Trends in Food Science & Technology 62 (2017) 13e22

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Trends in Food Science & Technology

journal homepage: http://www.journals.elsevier.com/trends-in-food-science-
and-technology

Review

An integrated strategy between food chemistry, biology, nutrition,

pharmacology, and statistics in the development of functional foods: A
proposal
Daniel Granato a, *, Domingos Sa

vio Nunes b, Francisco J. Barba c
a
Department of Food Engineering, State University of Ponta Grossa, Ponta Grossa, Brazil
b
Department of Chemistry, State University of Ponta Grossa, Ponta Grossa, Brazil
c
Nutrition and Food Science Area, Faculty of Pharmacy, Universitat de Val
encia, Avda. Vicent Andr

es Estell

es, s/n., 46100 Burjassot, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Background: Much research has been conducted to attest that different food and herbal extracts display
Received 1 August 2016 functional properties in humans when consumed regularly as part of a balanced diet. However, most
Received in revised form studies only relate one or some in vitro and/or in vivo functionalities of these extracts, without per-
18 December 2016
forming clinical trials to attest the alleged functionality. For instance, while some studies focus on the
Accepted 20 December 2016
Available online 1 February 2017
existence of statistical correlation between antioxidant activity and antidiabetic properties and the
phenolic composition of a certain herb, others aim to assess the effects of different extraction methods
(i.e., pulsed electric fields, ultrasound, and supercritical fluid extraction) on some selected in vitro
Keywords:
Experimental nutrition
functional properties.
Chemical compounds Scope and approach: Although these studies are essential for a better understanding of the extracts and
Phenolic compounds foods consumed worldwide, they lack depth and, principally, practical application on consumer's health
Antioxidant activity and well-being. In this scenario, in this article we propose an integration of multiple interlinked disci-
plines and between academia and food companies to elucidate the health-promoting properties of foods
and extracts. In addition, we discuss and propose a multidimensional team to develop new functional
foods.
Key findings and conclusions: Researchers should perform a wide variety of in vitro and in vivo tests to
determine the toxicological effects, therapeutic dosage and to assess the physicochemical, chemical, and
sensory properties of a developed food before stating its functionality. Complementarily, food scientists
should never develop and attest in vivo functionality alone; rather, a highly active interconnection with
related fields is required.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
As a basic definition, functional foods are foods that have a
potentially positive effect on health beyond basic nutrition, helping
the promotion of optimal health conditions and reducing the risk of
non-communicable diseases. It is widely known that a huge
number and varieties of plants, including herbs, fruits, leafs, and
barks, and their homemade and industrial preparations, have been
used worldwide to decrease the risk (prevention) and to treat
diseases and/or health complications (Guarrera & Savo, 2013). Such
applications may come from tradition use (Junior & Morand, 2016;
* Corresponding author.
E-mail address: granatod@gmail.com (D. Granato).
Rastogi, Pandey, & Rawat, 2016; Silva, Braga, Lemos, & Cortes, 2016;
Xing et al., 2015) or by scientific-based evidences derived from
experimental protocols (Bao, Hu, Zhang, & Wang, 2016; Chen et al.,
2016a; Dludla et al., 2014; Margin a et al., 2015). In a quick search
conducted on 10 November 2016 at Wiley and Sciencedirect data-
bases (1998e2017) using the index term ‘functional foods’, a total of
210,226 and 382,852 articles, chapters and books were found,
respectively. If we consider the Sciencedirect database, from 1998
until December 2016, an ascendant number of published articles/
chapters is observed (Fig. 1), showing that the scientific interest for
functional foods is increasing consistently.
Although the concept of functional foods is not new (it started in
the 80s in Japan), food companies and research institutes still
develop foods with potential functional properties without

http://dx.doi.org/10.1016/j.tifs.2016.12.010
0924-2244/© 2017 Elsevier Ltd. All rights reserved.
14 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22
Fig. 1. Total number of articles published from 1997 to 2016 (December 18) at
sciencedirect.com using the index term ‘functional foods’.
assessing their chronic and acute effects in humans to attest the
alleged potentiality. In a related field, namely ethnopharmacology,
the development of new products with pharmacological activity in
certain organs/glands and/or biomarker in humans, the study of
traditional herbs and foods, which are known to be safe from the
toxicological standpoint, is generally the first step to be taken after
some in vitro measurements (Abbasi et al., 2015; Satoh, Igarashi,
Yamada, Takahashi, & Watanabe, 2015). However, precaution
should always be taken when evaluating natural products: for
example, Abd-ElSalam et al. (2016) evaluated the antioxidant ac-
tivity in vitro (test tubes) and in vivo (rats previously subjected to
oxidative stress) of green tea-derived catechins, including epi-
gallocatechin gallate (EGCG). Authors verified no correlation be-
tween free radical scavenging activity and the content of EGCG
in vitro and the in vivo data showed not only loss of antioxidant
activity but also the formation of harmful pro-oxidants in hepato-
cytes, corroborating the fact that a careful design of quality control
protocols requires correlation between chemical assays and bio-
assays to verify efficacy, stability, and most importantly safety of
nutraceuticals.
