Food Chemistry 224 (2017) 160–171

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Fish viscera protein hydrolysates: Production, potential applications and

functional and bioactive properties
Oscar Villamil, Henry Váquiro, José F. Solanilla ⇑
Facultad de Ingeniería Agronómica, Universidad del Tolima. Ibagué, Colombia

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

Article history: The aquaculture and fishery chain is an important part of the economy of many countries around the
Received 12 July 2016 world; in recent years it has experienced significant growth that generates more and more quantities
Received in revised form 21 November 2016 of waste, which are mostly discarded, impacting the environment, despite having a useful chemical com-
Accepted 20 December 2016
position in various industrial sectors. This article presents a review of the agroindustrial potential of fish
Available online 21 December 2016
wastes, especially viscera, as a source for obtaining native protein and hydrolysates, explaining their pro-
duction process, chemical composition and functional and bioactive properties that are important to the
Keywords:
agricultural, cosmetic, pharmaceutical, food and nutraceutical industry.
Protein hydrolysates
Fish viscera
Ó 2016 Elsevier Ltd. All rights reserved.
Bioactivity
Functional properties
Peptides

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
2. Fish processing co-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3. Production of fish viscera protein hydrolysates (FVPH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.1. General. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.2. Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.3. Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.4. Recovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
4. Chemical characteristics of fish viscera protein hydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5. Functional properties of FVPH and food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
6. FVPH as source of bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7. FVPH as microbial growth media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

1. Introduction mary processing because they contain microorganisms and

The continuous growth of fish production, at an average annual
rate of 3.2%, and the increase in per capita fish consumption from
9.9 kg in 1960 to 19.2 kg in 2012 (FAO, 2014) have generated
greater amounts of organic wastes that are discarded during pri-
⇑ Corresponding author at: Facultad de Ingeniería Agronómica, Universidad del
Tolima, B. Santa Helena A.A. 546, Ibagué, Colombia.
E-mail address: jfsolanilla@ut.edu.co (J.F. Solanilla).
enzymes responsible for autolysis and high perishability of comes-
tible tissues (Feltes et al., 2010). These wastes generally consist of
viscera, carcass, head, skin and bones which have a high agroindus-
trial potential as a source of co-products; their percentage can vary
between 50% and 70% of the fresh weight, according to each spe-
cies (Guerard, Dufosse, De La Broise, & Binet, 2001; He, Franco, &
Zhang, 2013; Silva, Ribeiro, Silva, Cahú, & Bezerra, 2014). 50% of
the aforementioned waste is discarded and only 30% is used in

http://dx.doi.org/10.1016/j.foodchem.2016.12.057
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 O. Villamil et al. / Food Chemistry 224 (2017) 160–171 161

activities that have low value added, such as animal feed, fertilizer
agents and silage production. (Arvanitoyannis, 2010; He et al.,
2013; Hsu, 2010; Je, Qian, Byun, & Kim, 2007). This is due to igno-
rance about the effective management of waste and the benefits
that it provides, leading to its disposal in rivers, streams, seas
and many more (Ezejiofor, Enebaku, & Ogueke, 2014).
Fish co-products are a significant source of protein, and other
components, such as polyunsaturated fatty acids, phospholipids,
soluble vitamins and bioactive compounds (Shirahigue et al.,
2016), which make them attractive for various uses in technologi-
cal applications that promote product development and significant
advances in the fish industry (Feltes et al., 2010).
Based on the above, there have been studies and investigations
directed towards finding new forms of exploitation of fish waste in
order to mitigate environmental problems (Sheriff, Sundaram,
Ramamoorthy, & Ponnusamy, 2014). Protein hydrolysates have
been gaining great interest in recent years (Šližytė, Daukšas,
Falch, Storrø, & Rustad, 2005) since the modification by enzymes
or chemicals improves functional properties of native proteins
and their usefulness as intermediate ingredients in the cosmetics,
pharmaceutical, food and nutraceutical sectors (Klompong,
Benjakul, Kantachote, & Shahidi, 2007; Kristinsson & Rasco, 2000;
Sarmadi & Ismail, 2010).
Accordingly, the aims of this study are to present a review of the
agroindustrial potential of fish waste, especially viscera, as a source
for obtaining native protein and hydrolysates, and to explain their
production process, chemical composition and functional and
bioactive properties.
2. Fish processing co-products
Fish processing begins with size classification and removal of
scales, carcass and fins by washing; viscera is removed when fish
is not marketed fresh or frozen, and the process then continues
to produce fillets, canned fish and others (Feltes et al., 2010). The
co-products obtained in percentage terms are composed of muscle
cuts (15–20%), skin and fins (1–3%), bones (9–15%), heads (9–12%),
viscera (12–18%) and scales (5%) (Martínez-Alvarez, Chamorro, &
Brenes, 2015), all of which are susceptible to microbial degradation
(Arvanitoyannis, 2010; Benjakul, Yarnpakdee, Senphan,
Halldorsdottir, & Kristinsson, 2014; Hsu, 2010). Lately, there is a
particular interest in new uses of viscera, whose composition is
shown below in Table 1.
As seen in Table 1, fish viscera have both high protein and lipid
content whose variability in composition depends on the species,
seasonality, age, sex, nutrient intake and other factors (Huss,
1995). Different fish muscles and tissues are composed by struc-
tural, myofibrillar and sarcoplasmic proteins that have all the
essential amino acids, among which predominate lysine, pheny-
lalanine and valine (Hayes & Flower, 2013). They also have endoge-
nous enzymes, such as pepsin, trypsin, chymotrypsin, collagenase
and elastase, which can be used to hydrolyze proteins
(Vannabun, Ketnawa, Phongthai, Benjakul, & Rawdkuen, 2014).
Furthermore, collagen and gelatin, in tissues, such as bone and
skin, have recently gained interest in the field of research as an
alternative to mammalian collagen for food and pharmaceutical
applications (Karim & Bhat, 2009). In addition, the lipid fraction
of waste contains omega-3, squalane, phospholipid, cholesterol
and fat-soluble vitamins (Rai, Swapna, Bhaskar, Halami, &
Sachindra, 2010). Its utilization has been directed towards the food
and nutraceutical industries and the energy sector in the produc-
tion of biodiesel (Feltes et al., 2010).
3. Production of fish viscera protein hydrolysates (FVPH)
3.1. General
The aim of producing protein hydrolysates is the solubilization
of the protein source to improve its biological and nutritional value
to obtain products of high added value and commercial interest
(Nilsang, Lertsiri, Suphantharika, & Assavanig, 2005), forming dif-
ferent size peptides either by chemical or enzymatic methods
(Silva, dos Santos da Fonseca, & Prentice, 2014). Protein hydrolysis
has shown continuous development over time, but in a general
context, this process is still in the early stages of discovering pep-
tides and individual amino acid combinations to produce desired
effects for different applications (Pasupuleti & Braun, 2010).
Obtaining hydrolysates from co-products of the fishing and aqua-
culture industries, such as viscera (Fig. 1), involves processes of iso-
lation or pretreatments, followed by hydrolysis and protein
recovery (He et al., 2013).
3.2. Pretreatments
Pretreatments comprise a group of processes whose main
objective is to concentrate proteins. Additionally, their objective

Table 1
Proximate composition of fish viscera and fish viscera protein hydrolysates.

