high PER (Hsu et al., 1978). The NPU and NPR was significantly
(p<0.05) higher in CD and WASPI in comparison to WASPC and WASF (Table 4.31).
The NPU of WASF and WASPC was higher in comparison with Amaranthus
cruentusflour and protein concentrate (51.50 and 57.45) respectively (Escudero et al.,
2004).
The TD, AD and BV of CD and WASPI was significantly (p<0.01) higher than
WASPC and WASF (Table 4.33). The greater digestibility of the casein diet resulted in
a higher retention of dietary nitrogen as indicated by the lower fecal nitrogen output
of the rats consuming that diet reported by Sarwar & Peace (1986). The nutritional
quality of any protein relates to its amino acid composition, digestibility, and ability
to supply the essential amino acids in the amounts required by the species consuming
the protein (Endres, 2001). For plant protein, specifically soy protein is high in
protein value, however the protein efficiency ratio is lower compared from animal
protein (casein and buffalo meat). The PER for soy protein concentration is 2.1
(Richard, 2005), sodium caseinate is 2.5 (Sindayikengera & Xia 2005), casein is 2.5
(Hambraeus, 1982) and meat is 3.12 (Piva et al., 2000). Similar PER value was noted
for casein in the present study. Tempeh displayed the highest percentage of
digestibility (91.41%), followed by casein (91.34%), buffalo meat (90.79%), soy
protein isolate (89.52%) and sodium caseinate (89.47%) (Babji et al., 2010). The TD
of casein and WASPI were similar to that of reference protein (100% Nutrend from
Nestle food Nigeria) (86.18%) and lower than African yam bean meal (97.7%)
(Onwuka et al., 2009). The BV of WASPI (97.45%) was similar to the BV of casein
148
(97.28%) protein and greater than 100% Nutrend from Nestle food Nigeria (71.69%)
(Onwuka et al., 2009).
4.3.9.4 Organ Weight
Organ weight is also an important index of physiological and pathological
status in animals. The relative organ weightis fundamental to establish whether the
organ was exposed to the injury or not. The heart, liver, kidney, spleen, and lungs are
the primary organs affected by metabolic reaction caused by toxicant. The liver, being
a key organ in the metabolism and detoxification of xenobiotics, is vulnerable to
damage induced by a huge variety of chemicals (Jothy et al., 2011). The organ weight
of PFD, CD, WASF, WASPC and WASPI was depicted in Table 4.34. The absence of
significant differences in the weight of kidney and heart of five diets groups might
explain the protein sources did not show any significant influence on the tissues of
these organs within 28days of study period.
TABLE 4.34: RELATIVE WEIGHT (g/100 g OF BODY WEIGHT) OF LIVER, KIDNEY AND
HEART OF RATS FED WITH FIVE DIETS
Diet
Liver
Kidney
Heart
PFD
1.470.21 ab
0.240.00 a
0.230.01 a
CD
2.090.20 ab
0.260.02 a
0.260.01 a
WASF
2.060.31 a
0.280.06 a
0.300.10 a
WASPC
2.160.24 ab
0.260.02 a
0.360.19 a
WASPI
2.270.47 b
0.260.02 a
0.360.06 a
between groups at p<0.01. Average in columns with same superscript do not differ
significantly from each other p<0.05.
Similar observation was noted by Ekeanyanwu & Njoku (2014) that no