According to the World Health Organization (WHO, 2000),
traditional medicine is the sum total of the knowledge, skills, and
practices based on the theories, beliefs, and experiences indigenous
to different cultures, whether explicable or not, used in the main-
tenance of health as well as in the prevention, diagnosis,
improvement or treatment of physical and mental illness. In addi-
tion, traditional use of herbal medicines refers to the long historical
use of these medicines. Their use is well established and widely
acknowledged to be safe and effective (from the toxicological
standpoint), and are usually accepted by health authorities
(Elisabetsky, 1986; Elisabetsky & Wannmacher, 1993). The basic
team used in ethnopharmacology studies involves a chemist, a
biologist (botanist), and a pharmacist (pharmacologist) (Souza
Brito, 1996), who work together to identify the plants, to extract
the bioactive compounds using similar (preferably identical) con-
ditions that of the indigenous populations, to test and validate
biomarkers related to the disease, to assess the extract's toxico-
logical effects in vivo (animals and humans), to identify dose-
response effects, and to propose a therapeutic dose for the
extract (Ma, Wang, & Gao, 2017; Maimoona, Naeem, Saddiqe, &
Jameel, 2011; Wang, Qiu, Wu, Wei, & Xu, 2015). Although this
task seems to be easy and straightforward, much effort from all
professionals is required and companies still strive to look for
‘traditional medicine’ worldwide.
This holistic and multidisciplinary concept cannot be literally
translated into the food science field, as there is no subdivision of
professionals dedicated to develop new functional foods. What we
constantly observe, in practice, is food and chemical engineers,
dietitians, and technologists involved in the process of developing
food products, including beverages, and making health claims
based on the content of bioactive compounds (carotenoids, phe-
nolics, terpenoids, dietary fibers, high-protein foods and beverages,
among others) or on the level of probiotic bacteria that the final
product contains. This practice per se is not well accepted as it does
not include the bio-efficacy in vivo and usually studies do not
contain any information of the dose-dependency, toxicological and
biochemical effects of such components. In this view, Heywood
(2011) emphasized that ethnopharmacology is a science that
cannot be dissociated from human nutrition, food production, and
manufacture of functional foods.
Although in the last 20 years consumers have sought for func-
tional foods that help in the maintenance of the good health con-
ditions and also to decrease the risk of non-communicable diseases,
especially type II diabetes, cardiovascular diseases, and hyperlip-
idemia, there is no standardization on the steps and the qualifica-
tion of professionals involved in the functional food development.
In this scenario, in this article we summarize the current research
on the integration of multiple interlinked disciplines to elucidate
the health-promoting properties of food and herbal extracts aiming
to broaden the application of such studies on the assessment of
functional properties of food products. In addition, we discuss and
propose a multidimensional team to develop new functional foods.
2. Functional properties of herbal/food extracts
Nutritional labeling regulations and definitions of functional
foods vary considerably from country to country and is not the
main focus of the current research. The regulations and policies
regarding functional foods and natural health products, including
nutrient labelling regulations and novel food registration in Canada
and around the world were comprehensively reviewed by Malla,
Hobbs, and Sogah (2013) and by Verhagen and van Loveren
(2016). Below, we will focus on some insights and steps to take
into consideration to develop new potentially functional foods.
2.1. Use of plants with traditional use
Plants with traditional use are those that have medicinal prop-
erties and are used by a certain group of people, from one or more
locations, that are thought to be safe for humans (Junsi &
Siripongvutikorn, 2016). According to Uprety, Asselin, Dhakal, and
Julien (2012), annual sales of herbal-based herbs ranged between
US$ 7.5 (herbs) and US$ 108 billion (herbal preparations/processed
products) worldwide, proving that millions of people still look for
traditional plants to prevent, ameliorate, and treat diseases.