Species Protein (%) Lipids (%) Moisture (%) Ash(%) Reference

Proximate composition of fish viscera
Yellowfin Tuna (Thunnus albacares) 21.5 ± 0.50 5.08 ± 1.53 69.66 ± 2.32 4.46 ± 1.21 Ovissipour et al. (2012)
Tuna (Euthynnus affinis) 65.04 ± 1.40 11.77 ± 1.41 75.73 ± 0.33 3.12 ± 0.11 Salwanee, Wan Aida, Mamot, and Maskat (2013)
Catla (Catla catla) 8.52 ± 0.95 12.46 ± 1.45 76.25 ± 0.25 2.50 ± 0.04 Bhaskar et al. (2008)
Cod (Gadus morhua) 14.90 ± 2,30 2.00 ± 0.50 60.00 ± 0.00 4.40 ± 0.30 Šližytė et al. (2005)
Sardine (Sardina pilchardus) 15.76 ± 1.10 4.89 ± 0.11 77.46 ± 0.02 1.90 ± 0.00 Kechaou et al. (2009)
Rainbow Trout (Onchorhynchus mykiss) 15.00 ± 0.06 13.00 ± 0.76 71.65 ± 0.89 2.73 ± 0.89 Taheri, Anvar, Ahari, and Fogliano (2012)
Pink Salmon (Oncorhynchus gorbuscha) 15.30 ± 0.40 2.00 ± 0.30 81.20 ± 0.70 1.70 ± 0.10 Bechtel (2003)
Parastromateus Niger 14.40 ± 0.50 3.90 ± 0.30 74.00 ± 0.50 3.40 ± 0.40 Nazeer and Kumar (2011)
Tilapia (Oreochromis niloticus)* 14.62 ± 0.79 10.75 ± 0.97 60.44 ± 0.27 4.90 ± 0.61 Shirahigue et al. (2016)
Fish viscera protein hydrolysate
Persian sturgeon (Acipenser persicus) 65.82 ± 7.02 0.18 ± 0.40 4.45 ± 0.67 7.67 ± 1.24 Ovissipour et al. (2009)
Yellowfin tuna (Thunnus albacares) 72.34 ± 3.20 1.43 ± 0.57 2.82 ± 2.74 22.34 ± 1.38 Ovissipour et al. (2012)
Sardinella (Sardinella aurita) (6.62% hydrolysis)** 75.01 ± 1.72 8.53 ± 1.11 1.35 ± 0.55 14.81 ± 0.14 Souissi et al. (2007)
Sardinella (Sardinella aurita) (9.31% hydrolysis)** 72.99 ± 1.82 10.21 ± 1.58 2.83 ± 0.42 13.06 ± 0.13
Sardinella (Sardinella aurita) (10.16% hydrolysis)** 73.05 ± 1.91 10.29 ± 1.76 4.56 ± 0.27 12.10 ± 0.12
Rainbow trout (Onchorhynchus mykiss) 88.32 ± 0.07 0.80 ± 0.60 3.45 ± 0.02 1.14 ± 0.88 Taheri et al. (2012)
*
Total co-products (viscera, bones, head, scales),
**
Viscera and heads.
162 O. Villamil et al. / Food Chemistry 224 (2017) 160–171
Fig. 1. Flow chart of the generic process of obtaining fish viscera protein
hydrolysates (FVPH).
is to prepare homogeneous mixtures of water and chopped viscera
with the lowest possible percentage of fat and other undesirable
components for subsequent hydrolysis (He et al., 2013), since these
components contribute to oxidation and coloration of the final pro-
duct (Kristinsson & Rasco, 2000) and the formation of unpleasant
smells and tastes and highly toxic compounds (Dong et al., 2008).
Several methods have been used to remove fat from different
tissues of fish. In the case of viscera, heat treatment is a very con-
ventional practice, because it has the dual purpose of inactivating
enzymes and removing fat (Guerard et al., 2001). Then the solid
fraction is mechanically separated by centrifugation (Bhaskar,
Benila, Radha, & Lalitha, 2008). On the other hand, pressing has
also been used for this purpose; it is followed by 2–3 h of heat
treatment to yield a solid which is dried to a moisture percentage
lower than 10% (Valenzuela, Sanhueza, & de la Barra, 2012).
The use of solvents is another useful defatting procedure that
involves the heating of a mixture composed of minced raw mate-
rial and alcohols, such as ethanol or isopropyl alcohol, with con-
stant stirring during a certain period of time (Dong et al., 2008;
Klompong et al., 2007). This technique minimizes bacterial degra-
dation (Kristinsson & Rasco, 2000) and eliminates the characteris-
tic odour of fish and bitter flavours (Hoyle & Merritt, 1994).
However, protein solubility and dispersibility are usually affected,
whereby lower degrees of hydrolysis are obtained in comparison to
non-defatted protein (Klompong et al., 2007), which has a negative
effect on hydrolysis efficiency and properties of protein hydroly-
sates (Benjakul et al., 2014).
Some studies have used a new technology to isolate proteins
from fish tissues. It consists of solubilization of protein in acids
or alkaline solutions, in addition to centrifugation and filtration
in order to remove insoluble compounds; once removed, the pro-
teins are precipitated by adjusting the pH to the isoelectric point
and are recovered by centrifugation or decantation (Nolsøe &
Undeland, 2009).
3.3. Hydrolysis
Protein hydrolysis consists of the cleavage of peptide bonds to
obtain free amino acids and low molecular weight peptides, con-
suming a molecule of water for each broken bond. (Rutherfurd &
Gilani, 2009). The hydrolysis process (chemical or biochemical)
has recently been used in aquaculture and fisheries to produce
more acceptable and profitable products (Benjakul et al., 2014).
The conventional acid hydrolysis method is based on sample
treatment in excessively acidic solutions accompanied by high
temperatures and, in some cases, at high pressures over a given
time (Kristinsson & Rasco, 2000). It is a low cost, quick and simple
operation, which makes it applicable at the industrial level (Gao,
Hirata, Toorisaka, & Hano, 2006). However, essential amino acids,
such as tryptophan, methionine, cystine and cysteine, are usually
destroyed. Furthermore, asparagine and glutamine are converted
into aspartic acid and glutamic acid, respectively (Pasupuleti &
Braun, 2010). Additionally, obtained hydrolysates have poor func-
tional properties due to the formation of salts after the neutraliza-
tion process (Kristinsson & Rasco, 2000). Therefore, several
removal methods, such as nanofiltration and use of ion exchange
resins, have been proposed with excellent results (Pasupuleti &
Braun, 2010). Acid hydrolysis of fish co-products has been widely
studied to produce low cost fertilizers (Kristinsson & Rasco,
2000), silages (Vidotti, Viegas, & Carneiro, 2003) and alternative
substrate for lactic acid production using improved hydrolysis in
order to enhance nutritive value (Gao et al., 2006).
Alkaline hydrolysis is a very simple process, the sample is solu-
bilized by heating and mixed with alkaline solutions, then the reac-
tion temperature is maintained until reaching the desired degree of
hydrolysis (Pasupuleti & Braun, 2010). Its main drawback is the
production of hydrolysates with low amino acids content, such
as cystine, lysine, arginine, serine, threonine, isoleucine and resi-
dues like lanthionine and lysinoalanine (Tavano, 2013). Alkaline
hydrolysis has been widely used in the industrial field, but little
in biotechnology (Pasupuleti & Braun, 2010).
Unlike chemical methods, enzymatic hydrolysis uses mild con-
ditions and is easy to control, and is more precise in the cleavage of
peptide bonds; furthermore, there are no side reactions or decrease
in the nutritional value (Tavano, 2013), and it exhibits greater ease
in protein recovery and purification of some peptides (Pasupuleti &
Braun, 2010). For these reasons, it has gained more and more
attention to produce hydrolysates with better and more defined
nutritional, functional and bioactive properties (Benjakul et al.,
2014). Nevertheless the majority of the studies on enzymatic
hydrolysis have not been scaled and its biggest drawback is the
high production costs compared to chemical hydrolysis (He,
Franco, & Zhang, 2015).
The enzymatic hydrolysis process is usually performed in a
reactor with temperature, pH, agitation and time controls
(Benítez, Ibarz, & Pagan, 2008). Initially, the temperature and pH