significant (p>0.05) difference in weight of kidney and heat for the groups treated
with flavonoid rich fractions of seed extract M.tenuifolia after the day 14 and 28 of the
study. Besides, the weight of liver in PFD and WASPI diet groups were significantly
different from those of the CD, WASF and WASPC diet group. Similarly there was
significance (p<0.05) increase in liver of rats treated with flavonoid rich fraction of
seed extract M.tenuifolia (Ekeanyanwu & Njoku (2014). The decreased body weight
and organ weight in PFD could be due to reduced diet intake or necrotic changes in
different body tissues (Fukamachi et al., 2004). It was also evident that the
consumption of WASF, WASPC and WASPI would not cause any adverse effect on the
weight of these major organs which also suggested by Wong et al (2004). As reported
by Beynen et al (1987) and Dabai et al (1996) the variations in the weight and
149
appearance of livers in rats fed with different diets were related to the difference in
the amount of cholesterol and oil added into the diets.
4.4.9.5 Biochemical Parameters
Analysis of blood parameters is relevant to risk evaluation and the changes in
the haematological system have a higher predictive value for human toxicity, when
the data are translated from animal studies (Ibrahim et al., 2010). The assessment of
haematological parameters could be used to reveal the deleterious effect of foreign
compounds including seed extract on the blood constituents of animals. They can also
be used to determine possible alterations in the levels of bio molecules, metabolic
products, haematology, normal functioning and histomorphology of the organs (Jothy
et al., 2011). Urea and creatinine are considered as a suitable prognostic indicator of
renal dysfunction and kidney failure for any toxic compounds (Gnanamani et al.,
2008). Measurements of the activities of serum marker enzymes like ALT, AST and
ALP have provided a powerful tool for the assessment of liver function (Aliyu et al.,
2007). Alkaline phosphatase is membrane bound and its alteration is likely to affect
the membrane permeability and produce derangement in the transport of
metabolites (Ekeanyanwu et al., 2014). An elevated serum ALP level is often
associated with various disorders such as extra hepatic bile obstruction, intra hepatic
cholestasis, infiltrative liver disease, as well as hepatitis and bone disease (Jothy et al.,
2011).
The level of biochemical parameters (urea, BUN, creatinine and uric acid)
were normal in PFD, CD, WASF, WASPC and WASPI, whereas the blood glucose level
was elevated in PFD, CD, WASF, WASPC and WASPI (Table 4.35). The lipid profile
values were normal in PFD, CD, WASF, WASPC and WASPI. Liver function values were
normal other than bilirubin (direct) which was significantly greater than reference
value. The low in blood glucose might be due to starvation before the dissection of
rats. It was also highly correlated with increased bilirubin (direct); SGOT and SGPT
values of PFD, CD, WASF, WASPC and WASPI. This result was supported by the
histopathology of liver.
The total protein of CD, WASF, WASPC and WASPI were normal other than
PFD. The low protein level in PFD was due to low food intake and protein utilization,
which led to weight loss and low Hb in PFD. The other haematology values were
normal in PFD, CD, WASF, WASPC and WASPI when compared to the reference value.
150
TABLE 4.35: LEVEL OF BIOCHEMICAL PARAMETERS OF RATS FED WITH FIVE DIETS
Parameters
Unit
Ref value
PF
Casein
WASF
WASPC
WASPI
Blood glucose
mg/dl
50-160
45.90.12
9.30.02
80.00.03
77.50.10
68.40.02
Urea
mg/dl
10-50
38.10.01
39.20.01
42.30.09
31.10.01
40.30.02
BUN
mg/dl
10-21
17.80.03
13.60.00
19.60.02
14.50.01
20.40.00
Creatinine
mg/dl
0.2-0.5
0.160.01
0.170.01
0.210.02
0.160.04
0.130.07
Uric acid
mg/dl
3.4-7.0
1.60.0.02
2.20.05
3.50.06
2.90.03
3.00.01
Cholesterol
mg/dl
35-85
50.20.01
65.570.02
52.200.03
49.840.10
47.010.01
Triglycersides
mg/dl
20-114
68.90.02
79.00.10
89.00.01
78.00.02
74.00.01
HDL
mg/dl
40-59
44.370.00
64.700.01
45.450.01
48.040.00
43.890.02
LDL
mg/dl
<129
28.30.03
40.50.01
38.80.12
25.360.03
23.800.12
VLDL
mg/dl
10-30
13.60.01
15.80.01
17.80.03
15.60.11
13.80.05
Total Cho/HDL ratio
Ratio
1.0-3.0
1.330.00
1.010.02
1.140.01
1.030.00
1.070.01
Bilirubin (Total)
mg/dl
0.05-0.15
0.130.01
0.100.03
0.100.02
0.120.03
0.100.01
Bilirubin (Direct)
mg/dl
0.03-0.05
0.100.00
0.070.05
0.090.0
0.110.01
0.080.02
Bilirubin (Indirect)
mg/dl
0.01-0.12
0.030.04
0.030.02
0.010.01
0.090.05
0.020.00
Biochemistry
Lipid Profile
Liver Function
151
Total protein
Total protein
g/dl
6.6 8.3
3.20.01
6.80.04
6.680.01
6.670.01
6.700.01
albumin
g/dl
3.5 -5.2
2.40.01
4.40.01
4.00.00
4.20.00
4.20.02
globulin
g/dl
2.3 3.5
0.80.00
2.40.00
2.60.01
2.40.03
2.50.03
A/G ratio
Ratio
1.58-2.67
3.00.01
1.830.00
1.530.04
1.750.03
1.680.03
SGOT (AST)
U/L
5-50
262.70.01
220.70.02
217.10.03
201.60.03
214.60.03
SGPT (ALT)
U/L
7-40
47.40.02
60.70.03
38.20.04
42.50.03
41.30.03
Alkaline Phosphatase
U/L
40-130
1090.01
101.90.01
66.30.03
99.10.03
Gamma GT
U/L
10-71
6.10.00
10.30.06
11.920.07
11.50.03
8.30.03
Iron
g/dl
59-158
145.20.01
129.60.09
135.100.00
147.50.03
123.80.03
TIBE
g/dl
171-504
478.20.02
460.90.01
408.50.03
496.60.03
488.90.03
Calcium
mg/dl
10.9-12.3
11.120.00
10.930.01
11.670.03
12.160.03
12.210.03
Phosphorus
mg/dl
2.5-4.5
3.360.01
3.040.00
3.90.02
3.490.03
4.030.03
Magnesium
mg/dl
1.6-2.6
2.040.00
2.090.01
2.320.01
1.800.03
1.890.03
Copper
mg/dl
50-80
31.090.01
38.20.03
33.50.00
42.840.01
40.340.03
Amylase
U/L
20-90
290.01
310.01
400.02
840.02
820.00
Haemoglobin
g/dl
13-18
110.02
14.5.20.01
13.20.00
14.20.03
14.20.00
Haematocrit (PCV)
40-52
42.60.02
44.30.01
46.70.01
45.80.03
48.50.02
Total WBC count
(cells/cumm)
4000-11000
80000.00
106000.08
100000.04
98000.01
102000.00
68.30.05
Haematology
152
Differential count
Neutrophilis
40-75
62.70.08
62.20.03
51.50.01
520.00
67.10.07
Lymphocytes
20-45
30.10.00
31.20.01
430.03
43.80.02
28.90.03
Eosinophils
1-6
4.20.01
4.30.01
4.20.02
2.30.00
1.20.00
Monocytes
2-10
2.60.01
2.80.02
2.10.01
2.60.00
2.50.00
Basophils
0-1
0.40.06
0.50.00
0.20.09
0.30.00
0.30.01
Platelet count
cells/cumm
3850000.02
3820000.01
3320000.03
2770000.01
3770000.02
RBC count
millions/cumm
4.5 5.9
4.510.05
5.300.03
5.270.01
5.140.01
5.740.00
MCV
fl
80-100
86.80.01
87.10.01
85.60.04
85.60.03
87.20.01
MCH
pg
27-31
34.50.02
32.60.01
35.40.01
36.50.00
37.50.00
MCHC
g/dl
32-36
30.50.01
33.80.00
32.50.00
32.90.00
33.60.03
RDW-SD
fl
37 54
38.80.00
37.70.03
45.80.03
41.90.02
47.80.01
RDW-CV
11-16
19.30.01
14.10.00
12.80.01
15.00.01
15.80.05
PDW
fl
9-17
14.40.02
13.20.01
13.40.04
14.10.01
14.40.04
MPV
fl
9-13
9.30.01
11.30.01
12.40.03
12.60.01
12.80.03
PCT
0.17-0.35
0.3190.05
0.3020.02
0.2900.01
0.2160.00
0.2940.01
150000450000
Values are average of six rats in each group.
153
4.3.9.6 Histopathological Examination of Liver and Kidney