In 2004, the Food and Agriculture Organization (FAO, 2004)
discussed the various themes involving the development and
analysis of functional foods, and focused on the application of a
history of safe use of food ingredients. Distinctions should be made
between the country of origin and the country of use, historical and
modern uses of products, and clinical compared to cultural use of
products. Two good examples of this factor are green and red
rooibos teas (Aspalathus linearis from South Africa) and mate teas
(Ilex paraguariensis from Brazil, Argentine, and Uruguay) that have
being widely consumed in Europe and in North America.
In fact, the use of traditional foods and herbs in the development
of new potentially functional foods is highly encouraged (Caleja
et al., 2015; Hacke et al., 2016; Park, Choi, Park, Oh, & Ha, 2016;
Soltani et al., 2017; Ramos et al., 2017). In food science and tech-
nology, especially in those studies that relate the chemical
composition and functional properties (i.e., inhibition of digestive
enzymes in vitro, antioxidant activity, and antibacterial effects), the
 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22
identity of plant extracts is not always given in details (correct
identification of the species, deposit of a sample in a herbarium,
etc). It implies that the correct identification is not always per-
formed by a botanist, which hinders the identification of correla-
tion between species and biological activity. In real life practice, it is
very common to read reports in which the identification of the
plant is not made by a biologist and it is very erroneous not to
perform this basic step in the study of the plant as the link between
chemical composition, biological activity is totally dependent on
the species (Nunes, 1996). In ethnopharmacology and in all other
studies conducted with plants, their botanical identification is a
mandatory step in the study, that is, without this premise no
serious study can be properly performed, interpreted and
comprehensively understood (Giovannini, Howes, & Edwards,
2016).
For example, Guarrera and Savo (2013) reviewed the most
common wild and cultivated foods perceived as healthy in Italy and
their beneficial effects on human health. Most of these plants are
conventional foods used by a large group of people, such as Allium
species, Aspargus sp., Beta vulgaris, Brassica sp., Echium italicum,
Malva sp., Lens culinaris, Rumex sp., Vicia faba, among many others.
If a food company aims to develop a new food product with func-
tional actions using these plants, consumers will recognize more
easily the food as a ‘good product’ compared to a food made with an
unknown species. A typical example of this approach related to
development of dairy foods (i.e., yogurts and fermented milks)
added with herbal extracts. Muniandy, Shori, and Baba (2016)
analyzed the effects of green, white, and black teas (Camelia
sinensis) on the antioxidant activity of yogurts and verified that tea
yogurts presented higher antioxidant activity (i.e., FRAP and DPPH)
and total phenolic content compared to the natural yogurt
(control).
2.2. Integration of multiple disciplines in assessing the functional
properties of extracts: where we are and where to focus
It is too speculative to state that a food added with a bioactive
compound or a herbal extract, at a certain concentration, is a
functional food per se. As mentioned by Hu, Zou, He, Chen, and
Deng (2016), polyphenols usually show interesting and ‘high’
(bio)chemical characteristics and functional properties using
in vitro assays (Siucin
ska, Mieszczakowska-Fra˛c, Połubok, &
Konopacka, 2016); however, the assessments of their clinical ap-
plications in vivo are restricted by their limited bioavailability and
strong resistance to 1st-pass effects during absorption. Without
performing in vitro tests, checking the interaction of the bioactive
compounds with the intrinsic chemical composition of the food
matrix, their bioavailability and the in vivo functional properties, it
is erroneous to state any beneficial effect of the food (Guama
n-
Balca
zar, Setyaningsih, Palma, & Barroso, 2016; Paz et al., 2015).
Nonetheless, it is still common to see in researchers incorporating
phytosterols, vitamins, extracts rich in polyphenols and carotenoids
and stating the healthiness/functionality of the developed product
without performing human studies (Caleja et al., 2015; Herna
ndez-
Carrio
n, Varela, Hernando, Fiszman, & Quiles, 2015). This type of
practice should be avoided as recommended by Silva, Barreira, and
Oliveira (2016), Granato, Carrapeiro, Fogliano, and van Ruth (2016),
and Martins, Barros, and Ferreira (2016). In this aspect, in a recent
essay, Weaver (2016) stated that interdisciplinary interfaces are the
pathways to the improvement and interconnection between food
science and nutrition because the problems that need to be
addressed are complex and require multiple professionals, from
many basic and applied fields.