of the homogeneous mixture obtained from pretreatment are
adjusted according to the ideal working conditions of the enzyme.
When protease is added, the reaction between enzyme and sub-
strate causes changes in the pH of the solution due to the cleavage
 O. Villamil et al. / Food Chemistry 224 (2017) 160–171
Table 2
Species, enzymes and parameters used for obtaining protein hydrolysates from fish viscera.
Specie Enzyme
Yellowfin tuna (Thunnus albacares) Alcalase 2.4L
Cod (Gadus morhua L.) Flavorzyme 500L
Neutrase 0.8L
Sardinella (Sardinella aurita) Alcalase
Catla (Catla catla) Alcalase (0.6 AU/g)
Persian Sturgeon (Acipenser persicus) Alcalase 2.4L
Sardine (Sardina pilchardus) Protamex
Alcalase 2.4L
Flavorzyme 500MG
Black scabbardfish (Aphanopus carbo) Protamex
Rohu (Labeo rohita) Alcalase (0.6 AU/g)
Catla (Catla catla) Neutrase (1.5 AU/g)
Protex 7L
Protease-P-Amano (60000 U/g)
Parastromateus Niger Pepsin
Trypsin
a-Chymotrypsin
Tuna (Euthynnus affinis) Alcalase
Tilapia (Oreochromis niloticus) Neutrase
of peptide bonds to form new amino or carboxyl groups able to
release or accept protons. Faced with the above, some methods rely
on the addition of buffer solution in order to moderate pH changes
(Ovissipour, Kenari, Motamedzadegan, & Nazari, 2012; Ovissipour
et al., 2009; Shirahigue et al., 2016). However, it is considered that
the presence of salts in buffer solutions can affect functional prop-
erties of interest, such as the emulsifying and foaming capabilities
(Kristinsson & Rasco, 2000). Meanwhile, other studies choose to
maintain the optimal pH of enzyme activity by constant addition
of neutralizing solution during the hydrolysis process (Dong
et al., 2008; Hsu, 2010; Kechaou et al., 2009; Klompong et al.,
2007; Silva et al., 2014). In both processes, when the expected
degree of hydrolysis is obtained, peptidase is deactivated by
changes in temperature, pH or both variables simultaneously
(Benítez et al., 2008).
The choice of enzyme depends both on the final product and the
price (Benjakul et al., 2014). It is also important to consider the
amino acid composition of the protein because some proteases
have preferences for the cleavage of certain peptide bonds
(Pasupuleti & Braun, 2010). It bears mentioning that the medium
in which the enzymes work is a factor of choice, for example those
whose optimum pH is acidic can inhibit bacterial growth, but have
low recovery percentages of protein and decreased nutritional and
functional value in comparison to alkaline and neutral proteases
(Kristinsson & Rasco, 2000). This is the reason why microbial pro-
teases with high proteolytic activity are the most commonly used
and suitable in the production of hydrolysates from fish tissues
(Benjakul et al., 2014) as can be seen in Table 2.
Protein hydrolysates can also be obtained by use of proteases
present in the digestive system of fish, such as pepsin, trypsin, chy-
motrypsin, collagenase and elastase (Vannabun et al., 2014). Autol-
ysis has been widely used to obtain fish sauce and silages
(Kristinsson & Rasco, 2000). Bougatef et al. (2008) produced pro-
tein hydrolysates from heads and viscera of sardinella Sardinella
aurita by autolysis temperatures ranging from 40 °C to 50 °C and
pH 8, finding that the addition of an external enzyme can acceler-
ate reaction and increase the degree of hydrolysis.
Motamedzadegan, Davarniam, Asadi, Abedian, and Ovissipour
(2010) optimized the autolytic sequential process with neutrase
applied to yellowfin tuna, concluding that enzymatic activity of
39.61 AU/kg protein, 53 °C and 141 min are the recommended
parameters to achieve a hydrolysis degree of about 30%.
Vannabun et al. (2014) characterized acidic and alkaline proteases,
which showed better activity in acidic and alkaline media and an
163
Parameter Reference
E/S=0.2–3%; T=50 °C; pH=8.0 Guerard et al. (2001)
E/S=0.1%; T=50 °C; pH=7.0 Šližytė et al. (2005)
E/S=0.3%; T=50 °C; pH=7.0
E/S=727.26 U/g; T=50 °C; pH=8.0 Souissi et al. (2007)
E/S=1.5%; T=55 °C; pH=8.5 Bhaskar et al., 2008
E/S=0.1 AU/g; T=55 °C; pH=8.5 Ovissipour et al. (2009)
E/S=0.1% w/w; T=50 °C; pH=8.0 Kechaou et al. (2009)
E/S=0.1% w/w; T=50 °C; pH=8.0
E/S=0.1% w/w; T=50 °C; pH=8.0
E/S=0.5–4%; T=50 °C; pH=7.5 Batista, Ramos, Coutinho, Bandarra, and Nunes (2010)
E/S=0.5% w/v; T=40 °C Hathwar, Bijinu, Rai, and Narayan (2011)
E/S=0.5% w/v; T=40 °C
E/S=0.5% w/v; T=40 °C
E/S=0.5% w/v; T=40 °C
E/S=1.0% w/v; T=37 °C; pH=2.5 Nazeer and Kumar (2011)
E/S=1.5%; T=40 °C; pH=8 Salwanee et al. (2013)
E/S=0.5%; T=55 °C; pH=7.0 Shirahigue et al. (2016)
optimal temperature of 40 °C and 60 °C, respectively. However,
the enzymes did not show high stability in media with high salt
concentration. Autolysis is an economical procedure, but is difficult
to standardize and control because endogenous enzymes depend
on several factors, including seasonality, type and amount of
enzymes, fish species and others (Bhaskar et al., 2008).
The process efficiency is measured by quantifying the degree of
hydrolysis, which is defined as the amount of peptide bonds
cleaved out of the total amount of peptide bonds in native protein
(Benítez et al., 2008; He et al., 2013). Degree of hydrolysis is influ-
enced by different parameters, such as E/S ratio, pH, temperature
and incubation time; the first three have an effect on reaction rate,
while the latter only impacts the degree of hydrolysis (Benítez
et al., 2008). It may be the most suitable measuring method as
the interaction of the aforementioned factors with the choice of
substrate and enzyme are directly related to the amount of low
molecular weight peptides, protein recovery (Ovissipour et al.,
2009) and bioactive and functional properties of hydrolysates
(Klompong et al., 2007).
3.4. Recovery
FVPH can be purified by various methods depending on the final
use of the hydrolysates, which are centrifugation, nano-filtration,
ultrafiltration, microfiltration and ion exchange chromatography
(Pasupuleti & Braun, 2010). Centrifugation has been the most used
of the above, from which four phases are obtained: oil fraction,
emulsion layer, FVPH and sludge (Ramakrishnan, Ghaly, Brooks,
& Budge, 2013; Šližytė et al., 2005). After separating FVPH by
decantation, it is dehydrated through lyophilization or spray-
drying in order to increase shelf life and to provide greater ease
in handling, transportation and storage. The final product is usually
a creamy white powder with enhanced functional and bioactive
properties (He et al., 2013). All kinds of hydrolysates must be
stored taking into account parameters, such as moisture content
and glass transition temperature, which generally tends to decline
while the degree of hydrolysis is increasing (Rao, Klaassen Kamdar,
& Labuza, 2016). Therefore FVPH are commonly stored below 0 °C
to ensure stability over time (Ambigaipalan & Shahidi, 2015; Dong
et al., 2008).
It is noteworthy that laboratory scale subsequent processes
have been proposed to separate different peptide fractions accord-
ing to the molecular weight (He et al., 2013), because the relation-
ship between peptide size and certain bioactive and antioxidant
164 O. Villamil et al. / Food Chemistry 224 (2017) 160–171