The histopathology of liver sections of PFD, CD, WASF, WASPC and WASPI are
depicted in Table 4.36 and Figs. 4.20 (a-e). The PFD (Fig. 4.20a) liver showed a
normal lobular architecture; no changes in individual hepatocytes remarkable. The
portal tract was normal with bile duct hyperplasia. In CD (Fig. 4.20b), the liver
showed normal lobular architecture; individual hepatocytes showed microvesicular
fatty change and binucleation. There was no evidence of necrosis. In WASF (Fig.
4.20c), the liver showed hepatocytes microvesicular fatty change and cytoplasmic
vacuolation. There was no evidence of necrosis. In WASPC (Fig. 4.20d), the liver
showed normal lobular architecture and heptocytes; no remarkable changes in
sinusoids and portal tract. In WASPI (Fig. 4.20e), the liver showed normal lobular
architecture; individual heptocytes were with cytoplasmic vacuolation and
binucleation. The portal tract showed bile duct hyperplasia. The vacuolated
appearance of the cells was due to accumulation of glycogen which further
substantiated by the findings in rats starved for 24 h before sacrifice. It was known
that during starvation glycogen was rapidly removed from liver cells (Bannasch,
1968; Babcock & Cardell, 1974; Scherer & Emmelot, 1976; Cardell & Cardell, 1990).
TABLE 4.36: RESULT OF HISTOPATHOLOGICAL STUDIES OF LIVER
Inflammation
Cytoplasmic
Vacuolation
Bile duct
Hyperplasia
Binucleation
PFD
CD
WASF
++
WASPC
WASPI
Sample
TABLE 4.37: RESULT OF HISTOPATHOLOGICAL STUDIES OF KIDNEY
Inflammation
Glomeruli
Abnormality
Tubular
Injury
Cast formation
PFD
CD
WASF
WASPC
WASPI
Sample
154
The histopathology of kidney sections of PFD, CD, WASF, WASPC and WASPI
are depicted in Table 4.37 and Figs. 4.21(a-e). The PFD (Fig. 4.21a) kidney showed
normal cortex and medulla; glomeruli and both tubules showed normal morphology;
no significant pathology of interstitium; blood vessels showed congestion. The CD
(Fig. 4.21b) kidney showed normal cortex and medulla; glomeruli showed normal
with mild mesangeal hypercellularity; no remarkable change in tubules and
interstitium. The WASF (Fig. 4.21c) kidney showed normal cortex and medulla;
glomeruli showed normal morphology; tubules showed mild focal tubular injury and
tubular cast (hyaline cast); no remarkable change in interstitium and blood vessels.
The WASPC (Fig. 4.21d) and WASPI (Fig. 4.21e), kidney showed normal cortex and
medulla; glomeruli and both tubules showed normal morphology; normal
morphology of interstitium and showed congestion of blood vessels. The glomeruli
abnormality and tubular loss occurs due to protein load in kidney as reported by
(Brenner & Chertow, 1994). Hyaline cast are typically composed of proteins. They are
associated with increased protein in glomerular filtrate, such as albumin, or with
secretion into lumen from tubule cells. Hyaline cast are characteristics of chronic
progressive nephropathy (Hard et al., 1999).
155
(i)
(ii)
(iii)
(iv)
(v)
Fig.4.20a : PFD photomicrograph of liver section; (i)- Hepatocytes with
sinusoidal dilatation; (ii)- Bile duct hyperplasia; (iii)- Normal obular
architecture; (iv)- lobular architecture; (v)- sinusoidal dilatation and central
vein congestion
156
(i)
(ii)
(iii)
(iv)
(v)
Fig.4.20b: CD photomicrograph of liver section; (i)- Lobular architecture; (ii)Central vein congestion; (iii)- Normal obular architecture; (iv)- Shows
microvesicular fatty change (v)- Normal portal tract
157
(i)
(ii)
(iii)
Fig. 4.20c: WASF photomicrograph of liver section
(i)- Normal lobular architecture; (ii) Microvesicular steatosis;
(iii)- Mild cv congestion
158
(i)
(ii)
(iii)
Fig. 4.20d: WASPC photomicrograph of liver section