It is common practice to perform a scientific study aimed at
assessing some functional properties and even the toxicity level of
15
unknown plant/food extracts using chemical and enzymatic re-
actions that occur in glass tubes, cuvettes and microplates (in vitro
and in silico assays) (Sui, Zhang, & Zhou, 2016). The most common
assays include the antioxidant activity using different experimental
protocols and chemical reagents (Margraf, Santos, de Andrade, van
Ruth, & Granato, 2016; Zielinski et al., 2014), inhibition of a-
amylase and a-glucosidase enzymes (Mojica, Meyer, Berhow, & de
Mejía, 2015; Jin, Yang, Ma, Cai, & Li, 2015; Tan, Chang, & Zhang,
2017), inhibition of the anticholinesterase enzyme (Girone
s-
Vilaplana et al., 2015), antimicrobial properties (Makwana,
Choudhary, Haddock, & Kohli, 2015), antihemolytic (Phan-Thi,
Durand, Prost, Prost, & Wache
, 2016), antiproliferative effects on
different cancer/tumor cells (Danciu et al., 2015; de Lima, Azevedo,
de Souza, Nunes, & de Barros Vilas Boas, 2015; de Lima, Kanunfre,
de Andrade, Granato, & Rosso, 2015; Zhan, Pan, Tu, & Jiao, 2016),
nitrite scavenging effects in vitro (Zhan et al., 2016), and even
cellular rejuvenation (Fujita et al., 2015), neuroprotective (Bhullar &
Rupasinghe, 2015) and anti-inflammatory effects (Ku, Kwak, Kim, &
Bae, 2015), among others.
Although these studies are essential for a better understanding
of the potential functional properties of plant/food extracts
consumed worldwide, some lack depth and, principally, practical
application on consumer's health and well-being. One fact that may
explain this complex reason: as reviewed by Martins et al. (2016),
the in vitro observations are not always translated into in vivo
bioavailability and effectiveness (bio-efficacy). This means that
factors with influence on important clinical outcomes usually
cannot be predicted by the results of in vitro tests, showing that a
solid understanding of the mechanisms of action as well as the
pharmacodynamic and pharmacokinetic parameters in vivo is
highly demanded and necessary to assure bioefficacy (Huang et al.,
2016; Shao et al., 2016; Sun et al., 2016a).
Part of discrepancies between in vivo and in vitro data comes
from not only the effects of hormones, enzymes, temperature,
buffering system, pH and overall intrinsic physiological conditions
of the living organism being tested but also to the effects of the diet
(Tan & O'Toole, 2015), microbial metabolization of bioactive com-
pounds in the digestive system, and gastrointestinal digestion of
the matrix containing the bioactive compounds (Rashidinejad,
Birch, Sun-Waterhouse, & Everett, 2015). For instance, recently
Lin, Wang, Yang, Wang, and Ling (2016) used an in vitro fermen-
tation model using mouse gut microflora to assess the metabo-
lization of pure flavonoids (i.e., flavonols, flavones, and flavanones)
and mice were used to understand how the flavonoids were
metabolized in vivo. Authors concluded that the intestinal micro-
biota are not necessary for the absorption of flavonoids, but are
required for their transformation into phenolic acids, such as pro-
tocatechuic acid, that can be used as a marker of flavonoid
consumption.
In the field of absorption and metabolism of bioactive sub-
stances, some authors have stated that most food-derived bioactive
compounds do not sufficiently solubilize and permeate the small
intestine to yield efficacy (Wang et al., 2016). Therefore, delivery
systems based on esterification/lipophilisation and encapsulation
of phenolic compounds (Hu et al., 2016; Pagnussatt et al., 2016;
Yang, Guo, & Xu, 2012), heterogeneous catalytic E/Z isomerization
of carotenoids (Sun, Yang, Li, Raza, & Zhang, 2016b), design of
stimuli-sensitive nano-sized delivery systems of omega-3 and
omega-6 polyunsaturated fatty acids (Semenova et al., 2016),
development of chitosan-coated liposomes for delivery of grape
seed polyphenols (Gibis, Ruedt, & Weiss, 2016), and the develop-
ment of nanoparticles and intestinal permeation enhancers have
been created to increase the chemical stability, bioaccessibility, and
absorption of bioactive compounds (Gleeson, Ryan, & Brayden,
2016). In addition, micro and nano-encapsulation of plant
16 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22
extracts, bioactive compounds and vitamins have also been tested
(Katouzian & Jafari, 2016; Faridi Esfanjani and Jafari, 2016) and
implemented in food products that are widely consumed world-
wide (Ghorbanzade, Jafari, Akhavan, & Hadavi, 2017; Martins et al.,
2014).