Table 3
Fish viscera protein hydrolysates amino acid composition.

Amino acids (g/100 g) Fish specie

Persian Sturgeon Catla (Catla Tuna (Thunnus Atlantic cod (Gadus Atlantic cod Atlantic cod
(Acipenser persicus) catla) (Bhaskar albacares) morhua) (Horn, (Gadus morhua) (Gadus morhua)
(Ovissipour et al., 2009) et al., 2008) Alc (Ovissipour et al., Aspmo, & Eijsink, (Horn et al., (Horn et al., 2005)
Alc 2.4L 2.4L 2012) Alc 2.4L 2005) Pap 2005) Alc Endo
Essential Histidine 2.08 2.06 8.45 1.20 1.00 1.10
Isoleucine 3.80 3.60 6.93 3.30 2.90 3.10
Leucine 7.13 7.17 7.70 5.30 5.10 5.10
Lysine 6.80 7.07 1.87 3.30 3.70 3.70
Methionine 10.30 2.02 1.48 2.20 2.00 2.10
Phenylalanine 3.14 3.53 3.85 2.70 2.60 2.60
Threonine 3.50 4.02 5.90 4.10 3.40 3.70
Tryptophan – – – N.R. 0.50 0.50
Valine 5.79 4.79 8.93 4.00 3.60 3.80
Arginine 7.28 10.82 8.81 3.40 3.90 3.30
Non-essential Tyrosine 2.34 2.57 1.31 2.40 2.40 2.50
Aspartate/ 8.30 8.50 11.83 6.30 6.10 6.00
Asparagine
Glutamate/ 13.70 15.01 15.31 9.70 9.40 9.70
Glutamine
Glycine 5.40 10.99 5.87 7.10 7.40 6.60
Alanine 6.30 7.04 2.23 5.10 4.90 4.80
Proline/ 3.46 6.24 N.R. 4.30/1.40 4.20/1.60 4.00/1.20
hydroxyproline
Cystine N.R. 0.23 N.R. N.R. N.R. N.R.
Taurine N.R. N.R. N.R. 2.70 2.50 3.30
Serine 4.20 4.34 6.81 4.10 4.00 4.10

N.R.: No report, Alc: Alcalase, Pap: Papain, Endo: Endogenous enzymes.

properties has been demonstrated in several studies (Robert et al.,
2015). However, there are difficulties for industrial scaling at
present.
4. Chemical characteristics of fish viscera protein hydrolysates
Proximate composition of FVPH is shown in Table 1. These
products have high protein content due to protein solubilization
during hydrolysis and removal of insoluble material and most of
the fat layer during recovery processes (Chalamaiah, Hemalatha,
& Jyothirmayi, 2012). Moisture content, generally below 10%, is
caused by the concentration and drying processes, while ash con-
tent is linked to the neutralization process during hydrolysis
(Dong et al., 2008; Gbogouri, Linder, Fanni, & Parmentier, 2004;
Kristinsson & Rasco, 2000).
Amino acids have an important role in protein synthesis as
compound carriers of hydrogen, vitamins, carbon dioxide, enzymes
and structural proteins (Chalamaiah et al., 2012) and they also
influence bioactive and functional properties. Table 3 shows amino
acid composition of several FVPH whose variation depends on raw
material, enzyme type and hydrolysis parameters (Bhaskar et al.,
2008; Klompong et al., 2009). FVPH have all the essential and
non-essential amino acids, which is why they are considered to
be high nutritional sources.
5. Functional properties of FVPH and food applications
Functional properties are physicochemical characteristics that
influence the performance of the protein in different food systems
during different stages from processing to consumption
(Kristinsson & Rasco, 2000; Šližytė et al., 2005) (Table 4). Native
proteins present good functionality, however their use is limited
because most foods have pH close to the isoelectric point, therefore
protein hydrolysis has become an increasingly common technique
as it significantly improves the functionality of proteins to a wide
range of pH levels (de Castro, Bagagli, & Sato, 2015; de Castro &
Sato, 2014).
The parameters that affect protein functionality are: substrates,
enzymes used and degree of hydrolysis. Protease specificity influ-
ences molecular weight and hydrophobic character and, as a result,
protein behaviour (Kristinsson & Rasco, 2000). Thus, hydrolysates
with different molecular weights can be obtained by appropriately
selecting enzymes and substrates according to desired require-
ments. Proteins with a lower degree of hydrolysis show excep-
tional functional properties because they still retain amphiphilic
quality, which does not happen with those whose hydrolysis has
been excessive despite the fact that solubility, in most cases, has
increased (Klompong et al., 2007). Additionally, functional beha-
viour of proteins can also vary by extrinsic conditions, such as
the nature of the matrix, along with storage and processing param-
eters (Benjakul et al., 2014).
Protein functional properties are classified in hydrodynamic
properties, such as solubility, viscosity, gelling capacity and water
absorption, and surface active properties, such as emulsifying
capacity, foaming and film formation (de Castro et al., 2015). Solu-
bility is considered by many researchers as the most important
functional property because it significantly affects all others
(Benjakul et al., 2014). There is direct correlation between solubil-
ity and degree of hydrolysis; it can be explained by the fact that
longer hydrolysis times produce proteins and peptides with smal-
ler molecular weight generating greater exposure of ionizable and
polar groups on the protein surface, which influences the improve-
ment of their ability to form hydrogen bonds with water (de Castro
& Sato, 2014; He et al., 2013; Yin, Tang, Wen, Yang, & Li, 2008).
Fish protein hydrolysates present improved solubility in com-
parison with native proteins (Dong et al., 2008; Klompong et al.,
2007). Protein hydrolysates made with trypsin, pepsin and chy-
motrypsin from muscle, skin, bones and viscera of horse mackerel
and croaker showed solubility greater than 65% over a wide pH
range (Kumar, Nazeer, & Ganesh, 2012). Similar results were
obtained by Liu et al. (2014), who studied solubility in surimi
 O. Villamil et al. / Food Chemistry 224 (2017) 160–171 165