(i)- Central vein congestion; (ii) normal lobular architecture;
(iii)- Mild bile duct hyperplasia
159
(i)
(ii)
(iii)
(iv)
Fig. 4.20e: WASPI photomicrograph of liver section (i)- Normal lobular

architecture; (ii) blood vessels congestion; (iii)- central vein normal
(iv)- normal portal tract
160
(i)
(iii)
(ii)
(iv)
Fig.4.21a: PFD photomicrograph of kidney section;

(i)- Normal kidney (ii)- Normal glomeruli;
(iii)- Normal tubular compartment; (iv)- Normal tubules
161
(i)
(ii)
(iii)
(iv)
(v)
Fig. 4.21b: CD photomicrograph of kidney section; (i)- Normal cortex and
medulla; (ii)- Normal kidney; (iii)- Normal glomeruli; (iv)- Normal measngeal
hyperplasia; (v)- Normal tubules
162
(i)
(iii)
(ii)
(iv)
Fig. 4.21c: WASF photomicrograph of kidney section (i)- Normal kidney;

(ii)-Mild focal tubular loss; (iii)- Normal glomeruli and tubules;
(iv)- Hyaline cast
163
(i)
(ii)
(iii)
(iv)
Fig. 4.21d: WASPC photomicrograph of kidney section

(i)- Cortex and medulla; (ii)- Normal medulla;
(iii)- Normal glomeruli ;(iv)- Normal interstitium
164
(i)
(ii)
(iii)
Fig. 4.21e: WASPI photomicrograph of kidney section (i)- Normal kidney;
(ii)- Normal tubules; (iii)- Normal glomeruli
165
Summary and Conclusion
5. SUMMARY AND CONCLUSION