Allying food technology and nutrition, Fu et al. (2016) added
curcuminoids (300 mg/100 g) in buttermilk for the manufacture of
a fortified yogurt. Curcuminoids were quantified after the techno-
logical process and after in vitro simulated gastrointestinal condi-
tions and verified that about 89% of the total curcuminoid content
remained stable in yogurt and their bioaccessibility was 15-fold
more than that of curcuminoids in aqueous dispersion,
concluding that yogurts may be suitable vehicles of these func-
tional substances. Similarly, Verrijssen et al. (2016) studied the ef-
fect of food ingredients (citrus pectin and sugar beet pectin with
different degree of methyl-esterification) on the in vitro bio-
accessibility and lipid digestion of b-carotene, a carotenoid with
recognized antioxidant efficacy in vivo. Authors concluded that
pectin with different degrees of methyl-esterification cab ne engi-
neered aiming carotenoid bioaccessibility in food products. As a
final remark on this topic, the food scientist probably will not work
alone in this field, therefore the alliance with a pharmacist/
biochemist is necessary.
In food sciences, research is focused on to study the comparison
and effect of different processing methods on the chemical
composition and some limited in vitro functional properties of a
wide variety of foods and beverages (Chen et al., 2015; Markowski,
Baron, Le Que
re

, & Płocharski, 2015; Okumura, Hosoya, Kawarazaki,
Izawa, & Kumazawa, 2016; Sogi, Siddiq, & Dolan, 2015; Szwajgier &
Gustaw, 2015; Tan, Chang, & Zhang, 2016; Yang et al., 2015),
extraction optimization of bioactive compounds (Belwal, Dhyani,
Bhatt, Rawal, & Pande, 2016; Pedro, Granato, & Rosso, 2016;
Santos et al., 2016), and assessment of adding different in-
gredients rich in some bioactive component in traditional foods
(Bertolino et al., 2015; Jung et al., 2016a; Martins et al., 2014;
Szwajgier & Gustaw, 2015). In vivo studies related to the toxicology,
bioavailability and bioactivity of food and herbal extracts are usu-
ally performed by professionals located in research laboratories
with a background in biology, pharmacology, medicine, and
nutrition, in which the main aim is to find out the mechanisms of
action in a target organ and to establish therapeutic and prophy-
lactic doses to obtain the bio-efficacy in humans (Jung, Ok, Park,
Kim, & Kwon, 2016b; Sui et al., 2016; Zhang & Tsao, 2016; Zou
et al., 2016). Additionally, in experimental protocols related to the
nutrition, the impact of chemical compounds on some selected
markers in specific cells/organs (animals) or biological fluids
(humans) and the dose-dependency on the biomarker(s) related to
the functionality are the focus of research (Weber, Nguyen, &
Hanrahan, 2014; Yao et al., 2016; Li et al., 2016). In addition, the
use of biomarkers for deficient and excess exposures should also be
assessed and determined with precision.
Regarding the protocols needed to attest functionality of foods
and herbal extracts in humans, some guidelines are available both
to support nutrition science and to facilitate the substantiation of
health claims (Welch et al., 2011). This publication contains the pros
and cons of current methodologies (i.e., design of the study, selec-
tion of participants and conduction of the experiment, data anal-
ysis, and interpretation/report of results) used to assess the
functional properties of foods/herbal extracts in humans.
In all scientific areas, the use of multiple statistical techniques is
required not only to help in the study design but also to collect,
organize and analyze the results is essential (Nunes, Alvarenga,
Sant'Ana, Santos, & Granato, 2015). In food chemistry, the most
common uses of applied statistics are related to comparison of
methods and samples (Fidelis et al., 2017; Santos, Brizola, &
Granato, 2017), while in biology, it is common practice to assess
the effects of certain compounds on the growth of a microorganism
on the medium or to compare groups of animals. Similarly, in
nutrition, applied statistics is vastly used and required to compare
the control group with the test group(s), enabling one to state
whether the treatment provoked any changes in the individuals
(Bao et al., 2016; Chen et al., 2016a, Chen et al., 2016b; Margina 
et al., 2015). In this view, Fig. 2 represents the major aims of each
area related to the assessment of functional properties of herbal
and food extracts and, more importantly, the interconnection of
these areas, in the study of functional foods.
The ideal situation would be to unite the expertise of each area
and perform one complete and detailed study on the functional
properties of herbal extracts or even new potentially functional
foods. However, this situation would demand much effort, part-
nerships between institutes, and principally, financial support to
conduct in vitro initial assessments, experimental studies in ani-
mals, preclinical studies and then a complete randomized placebo-
controlled (double-blind) study. In summary, ethnopharmacology,
food science, nutrition, biodiversity, are all inextricably inter-
connected in a way that scientists from these areas should establish
linkages to perform an organized and effective work (Heywood,
2011). Fig. 3 contains a summary on how the integration between
science fields should be interlinked aiming to assess the functional
properties of plant-based and herbal extracts and preparations.