by-products, including fish meat leftover on bones, head, skin, and 65% and more in protein hydrolysates made with protamex and
viscera; they reported an increase from 10% in native protein up to alcalase in a wide pH range. Souissi, Bougatef, Triki-Ellouz, and

Table 4
Functional properties and applications of fish wastes.

Skin and Gelatin extraction

Source Pre-treatment extracting conditions Yield (% on a weight Outcome Reference
basis)
Megrim (Lepidorhombus 0.05 M Acetic acid. 45 °C – overnight 7.4 Gelatins were obtained without Gómez-Guillén et al.
boscii) proteolytic digestion. The gelling ability (2002)
Dover sole (Solea vulgaris) 8.3 and thermal stability of the gels were
Cod (Gadus Morhua) 7.2 found to be influenced by extrinsic
Hake (Merluccius merluccius, 6.5 factors like fish species.
L.)
Black tilapia (Oreochromis 0.2% Sulfuric acid/1% citric acid. 45 °C – 5.39 Gelatins from black and red tilapia were Jamilah and
mossambicus) 12 h obtained. The results suggest that they Harvinder (2002)
Red Tilapia (Oreochromis 7.81 can be for applications different from
nilotica) cold water fish gelatin.
Pangas catfish (Pangasius 1% (1:3 b/v) citric acid (pH 3) for 12 h. 22 The fishes investigated were potential Ratnasari, Yuwono,
pangasius) 60 °C – 6 h alternative sources of gelatin in spite of Nusyam, and
Asian redtail catfish 21.28 showing lower physicochemical and Widjanarko (2013)
(Hemibagrus nemurus) rheological properties compared to the
Striped snake head (Channa 20.25 commercial gelatin.
striata)
Nile tilapia (Oreochromis 21.93
niloticus)
Squid (Dosidicus gigas) 0.05 M Acetic acid for 3 h. 65 °C – 12 h 7.5 D. gigas skin gelatin has potential as a Uriarte-Montoya et al.
source of functional component for (2011)
different industrial applications.
Collagen
Source Pre-treatment extracting conditions Yield (% on a weight Outcome Reference
basis)
Ocellate puffer fish ASC: Defatting: 10% butyl alcohol for 2 d. ASC: 10.7*PSC: 44.7* The obtained Pepsin-Solubilized collagen Nagai, Araki, and
(Takifugu rubripes) Extraction: 0.5 M acetic acid for 3d. is a heterotrimer with a chain Suzuki (2002)
Precipitation by the addition of NaCl composition of (a1)2a2.
(final concentration of 2.3 M) in 0.05 M Td: 28 °C.
Tris–HCl (pH 7.5). Dialysis against 0.1 M
acetic acid solution, distilled water and
then lyophilized.
PSC: Residue was suspended in 0.5 M
acetic acid and digested with 10% (w/v)
pepsin for 48 h at 4 °C. Dialysis 0.02 M
Na2HPO4 (pH 7.2) for 3d. Precipitation
by adding NaCl (final concentration of
2.2 M) in 0.05 M Tris–HCl (pH 7.5).
Dialysis against the same solution and
lyophilized.
Grass carp PSC: 46.6* The obtained collagen contained a1 and Zhang et al. (2007)
(Ctenopharyngodon a2 chains. Td: 28.4 °C
idella) Tmax: 24.6 °C
Tilapia (Oreochromis Method: fish skins were soaked in 0.1 M Molecular weight Results conclude that there might be a Tang et al. (2015)
niloticus) NaOH with a skin/solution ratio of 1:10 (MW) ranging from correlation between the film-forming
Grass carp (w/v) for 36 h, and alkali solution was 100 to 20 kDa (SDS– ability of collagen from freshwater fish
(Ctenopharyngodon changed every 12 h. Defatted using 10% PAGE) skins and the primary structures and
idella) butyl alcohol with a solid/solvent ratio of conformation of collagen molecules.
Silver Sarp 1:10 (w/v) for 36 h and the solvent was Td: 33 °C
(Hypophthalmichthys changed every 12 h. Was washed with
molitrix) cold tap water and subsequentially
extracted with 0.5 M acetic acid with a
skin/ acetic acid ratio of 1:30 (w/v) for
2 days.
Odonus niger Method I: (ASC) 10 volumes of 0.5 mol/L 50 The collagen from leather jacket skin Muralidharan,
of the acetic acid for 3 days showed good thermal properties and it Shakila, Sukumar, and
Method II: extraction of ASC was first 55–60 can be used effectively for Jeyasekaran (2013)
extracted. Then pepsin soluble collagen pharmaceutical and biomedical
(PSC) was extracted by using 0.1% (w/v) applications.
pepsin to 0.5 mol/L of acetic acid
Method III: PSC was extracted twice and 70
then Centrifugation. Precipitation of
collagen by using 2 mol/L NaCl for 24 h
at 4 °C. and centrifuging again, followed
by dialysis against 0.02 mol/L phosphate
buffer (pH 7.2) for 24 h at 4 °C.