The research finding on the present study entitled Extractability and
Characteristics of Wood Apple (Limonia acidissima) Seed Protein were
summarised under the following headings.
5.1 Summary of work performed
5.2 Summary of findings
5.3 Conclusion
5.4 Recommendation
5.1 SUMMARY OF WORK PERFORMED
Wood apple also known as elephant apple and monkey fruit, wood-apple is a
medium-sized deciduous tropical tree common to the dry districts of India as well as
the dry and (some of the wet) territories of Sri Lanka. The brown pulp has a strong
smell but an excellent, slightly sour flavour when ripe, at which stage it is dark brown.
When ripe, the fruits pulp can be consumed either fresh or processed into high value
and extremely popular products such as jams and cordials. Furthermore, wood apple
is also said to have various curative properties i.e. anti-diabetic, antimicrobial, antiinflammatory, anti-pyretic, analgesic, anti-tumour and antioxidant potential, much
used in Indian folk medicine as a liver and cardiac tonic with hepato-protective
activities. Although many beneficial effects of wood apple have been described, its use
as a plant material, especially the seeds, has not been widely studied. The insertion of
seeds in the human diet will be determined on their characteristics, hence the present
research work were made in three phases; in phase I, the whole fruit of wood apple
and seed were characterized for its agronomical features, anatomical characteristics
and physical properties. In phase II, protein from the wood apple seed were isolated
by five different methods (TCA/acetone precipitation, phenol extraction, ammonium
sulphate precipitation, Tris-HCl buffer precipitation and alcoholic precipitation and
efficient method of isolation was determined which resulted in high quality purified
WASPI. The framed hypothesis such that the method of isolation did not significantly
influence the quality of WASPI was tested through protein recovery, protein content,
functional properties-hydrophilic and hydrophobic properties, surface properties and
properties related to protein-protein interactions, fractions of proteins and 1D SDS
PAGE electrophorogram. In phase III, WASF, WASPC and WASPI were compared by
studying its physio-chemical properties, nutritional composition, anti-nutritional
composition, qualitative profile of phyto chemistry, total phenol content, antioxidant
166
activity profile, in-vitro protein digestibility, thermal properties, rheological

properties and in-vivo protein quality. Through these characteristics the framed null
hypotheses that there was no significant difference between the characteristics, of
seed flour, protein concentrate and protein isolate from wood apple seed and the
protein quality of wood apple seed was not significantly comparable with casein
protein were tested to predict the industrial exploitability of WASPI.
5.2 SUMMARY OF FINDINGS
The present investigation was performed in three phases and the test results
on hypotheses were described as follows.
5.2.1 Phase I
The wood apple seed was separated from whole fruit pulp in two methods
(under tap water and centrifugal juicer). The seed collected by juicer method was
quantitatively greater with highest seed to pulp ratio. The longitudinal dimension
(length) of the seed was higher than the width and thickness. The length of the seed
was positively related to its thickness and geometric mean diameter; thickness was
positively related to its geometric mean diameter. According to the low sphericity
and aspect ratio of the seed, wood apple seeds are likely to slide on their flat surfaces
rather then roll. The average projected area and surface area was found to be 16.47
(0.23) and 35.46 (0.60) mm2 while the mean volume determined for the seeds was
12.66 (0.45) mm3.
The wood apple (Limonia acidissima) seed were 5 or 6 2.55 mm in size. The
seeds are slimy and brown or dark brown in colour and the seeds are exalbuminous.
The seeds were oblong elliptical. There are two colyledons which are plano convex.
The flat sides of the cotyledons are juxtaposed and occur in close proximity within the
seed coat shown. In median longitudinal section, the full embryo with cotyledons and
radicle were seen. The cotyledons were 20 mm long and 8 mm thick, while the radicle
were thick and conical with 15 mm height and 10 mm thickness. Procambial strand
was seen in the radicle and plumule was minute and occurs in between cotyledons.
The cells of the cotyledons are thin walled, polyhedral and are darkly strained. The
cells contain many cell inclusions, the starch grains being the major content. The
wood apple seed coat (Testa) consists of three zones. The outer zone had one or two
layers brachy scelerids with lignified walls. From the scelerotic layer several conical
bundles of hairs arose which were tightly held and conical in shape. The surfaces of
the tufts of hairs were irregularly bulged. The trichome bundles were up to 20 mm
long. The middle part of the seed coat was thin and includes three or four layers of
167
tangentially elongated parenchyma cells and discontinuous small clusters of lignified