2.3. Developing a functional food: a theoretical and practical
approach
2.3.1. General scenario
According to the guidelines provided by FAO (2007), building a
strong and acceptable scientific basis for functional food claims
relies on the ability to demonstrate the bio-efficacy of functional
food components in vivo and not only by performing in vitro tests. It
is complex and costly task, but it is essential to acceptance of
functional food. Because of the number of bioactive compounds
and the diversity of likely biological effects, numerous and diverse
experimental approaches must be taken to increase the under-
standing of the biology of bioactive compounds. At the end, sub-
stantiation of a claim should be based on human data, especially
those come from clinical and epidemiological studies with a large
number of participants.
It is not easy and straightforward to develop and attest benefi-
cial (or not) health effects of a potentially functional food product.
In this aspect, the safety of intake must be considered and evalu-
ated when functional foods are developed (FAO, 2007; Lupton,
2009). For the majority of research studies, the optimal levels of
nutrients and other physiologically active components in functional
foods have yet to be determined in humans (Aggett et al., 2005).
The safety evaluation of dietary functional components must take
into account potential adverse effects of low intake (clinical defi-
ciency) as well as effects from intakes that are too high (clinical
toxicity) (FAO, 2007; Akindele, Unachukwu, & Osiagwu, 2015). The
dose-effect relationship might lead to considerations of physio-
logical/nutritional disturbances that are irrelevant to standard
safety assessment (Martirosyan & Singh, 2015; Nghonjuyi et al.,
2016).
After checking the functionalities of a food matrix, it is of
paramount importance to check for its acceptance regarding its
sensory properties, such as, acidity, creaminess (i.e., yogurts and
processed cheeses), overall appearance, bitterness (some juices and
teas), and odor (Bertolino et al., 2015). For that purpose, usually
either a large number of consumers (usually n > 75) or a trained
panel (n ¼ 8e15) is used to perform the sensory analysis. Both
descriptive and hedonic sensory methods can be used in the
 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22 17

Fig. 2. Representation of the major aims of each area related to the assessment of functional properties of herbal and food extracts.

Fig. 3. Summary on how the integration between science fields should be interlinked aiming to assess the functional properties of plant-based and herbal extracts and preparations.

analysis. When the product receive a satisfactory rating for the
attributes evaluated (scores 6 when a 9-poin hedonic scale is
used), then the product has a potential to be successful from the
sensory standpoint. If the score is not satisfactory, then reformu-
lation and optimization of the product should be considered and
performed (Tuorila, 2015).
In addition to the sensory aspects of the potential functional
food or beverage, the chemical stability of the bioactive compounds
present in the food matrix should be evaluated aiming to maintain
the functionality for longer periods. As most bioactive compounds
can be detected and quantified analytically (UV/VIS
spectrophotometry, selective electrodes, HPLC, LC-ESI-MS/MS, GC-
FID, GC-MS, among others), a kinetic study should be carried out
using the conditions (packaging, air moisture, temperature) the
product will be subjected during storage. Having the data, the shelf
life of the final product should be calculated using proper statistical
methods and fully stated and described (Nunes et al., 2016).
Being part of any serious governmental organization, the regu-
lation of a potential functional food requires the conduction of a
proper assessment to attest their functionality not only in vitro but
also using cell lines, animals, and humans (preclinical and clinical
trials). The alleged functional property can only be present on the
18 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22
product's label if there is vast scientific evidence on the matter. In
Brazil, for instance, functional foods is a food category that should
have a scientific consensus from a wide range of published data
about the bioactive component responsible for the alleged func-
tional property. In the case of new products, clinical trials (Brasil,
1999). On the other hand, in the USA (FDA, 2016), depending on
the type of health claim that is projected on the product's label,
functional foods can be regulated in the categories: conventional
foods, dietary supplements, drugs, medical foods or food for special
dietary use. It is also advisable to note that the regulatory frame-
works for products containing health claims should be propor-
tionate and not overburdening and misleading. In this aspect, food
companies should bear in mind that clear information and
communication to consumers is highly required.
Fig. 4 contains a practical and systematic approach that can be
used to develop a functional food product according to various
factors (sensory, technological and regulation aspects, and data
coming from biological, nutritional, and chemical tests) and Fig. 5
contains details on the proposed workflow that should be con-
ducted in studies regarding the functionality of foods, beverages
and extracts. All this information should be taken into consider-
ation by both food companies and research institutes in designing
studies focused on the development and assessment of in vitro and
in vivo functionalities of food products.