(continued on next page)

166
Table 4 (continued)
Antioxidant and bioactive peptides
Source
Unicorn leatherjacket
(Aluterus monoceros)
Hoki (Johnius belengerii)
Skate (Okamejei kenojei)
Bones: Antioxidant and bioactive peptides
Source
Yellowtail fish
Tuna backbone
Alaska Pollack (Theragra
chalcogramma)
Viscera: Proteolytic Enzymes
Source
Giant catfish
(Pangasianodon gigas)
O. Villamil et al. / Food Chemistry 224 (2017) 160–171
Enzymes used Properties Outcome Reference
Alcalase DH 40%. Hydrolysates can be used as a source of Sai-Ut, Benjakul,
ABTS: 48 lmol TE/g natural antioxidants that are useful in Sumpavapol, and
solid the oxidation inhibition both food and Kishimura (2014)
FRAP: 8 lmol TE/g biological systems.
solid
Fe+2 Chelating activity:
19 lmol EE/g solid
Protease from B. amyloliquefaciens H11. ABTS: 66 lmol TE/g
solid
FRAP: 9.5 lmol TE/g
solidFe+2 Chelating
activity: 19 lmol EE/g
solid
Trypsin DPPH: 30% An antioxidant peptide sequence Mendis, Rajapakse,
Carbon centered: 40– identified to be His-Gly-Pro-Leu-Gly- and Kim (2005)
50% Pro-Leu (797 Da) was purified by
Superoxide: 40–50% consecutive chromatographic
a -chymotrypsin, DPPH: 10–20% separations of tryptic hydrolysate. It
Carbon centered: 20% showed positive effects on the
Superoxide: 30–40% Antioxidative Enzyme Activities in
Pepsin DPPH: 20–30% Hep3B Cells and on the inhibition of lipid
Carbon centered: 20– peroxidation.
30%
Superoxide:40%
Alcalase ACE inhibitory activity: Two peptides associated with ACE Ngo, Ryu, and Kim
72.8% at 2 mg/ml inhibitory activity were identified to be (2014)
MVGSAPGVL (829 Da) and LGPLGHQ
(720 Da), with IC50 values of 3.09 and
4.22lM, respectively. They could be used
as functional ingredients.
Pre-treatment extracting conditions Properties Outcome Reference
Pretreatment: 0.6 N HCl for 24 h (10% w/ IC50: 0.16 mg/ml The hydrolysate has suitable properties Morimura et al.
v). Drying at 60 °C, 76 mm Hg, 24 h. IPOX50: 0.18 mg/ml for the food industry. (2002)
Hydrolysis: Enzyme L, 200 rpm, pH: 8.0,
60 °C, 60 min.
Drying at 105 °C for 24 h.
Alcalase. Buffer 0.1 M Na2HPO4– DPPH: 4.82% An antioxidant peptide was purified and Je et al. (2007)
NaH2PO4. pH:7.0; T:50 °C Hydroxyl: 77.85% identified to be VKAGFAWTANQQLS
Superoxide: 22.53% (1519 Da). Results suggest that these
a -Chymotrypsin. Buffer 0.1 M DPPH: 17.38% peptides fractions could be used as diet
Na2HPO4–NaH2PO4. pH:8.0; T:37 °C Hydroxyl: 83.25% nutrients, food additives and
Superoxide: 32.25%. pharmacological agents.
Papain. Buffer 0.1 M Na2HPO4–NaH2PO4 DPPH: 36.72%
pH:6.0 T:37 °C Hydroxyl: 78.23%
Superoxide: 21.89%
Pepsin. Buffer: 0.1 M Glycine–HCl. DPPH: 35.82%
pH:2.0; T:37 °C. Hydroxyl: 80.91%
Superoxide: 31.92%
Neutrase. Buffer 0.1 M Na2HPO4– DPPH: 5.13%
NaH2PO4 pH:8.0 T:50 °C Hydroxyl: 65.23%
Superoxide: 27.63%
Trypsin Buffer 0.1 M Na2HPO4–NaH2PO4. DPPH: 6.89%
pH:8.0 37 °C Hydroxyl: 81.08%
Superoxide: 29.89%.
Pepsin. pH:2.0; T:37 °C; E/S:1/100; Calcium solubility: A low molecular peptide was recovered Jung et al. (2006)
Substrate concentration: 1% 32 mg/l at a from pepsinolytic hydrolysates and it
concentration of the was identified to be Val-Leu-Ser-Gly-
peptide of 250 mg/l Gly-Thr-Thr-Met-Ala-Met-Tyr-Thr-Leu-
Val (1442 Da). Its affinity to calcium
suggests that the peptides might be used
in nutraceutical products to oriental
people with lactose indigestion and
intolerance.
Pre-treatment extracting conditions Optimum conditions Outcome Reference
and parameters
Acid protease: 10 mM citrate/HCl pH 3.0 Acid proteases: pH 3.0; Proteases were successfully used to Vannabun et al.
Centrifugation at 10000g for 10 min at 40 °CAlkaline hydrolyze bovine muscle and farmed (2014)
4 °C.Alkaline protease: 10 mM Tris-HCl proteases: pH 9.0; giant catfish skin. Conclusions suggest
 O. Villamil et al. / Food Chemistry 224 (2017) 160–171 167

Table 4 (continued)