scelerids. The inner zone of the seed coat was parenchymatous (sarcotesta). It
includes six or seven layers of circular thin walled compact cells with dense cells
inclusions. The innermost layer of the parenchymatous seed coat forms a thin layer
of epidermis. The total thickness of seed coat was 200-300 m. Along the boundary of
the middle seed coat occurs a layer of calcium oxalate crystals. The crystals were
prismatic type. The crystalliferous layer consists of two or three rows of crystals and
the layer was continuous all along the seed coat.
5.2.2 Phase II
The amount of protein extracted by TCA/acetone as precipitating agent was
significantly (p<0.01) higher, which was followed by ammonium sulphate
precipitation, alcoholic precipitation, Tris-HCl extraction and phenol extraction
method in order. The TCA/acetone method was considered to be the efficient method
for extraction of protein from WAS in terms of both protein recovery and purity.
The WAC, OAC, dispersibility and solubility of WASPI extracted by
TCA/acetone as precipitating agent was significantly (p<0.01) higher than other
extraction methods. The maximum protein solubility of WASPI was noted at pH 12.0
in TCA/acetone method and alcoholic precipitation method, pH 6.0 in phenol method,
pH 5.0 in ammonium sulphate method and pH 7.0 in Tris-HCl buffer method. While
considering the LGC, weak gel was formed at 8% (w/v) in TCA extracted WASPI, good
gel was formed at 12% in alcoholic precipitated WASPI; at 16% in WASPI
precipitated by phenol, ammonium sulphate and Tris-HCl buffer methods.
The foaming capacity of WASPI extracted by TCA/acetone method was
significantly (p<0.01) greater than other extraction methods. In alcoholic
precipitation method more than 50% FC was noted. The lowest FC of 24% was noted
in ammonium sulphate as precipitating agent. After 30 minutes of foam formation,
52% stability was noted in WASPI extracted by TCA/acetone method (significant at
p<0.01). Irrespective of method of extraction, foaming capacity was directly
correlated with the FS. The foam was least stable (10.67%) in WASPI extracted by
ammonium sulphate method. The WASPI extracted by TCA/acetone method had
exhibited good intermolecular cohesiveness and elasticity which reflected 50% foam
stability after 30 minutes. Good foam stability of WASPI extracted by TCA/acetone
method denotes capacity to effectively reduce interfacial tension and favourable
molecular orientation at the oil and water interface.
168
The EC of WASPI extracted by TCA/acetone method was significantly greater