After all these details, it should be highlighted the partnership
between food companies and academic bodies in universities and
research centers is essential. It is important to mention that what
we see in practice is that researchers in university and/or research
centers are funded by governmental agencies and rarely include
human trials (i.e., multicenter, randomized, double-blind, and
crossover studies). They are responsible for discovering functional
food materials, ingredients, and specific chemical compounds via
in vitro and in vivo studies (usually sing small animals or preclinical
trials in some cases). In the same sense, food and pharmaceutical
companies usually develop and patent the related products after
performing clinical trials, studies regarding the safety and stability,
regulation, and administrative approvals. It seems that few com-
panies want to work together with researcher based in universities
to develop new technological strategies and products. This
Fig. 4. Practical and systematic approach that can be used to develop a functional food product according to various factors.
behavior should be avoided and, in a general optimistic view, both
academia and food companies should focus on a strong and effec-
tive alliance to accomplish the goal of developing new functional
foods with proved functional effects in vitro and in vivo. Allying the
expertise of academia and companies would allow a broader and
more comprehensive strategy to develop and to offer new func-
tional foods for consumers worldwide.
2.3.2. Application of integrated disciplines in developing potentially
functional foods: improving bioavailability and bioaccessibility
through food preservation, and extraction innovative processing
Due to the large scientific impact of functional food/extracts and
the wide acceptance of these products by consumers, over the last
years several efforts have been made by both the scientific com-
munity and the food industry to develop such products. For
instance, the application of new processing techniques for food
preservation can be a useful tool to develop new functional foods
with improved healthy properties. In this line, the potential of non-
thermal processing such as high pressure processing (HPP), pulsed
electric fields (PEF) to improve the bioavailability and bio-
accessibility of beneficial compounds from food products has been
investigated obtaining some promising results. Some of the most
relevant finding were recently reviewed (Barba, Terefe, Buckow,
Knorr, & Orlien, 2015; Barba et al., 2015). Additionally, other
innovative processing technologies such as high pressure homog-
enization (HPH) and microwaves (MW) have been also shown as
useful technologies to improve bioavailability and bioaccessibility
of bioactive compounds (Benlloch-Tinoco et al., 2015; Van
Buggenhout et al., 2010).
On the other hand, as it was previously stated, the most
commonly used strategy to obtain “functional” extracts and/or food
rich in bioactive compounds with well-known beneficial properties
consists on applying conventional (grinding, maceration, solvent)
and/or innovative (PEF, ultrasounds (US), MW, sub- and supercrit-
ical extraction (SC-CO2), high pressure, etc.) extraction technolo-
gies. Once the extracts are obtained, they can be used per se or to be
included in the food product (Da Silva, Barreira, & Oliveira, 2016).
However, one of the main drawbacks in the experimental design of
these studies is the lack of information about the in vivo
 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22
Fig. 5. Practical workflow to develop and test new potentially functional foods by food companies.
bioavailability, bioaccessibility, toxicological implications, etc. after
food preservation and/or extraction. The multidisciplinary team
should work together to address this issue prior to attest ‘func-
tional’ properties of those foods.
Some of the beneficial properties of the functional extracts have
been attributed to their high antioxidant properties (Carbonell-
Capella, Buniowska, Barba, Esteve, & Frígola, 2014). For instance,
in the last three decades, several studies have appeared evaluating
the in vitro antioxidant properties of the extracts. However, over the
last years, there is a growing tendency to reject the articles evalu-
ating only in vitro antioxidant tests (i.e., 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-
picrylhydrazyl (DPPH), oxygen radical absorbance capacity
(ORAC), ferric reducing ability of plasma (FRAP), etc.) mainly due to
the contradictory results, which showed that some antioxidants
exerting their antioxidant properties in vitro afterwards did not
present any antioxidant capacity in vivo and vice versa (Alam, Bristi,
& Rafiquzzaman, 2013; Mohamed & Al-Okbi, 2004; Tan & Lim,
2015). For example, in a study conducted by Mohamed and Al-Okbi
(2004), authors recommended the use of methanolic extract of
edible portion from date fruits as functional food ingredient after
evaluating the antioxidant activity in vivo. However, they did not
observe any antioxidant capacity in vitro. In addition, it is known
that MeOH is toxic to humans and any extract obtained using this
solvent should not be consumed.