Viscera: Proteolytic Enzymes

Source
Hybrid catfish (Clarias
microcephalus  Clarias
gariepinus)
Hybrid catfish (Clarias
microcephalus  Clarias
gariepinus)
Pirarucu (Arapaima gigas)
Monterey sardine
(Sardinops sagax
caerulea)
Golden grey mullet (L.
aurata)
Nile tilapia (Oreochromis
niloticus)
Scale: Collagen
Source
Sardine (Sardinops
melanostictus)
Red sea bream (Pagrus
major)
Japanese sea bass
(Lateolabrax japonicus)
*
Dry weight basis, Td: denaturation temperature, Tt: transition temperature, ASC: Acid-Solubilized Collagen, PSC: Pepsin-Solubilized Collagen, DH: Degree of hydrolysis.
Pre-treatment extracting conditions Optimum conditions Outcome Reference
and parameters
pH 8.0, 10 mM CaCl2 (1:5w/v) 60 °C that they could be used as
Centrifugation at 10000g for 10 min at biotechnological alternative for gelatin
4 °C. hydrolysate production and others.
10 mM Tris-HCl pH 8.0, 1 mM CaCl2 pH 9.0; 50 °C A protein hydrolysate derived from Klomklao, Kishimura,
(1:50 w/v). Centrifugation at 10000g for toothed ponyfish with desirable and Benjakul (2013)
10 min at 4 °C. composition of aminoacids and peptides
was obtained.
10 mM Tris-HCl pH 8.0, 1 mM CaCl2 pH 8.0; 60 °C A 24KDa purified trypsin was obtained. Klomklao, Benjakul,
(1:50w/v). Centrifugation at 10000g for Kishimura, and
10 min at 4 °C.Purification by using Chaijan (2011).
ammonium sulphate fractionation and a
series of chromatographies.
0.1 M Tris-HCl pH 8.0 (200 mg tissue/ml pH 9.0; 65 °C A thermostable alkaline protease with Freitas-Júnior et al.
buffer). Centrifugation at 10000g for great activity and stability over a wide (2012)
20 min at 4 °C.Purification by a four step alkaline pH range and high salt
procedure: heat treatment, ammonium concentrations was obtained from
sulphate fractionation, molecular size pyloric caeca of pirarucu. Its
exclusion chromatography and affinity characterization proved that this
chromatography. protease is a trypsin.
100 g sample was homogenized with pH 2.5; 45 °C Viscera extract from Monterey sardine Castillo-Yánez,
200 ml ice-cold distilled water and found to be a promising biotechnological Pacheco-Aguilar,
centrifuged at 26000g for 20 min at 2- alternative to the food industry due to its García-Carreño, & de
4 °C. high activity detected. los Ángeles
Navarrete-Del-Toro
(2005)
10 mM Tris-HCl buffer, pH 7.0, (1:2w/v). pH 3.0; 40 °C The crude acid protease was proven to Bkhairia, Mhamdi,
Centrifugation at 8000g for 15 min at be effective in gelatin extraction from Jridi, and Nasri (2016)
4 °C.Supernatant was collected and golden grey mullet skin.
adjusted at pH 2.0 with 1 M HCl, then
centrifuged for 30 min at 4 °C at 5000g.
Viscera homogenized 40 mg of tissue/mL pH 8.0; 50 °C Enzymes from Nile tilapia viscera could Bezerra et al. (2005)
(w/v) in 0.9% (w/v) NaCl Centrifugation be isolated at low cost due to the large
at 10000g for 10 min at 10.8 °C. amount of this co-product in industrial
Purification by a three step procedure: processing. It can be used in the
heat treatment, ammonium sulphate production of fish protein hydrolysates,
fractionation and gel filtration. fish sauce and as a laundry detergent
additive.
Pretreatment extracting conditions Yield (%) Outcome Reference
Decalcification 0.05 m Tris–HCl (pH 7.5) 50.9 Fish scales have potential as an Nagai, Izumi, and Ishii
containing 0.5 m EDTA-4Na for 2 d. alternative source of collagen for use in (2004)
Disaggregation 0.1 m Tris–HCl (pH 8.0) 37.5 the cosmetic and medical fields
containing 0.5 m NaCl, 0.05 m EDTA-2
Na and 0.2 m 2-mercaptoethanol (2-ME) 41.0
for 3 d and limited pepsin digestion.