which was followed by alcoholic precipitation, ammonium sulphate precipitation,
Tris-HCl buffer extraction and the least EC in phenol extraction method. Irrespective
of effect of ratio of water, extraction time and pH, the TCA/acetone method exhibited
greater protein extractability from WASPI. The highest yield of precipitated water
soluble wood apple seed protein was obtained at pH 4.5. The percentage of protein
precipitated was significantly (p<0.01) high in TCA/acetone method and low in
ammonium sulphate method. The highest extraction of albumins (66.03 g%),
globulins (24.86 g%), prolamins (5.67 g%) and glutelins (6.13 g%) was noted in TCA
method, Tris-HCl buffer method, Tris-HCl buffer method and alcoholic precipitation
method respectively. The high albumin extractability in TCA/acetone method was
highly correlated with protein solubility. The TCA/acetone method (14.2 mg/ml)
suggested the maximum amount of protein extracted and established as best
extraction method. A very low amount of protein was estimated in ammonium
sulphate method.
The relative MW of band was mainly ranged from 14.4 to 97.4 kDa. The band
of WASPI extracted by TCA/acetone method consisted of 27 bands with different
intensities. The number of bands in WASPI extracted by ammonium sulphate method
revealed 13 weak stained bands. In Tris-HCl buffer extracted and alcoholic
precipitation method only one band with strong intensity was noted between 43 kDa
and 66.2 kDa. In phenol extraction method nine bands with very weak stained were
identified. The polypeptide subunits confirmed the presence of albumin, globulin,
prolamin and glutelin fractions in WASPI.
While considering the above mentioned results, significant influence of
different extraction methods on quality characteristics of WASPI revealed the
rejection of null hypothesis (The method of isolation did not significantly
influence the quality of the wood apple seed protein isolate).
5.2.3 Phase III
The WAC and EC of WASF were greater than WASPC and WASPI. The OAC, FC,
FS, dispersibility and bulk density were observed to be significantly (p<0.01) higher
in protein isolate than the protein concentrate and seed flour. Wettability grade of
wood apple seed protein isolate was excellent since it wet slightly when it comes in
contact with water and after 30 min the wetted sample and the powder sunk to the
bottom. The WASF and WASPC also had good wettability property as 80% of sample
got wet in 30 mins.
169
The WASF was rich in moisture, fat, carbohydrate, fibre and energy content
than WASPC and WASPI. The WASPC was rich in ash, while WASPI was rich in
protein. The mineral content of the wood apple seed components increased on
defatting. The high amount of potassium was observed in the wood apple seed
protein concentrate. The WASPC had more Ca, P, Na and K when compared to WASF
and WASPI. The Ca/P ratio of WASF, WASPC and WASPI was medium since it is <1
and >0.05; Na/K ratio was <0.6. The essential amino acids (histidine, threonine,
valine, leucine, isoleucine and lysine) of WASF, WASPC and WASPI were in higher
percentage than the FAO/WHO reference, pattern for child and adult while it was
lower when compared to casein protein. According to FAO/WHO reference
tryptophan was the first limiting amino acid, phenyalanine was the second limiting
amino acid and methionine was the third limiting amino acid in WASPI. In
comparison with casein leucine, tryptophan, valine and isoleucine were the limiting
amino acids in ascending order in WASPI. As per the percentage of amino acid with
different functional group characteristics in WASF, WASPC and WASPI, hydrophobic
(alanine, isoleucine, leucine, methionine, phenylalanine, valine) and basic amino acid
(lysine, arginine, histidine) were found to be in highest percentage followed by acidic
(aspartic acid, glutamic acid) and uncharged polar (glycine, serine, threonine,
tyrosine) amino acid groups. According to EAAI and P-PER value, WASF, WASPC and
WASPI were said to be of good quality proteins and WASPI was comparable with
casein protein.
Anti-nutritional components (tannin, phytate, oxalate and saponin) were
significantly reduced on extraction and purification of wood apple seed protein as
isolate.
Among the screened phytochemicals flavonoids, phenolics, carbohydrate,
proteins and amino acids were present in high amount in aqueous extract of WASF;
flavonoids, amino acids and protein in aqueous extract of WASPC; flavonoids,
phenolics and protein in aqueous extract of WASPI. The intensity of glycosides and
saponins were reduced on extraction and isolation of protein from WASF. Steroids
and sterols were not identified in aqueous and ethanol extract of WASF, WASPC and
WASPI though it was observed in very low intensity in methanol extract of WASF and
WASPC. The changes in intensity of saponin and tannin reflecting the content in
WASF, WASPC and WASPI was highly correlated with quantitative profile of these
compounds. WASPI had significantly (p<0.01) high polyphenol than WASPC and
WASF.
170
The radical scavenging activity of WASF, WASPC and WASPI was considered
to be dose dependent irrespective of different extracts. Among the extracts methanol
revealed significantly (p<0.05) a high DPPH scavenging activity followed by aqueous
extract and then by ethanol extract. The DPPH scavenging activity was significantly
(p<0.05) low in ethanol extracts. WASPI scavenged 50% DPPH radical at the lowest
sample concentration followed by WASPC and WASF irrespective of extract. The
methanol extract revealed significantly (p<0.05) a high ABTS scavenging activity
followed by aqueous extract and then by ethanol extract. According to IC50 value,
WASPI showed the scavenging activity at the lowest concentration followed by
WASPC and WASF irrespective of extract. WASPI had evidenced the greater ABTS
activity than in WASPC and WASF. WASPI was more effective in reducing ferric ion to
ferrous ion at lowest concentration than WASPC and WASF. A high degree significant
(p<0.01) negative correlation was noted between TPC (as GAE) and DPPH; TPC (as
GAE) and ABTS activity. A low degree significant (p<0.05) negative correlation was
noted between TPC (as GAE) and FRAP assay.
The highest (significant at p<0.01) in vitro protein digestibility value was
obtained for WASPI than WASPC and WASF.
In thermal property the first peak between 55C and 75C in WASF (Td73.6C), WASPC (Td-59.1C) and WASPI (Td-56.9C) reflected the presence of albumin
and globulin I (7S globulin) which were of less thermostable; the denaturation
between 125C and 130C in WASF, WASPC and WASPI suggested the presence of
glutelin, globulin II or 11S globulin in WAS protein, which were more thermostable
than albumin and globulin I (7S globulin). The presence of albumin, globulin I (7S
globulin) and globulin II (11S globulin) was consistent with the SDS-PAGE result. The
amount of protein denatured in WASF below 80C was less than WASPC which was
followed by WASPI.
The pasting property of WASF, WASPC and WASPI was very low. Peak time is
a measure of the cooking time and it ranged between 2.07-4.47 mins for WAS. The
peak viscosity was significantly (p<0.01) high in WASPC than WASF and WASPI. The
trough viscosity, final viscosity and setback viscosity of WASPC was significantly
(p<0.01) greater than WASPI and WASF. WASPI had exhibited significantly (p<0.01)
greater breakdown viscosity than WASPC and WASF.
Since there was a significant difference between the characteristics of
WASF, WASPC and WASPI, the framed null hypothesis (There was no significant
171
difference between the characteristics of WASF, WASPC and WASPI) was