Several authors have studied the potential of other innovative
extraction technologies (PEF, MW, US, SC-CO2, high voltage elec-
trical discharges (HVED), high pressure, etc.) to obtain functional
juices and/or extracts. However, to the best of our knowledge, there
are no articles evaluating in vivo antioxidant and cytotoxic potential
of extracts/oils/juices obtained from any matter using these tech-
nologies. In addition, the literature evaluating the in vitro
19
bioprotective effects, bioavailability and bioaccessibility is also
scarce. Below we summarized some of the most relevant findings in
the available literature.
Alam et al. (2013) evaluated the bioaccessibility of polyphenols
obtained after US or conventional assisted extraction from dried
olive leaves using an in vitro simulated model. They did not find any
significant effect of dehydration treatment on polyphenol's bio-
accessibility. However, they found different behavior during in
digestion process according to the polyphenol's studied. Oleur-
opein and verbascoside were not significantly degraded during
gastric digestion but the concentration was significantly decreased
during intestinal phase. They also found that luteolin-7-O-gluco-
side presented the highest bioaccessibility (43%) during the study.
Leong, Burritt, and Oey (2016) used human intestinal Caco-
2 cells to evaluate the bioprotective effect of PEF-extracted Pinot
Noir juices (Vitis vinifera L.) against H2O2-induced oxidative stress.
For this purpose, they used some biomarkers (cell viability and
lactate dehydrogenase (LDH) leakage). They used these cells as it is
well known the correlation between the in vitro apparent perme-
ability across Caco-2 monolayers and the in vivo fraction absorbed.
The authors obtained that an increase in bioprotective capacity
(þ25% for cell viability and þ30% for LDH leakage), which was
mainly attributed to an increased release of malvidin-3-O-gluco-
side content when PEF pretreatment was used to extract the juice.
On the other hand, in a study conducted by Ben Rahal, Barba,
Barth, and Chevalot (2015), the authors first optimized supercriti-
cal carbon dioxide (SC-CO2) processing conditions to recover oil,
flavonolignans (silychristin, silydianin and silybinin) and fatty acids
from milk thistle seeds and subsequently the antioxidant and
cytotoxic properties of the selected oils were evaluated in Caco-2
carcinoma cells. The authors observed a high potential of SC-CO2
extract to inhibit the proliferation of Caco-2 cells and high
20 D. Granato et al. / Trends in Food Science & Technology 62 (2017) 13e22
antioxidant capacity.
Therefore, in order to consider innovative processing techniques
the following steps are necessary: First, good experimental designs
are necessary (i.e., using factorial designs, such as response surface
methodology, artificial neural networks, etc.) in order to evaluate
the different compounds present in the extracts and to optimize the
extraction processing conditions. Moreover, it is necessary to
identify which is the ideal technology thus reducing the cost of the
process, extraction time, the use of solvent and of course elimi-
nating the presence of impurities in the extracts. Then, after eval-
uating the various compounds, it is necessary to study the extracts
in vivo, alone and/or when they are added into the food, to see what
changes occur in the body and see the compounds responsible for
the beneficial effects.
All these observations on previous studies are necessary so we
can look ahead and make changes in the way research has been
conducted. In addition, it does not mean that all previous studies
are meaningless, but the time has come to study in depth the
mechanisms and transformations that make extracts obtained after
the extraction process having in vivo beneficial properties. It is
necessary to understand how the various innovative preservation
and/or extraction processes can affect the beneficial properties of
the functional extracts or foods and to compare the effects with
conventional methods.
3. Final comments
Researchers should perform a wide variety of tests, both in vitro
and in vivo, to determine the toxicological effects, therapeutic
dosage and to assess the physicochemical, chemical, sensory, shelf
life properties of a developed food before stating it is functional.
Here we presented some aspects that should be definitely taken
into consideration when designing such studies. One important
outcome must be clearly understood: Food scientists and technol-
ogists should never develop and attest in vivo functionality alone;
rather, a highly active interconnection with related fields, such as
nutrition, pharmacology, statistics, biology is highly required.
Additionally, a strong alliance between academia and food com-
panies should be highly considered worldwide.
As a conclusion and final remark, when a functional food is the
aim of a food company or research institute, it is highly recom-
mended that a multidisciplinary team composed of a food engineer
or technologist, a nutritionist, a pharmacologist, a statistician, and a
biologist (in case of products made of herbal preparations) work
together to assure all aspects related to the commercialization,
safety, and mechanism of action of the functional constituent are
covered, fully understood, and controlled. Without this alliance
between multiple areas, the fail in the functional food chain is
highly possible and expected.
Acknowledgements
Authors would like to express their gratitude to CAPES for the
research funding (PROAP/CAPES) provided in multiple studies of
the group.
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