Nasri (2007) found that alcalase hydrolysates obtained from heads
and viscera of sardinella showed solubility of up to 100% between
pH ranging from 6 to 10 with a degree of hydrolysis around 10%.
These results indicate that fish protein hydrolysates can be used
easily in food formulations.
Water-holding capacity consists of the ability of the protein to
capture water in the food matrix preventing its flow by gravita-
tional force (Kristinsson & Rasco, 2000), it is important to the food
industry because it influences texture (Pires & Batista, 2013). Fish
protein hydrolysates from cod Gadus morhua by-products function
as good water scavengers in food systems by adding them to com-
minuted fish and then freezing the fish in order to observe the
influence of these components on water retention during thawing
(Šližytė et al., 2005). Furthermore, Balti et al. (2010) observed the
increase of water-holding capacity with the extent of hydrolysis,
concluding that there is a direct correlation between such variables
probably due to the exposure of polar groups as the hydrolysis pro-
cess advances.
Oil holding capacity or fat absorption refers to the quantity of
oil absorbed and retained by the protein; it is related to emulsify-
ing capacity and may be affected by bulk density, degree of hydrol-
ysis and enzyme – substrate specificity (Kaur & Singh, 2007; Pires
& Batista, 2013). Oil holding capacity in fish by-product hydroly-
sates has been studied. FVPH obtained from viscera and heads of
sardinella Sardinella aurita presented improved fat absorption
compared to native protein and showed better results than casein
at a degree of hydrolysis of 9.33% (Souissi et al., 2007); similar
results were collected by Balti et al. (2010), hydrolysates produced
from skin and viscera of cuttlefish Sepia officinalis revealed excel-
lent oil holding capacity that enables their use in formulations
for the food and confectionery industries.
Proteins are good emulsifiers because they have an amphipathic
structure that facilitate their absorption at the oil–water interface
(Pires & Batista, 2013), this property in hydrolysates is mainly
influenced by the size and molecular weight of peptides, surface
hydrophobicity, enzyme, amino-acid composition (Liu et al.,
168 O. Villamil et al. / Food Chemistry 224 (2017) 160–171
2014), volume fraction of oil, protein concentration, equipment
used to form the emulsion (Šližytė et al., 2005) and solubility
(Klompong et al., 2007). Many studies have reported emulsifying
properties in fish co-products hydrolysates. Souissi et al. (2007)
showed that fish hydrolysates from heads and viscera of Sardinella
aurita, at a degree of hydrolysis of 6.67%, have emulsifying capacity
greater than casein, but this capacity decreases with the extent of
hydrolysis. These results coincide with those of Klompong et al.
(2007), however Balti et al. (2010) reported that protein hydroly-
sate from skin and viscera of cuttlefish Sepia officinalis at a degree
of hydrolysis of 5% showed lower emulsifying capacity than those
with a degree of hydrolysis of about 10 and 13.5%, probably due to
the liberation of peptides with medium weight as hydrolysis pro-
ceeds, which increased the flexibility of such peptides and conse-
quently their surface size and ability to form emulsions. The
emulsifying properties of FVPH and others obtained from different
co-products have proven to be more effective than commercial
food-grade emulsifiers, this represents a high potential in the use
of FVPH in food formulations (He et al., 2013).
At present, several fish protein hydrolysates have been success-
fully incorporated into food systems, such as cereals, cookies, des-
serts and meat products (Chalamaiah et al., 2012; Kristinsson &
Rasco, 2000), however there is insufficient information about the
performance of hydrolysates from viscera and other co-products
in food matrices and commercial formulations, although they were
found to be potential ingredients in food formulations according to
various laboratory assays (He et al., 2013).
6. FVPH as source of bioactive compounds
Current research is focused on bioactive peptides (Sarmadi &
Ismail, 2010) due to their biological roles which are beneficial to
human health and useful to the food industry (Sarmadi & Ismail,
2010; Wu et al., 2015), such peptides consist of short amino acid
chains that are inactive within the precursor protein. (Hayes &
Flower, 2013). The performance of hydrolysates from several fish
protein sources as antioxidant, antihypertensive and antimicrobial
agents has been reported (Ryan, Ross, Bolton, Fitzgerald, & Stanton,
2011). Souissi et al. (2007) evaluated hydrolysates obtained from
heads and viscera of sardinella Sardinella aurita in terms of scav-
enging effect on DPPH free radical and Inhibition of linoleic acid
autoxidation, the results indicate that the aforementioned peptides
exhibited more than 50% inhibition of linoleic acid peroxidation
and an antioxidant activity of about 41%. Kumar, Nazeer, and
Jaiganesh (2011) purified and characterized an antioxidant peptide
from horse mackerel Magalaspis cordyla viscera protein whose
sequence was determined as Ala-Cys-Phe-Leu (518.5 Da), it
showed 59.1 and 89.2 percentage of scavenging of hydroxyl radi-
cals and DPPH respectively, using a concentration of 0.2 mg/ml. It
also was found to be better than a-tocopherol in terms of inhibi-
tion of lipid peroxidation. Nazeer and Kumar (2011) evidenced that
fish viscera protein hydrolysates obtained from Parastromateus
niger have a protective effect on DNA against damage caused by
hydroxyl radicals. All those outcomes suggest the presence of
antioxidant peptides in hydrolysates obtained from fish co-
products.
Hypertension is one of the main risk factors associated with car-
diovascular disease; it is caused by angiotensin-I-converting
enzyme (EC 3.4.15.1; ACE) which plays an important role in the
blood pressure control producing angiotensin II, a powerful vaso-
constrictor and destroyer agent of vasodilators like bradykinin
(Barbana & Boye, 2010; Herpandi, Rosma, & Wan Nadiah, 2011).
The inhibition of this enzyme is the key factor to prevent and treat
hypertension. Currently, synthetized chemical compounds have
been used for that purpose, but their side effects have led to the
search for peptides with ACE inhibitory activity from several
sources (Harnedy & FitzGerald, 2012).
Many studies have evidenced the ACE inhibitory activity of fish
co-products peptides (Harnedy & FitzGerald, 2012; Hayes &
Flower, 2013; Herpandi et al., 2011; Kim, Ngo, & Vo, 2012;
Raghavan & Kristinsson, 2009; Wu et al., 2015). Bougatef et al.
(2008) produced sardinella Sardinella aurita head and viscera pro-
tein hydrolysates using alcalase, chymotrypsin, crude enzyme
preparation from Aspergillus clavatus ES1, alkaline proteases from
B. lincheniformis NH1 and crude enzyme extract viscera of sardine
Sardina pilchardus. The fish co-product hydrolysates were found to
be good ACE inhibitors, whose IC50 values ranged from 1.24 to
7.40 mg/ml. Balti et al. (2010) obtained cuttlefish Sepia officinalis
skin and viscera protein hydrolysates whose IC50 was about
1.00 mg/ml at 13.5% degree of hydrolysis. Both studies agree that
the enzyme plays a decisive role in the cleavage of peptide bonds
due to its specificity and that these results suggest that peptides
from such materials can be used as ingredients in functional prod-
ucts for the prevention and treatment of hypertension. However,
the bioactivity of fish co-products peptides has not been as exten-
sively studied as milk and plant peptides (Rustad & Hayes, 2012),
more research about anti-cancer, anti-atherosclerotic and anti-
inflammatory activities of peptides derived from different fish tis-
sues is needed (Halim, Yusof, & Sarbon, 2016). Also, there is limited
information about the behaviour of these peptides on several food
matrices and their stability throughout industrial processing
(Samaranayaka & Li-Chan, 2011), as well as during gastrointestinal
absorption (Sarmadi & Ismail, 2010). Additionally, the production
of FVPH is a huge challenge because of sensory problems, high
costs and difficulties to ensure reproducibility (Rustad & Hayes,
2012) which represent new fields to research.
7. FVPH as microbial growth media
The use of fish tissues as a source of nutrients for microorgan-
isms has been known for decades; several studies with the aim
of exploring the use of fish peptones as a component of microbial
growth substrates have been reported. Proteins obtained from
tuna, cod, salmon and unspecified fish were compared with casein
peptone by the simulation model Gompertz applied to microbial
growth of six species of bacteria, yeast and mold, and results
revealed that fish peptones were effective (Guerard et al., 2001).
Also, fish viscera peptones from tuna, yellow stripe, swordfish,
rainbow trout and squid were evaluated as growth medium for dif-
ferent types of microorganisms (Pseudomonas, Vibrio and
Roseobacter) that are of interest to aquaculture due to their patho-
genic or probiotic character. The outcomes showed that the effec-
tiveness of such culture media was even better than the
commercial ones (Vázquez, González, & Murado, 2004). In addi-
tion, the potential of peptones from Atlantic cod viscera for growth
of five different microorganisms (Escherichia coli, Bacillus subtilis,
Lactobacillus sakei, Saccharomyces cerevisiae and Aspergillus niger)
was also reported, verifying that it is a promising alternative nitro-
gen source against currently available commercial products
(Aspmo, Horn, & Eijsink, 2005). Peptones obtained from cod stom-
achs showed higher performance than commercial products for the
growth of pathogens like Vibrio anguilarum and Aeromonas
salmonicida (Gildberg, Dahl, Mikkelsen, & Nilsen, 2010). Similarly,
yellowfin tuna viscera hydrolysates were effective for such pur-
poses in Escherichia coli y Staphylococcus aureus (Klompong,
Benjakul, Kantachote, & Shahidi, 2012). It is worth highlighting
that the exploration of new resources is still open because each
peptone has its own characteristics and none by itself can
meet all the requirements of microorganisms during cell culture
(Dufossé, De La Broise, & Guerard, 2001; Herpandi et al., 2011).
 O. Villamil et al. / Food Chemistry 224 (2017) 160–171
On the other hand, seasonal variation influences composition of
biological resources like fish viscera and, as a result, affects the per-
formance of peptones as a culture media, especially for fastidious
microorganisms, such as Lactobacillus sakei (Horn, Aspmo, &
Eijsink, 2007). Likewise the influence of hydrolysis parameters,
such as enzyme used, type and degree of hydrolysis on its aptitude
as culture media was evidenced (Klompong et al., 2012). There are
still questions about the standardization of the raw material as it is
considered a critical point because its composition can vary from
one production batch to another. It is also necessary to investigate
about what degree of hydrolysis is suitable for the growth of cer-
tain microorganisms and how to scale an economical production
of peptones by enzymatic hydrolysis (He et al., 2013; Klompong
et al., 2012).
8. Conclusions
FVPH are promising high added value products due to their rel-
evant nutritional content, bioactive and functional properties that
can be applied in the food and pharmaceutical industries, as well as
other uses as microbial growth media. However, most experiments
about FVPH’s bioactivity have been carried out in vitro and further
investigation about other bioactivities and the performance and
stability of FVPH in food matrices and commercial formulations
are needed. The production and use of FVPH, whether chemical
or biological, is becoming popular as a sustainable alternative,
but there are huge challenges in raw material and product quality
assurance, development of low-cost processes, industrial scaling
and isolation and recovery of peptide sequences that are poten-
tially desirable during food processing and food supplementation.
Addressing these concerns could generate the commercial devel-
opment of FVPH products within the agricultural, cosmetic, phar-
maceutical, food and nutraceutical industry.
Acknowledgements
The authors acknowledge financial support from Research Fund
of Universidad del Tolima (Colombia) and the Administrative
Department of Science, Technology and Innovation of Colombia.
The authors have no conflicts of interest concerning this
research or its funding.
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