rejected.
In terms of in vivo quality protein parameters (weight gain, protein intake,
PER, C-PER, NPR and NPU, TD, AD and BV), biochemical paramaters and
histopathalogical examination of liver and kidney, WASPI was significantly (p<0.01)
comparable with casein protein.
Hence the framed null hypothesis (the protein quality of wood apple
seed was not significantly comparable with casein protein) was rejected.
5.3 CONCLUSION
Wood apple seeds can be used in balanced diets as functional foods, consumed
safely without any concern of health risk. In countries with high population where the
food requirements are not being fulfilled by seasonal fruits, wood apple seeds can be
used as a good substitute to overcome protein calorie malnutrition. The seed flour can
be an alternative protein rich product, stored and utilized for value addition. The
results of the study promotes increased consumption of wood apple seeds by general
public and offers opportunity to develop value added products and thereby determine
the feasibility of commercialising them. WASF, WASPC and WASPI are good source of
protein, minerals and phytochemicals especially flavonoids and antioxidants which
made appropriate to be used as a matrix or included in different preparations as food
fortificant. The functional characteristics of the WASPC and WASPI are highly
comparable to those of casein, legume seed meals and isolates, the WASPC and WASPI
may be explored industrially as protein supplement.
5.4 RECOMMENDATIONS
While highlighting the excellent and comparable quality characteristics of

WASPI with casein protein, the following recommendations could be considered for
further research.
1. Research efforts should be broadened to take into account the constraining
factors faced by the wide spectrum of wood apple growers to complement
the increase productivity.
2. Research on policy formulation should be initiated to form the stable, well
vertically integrated supply chain linking agro-processors, exporters, whole
sellers and retailers with growers.
3. Research identifying supplements incorporated with WASPI could be
encouraged through small and medium enterprises.
172
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Introduction

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

functional properties that influence the consumer acceptance of food products

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

conventional extraction methods. Hence there is a need to identify the best

To determine the physical and morphological characteristics of wood apple

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Picture 2.1: Bael (Aegle marmelos); Wood apple (Limonia acidissima)

Limonia acidissima (L.) of family Rutaceae (Citrus family) belongs to the

Limonia acidissima is a moderate sized deciduous tree grown throughout

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Picture 2.4 C: Wood apple available at local market of Salem

Picture 2.4 D: Evidence of Poor Mans Fruit and cheap availability

The number of traditional remedies attributed to wood apple is

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

carminative and digestive stimulant. The leaves possess a number of health

Pulp (ripe) (%)

Source: Singh et al (2009)

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

transaminase (SGPT); alkaline phosphatase (ALP) and serum bilirubin by which

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

brought about homeostasis in the biochemical parameters such as cholesterol, urea,

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

anti-breast cancer activity of methanolic extract of Limonia acidissima (MELA) on

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

antioxidant and in the development of functional food and raw materials of

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

dysentriae, V. cholerae and K.pneumoniae) at varying concentration (100 to 500

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

2.5.2.10 Antimicrobial Activity

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

The relative survival percentage after Aeromonas hydrophila challenge

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

In Indonesia, wood apple is mixed with honey and eaten in breakfast. In

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

treatments viz. T1 (sodium benzoate), T2 (Potassium metabisulphite) and T3

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

World demand for proteins is increasing and so more food protein is

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

There is a growing interest in the utilization of plant proteins for the

Other plant proteins used commercially are peanuts,

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Extractability and Characteristics of Wood Apple (Limonia Acidissima) Seed Protein

Saravanan & Rose

Zhen & Shi (2011)

Zhen & Shi (2011)

Wu & Wang (1984)

Zhen & Shi (2011)