Innovative Food Science and Emerging Technologies 37 (2016) 201–215

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Innovative Food Science and Emerging Technologies

journal homepage: www.elsevier.com/locate/ifset

Valorización sostenible de los subproductos del mar: Recuperación del colágeno y desarrollo de
nuevos ingredientes alimentarios funcionales a base de colágeno
Sustainable valorisation of seafood by-products: Recovery of collagen
and development of collagen-based novel functional food ingredients
Gaurav Kumar Pal, P.V. Suresh ⁎
Academy of Scientific and Innovative Research, Meat and Marine Sciences Department, CSIR-Central Food Technological Research Institute, Mysuru 570020, India

Ecosistema marino; reserva sin explotar de compuestos bioactivos, que tienen un potencial
a r t i c l e i n f o a b s t r a cpara
considerable t suministrar nuevos ingredientes para el desarrollo de productos alimenticios
funcionales comerciales.
Article history: Aquatic, especially marine ecosystem is still an untapped reservoir of bioactive compounds, which have consid-
Received 30 October 2015 erable potential to supply novel ingredients towards the development of commercial functional food products.
Received in revised form 8 March 2016 Seafood products are an important part of the diet in many nations. Moreover, as a source of protein, seafood
Accepted 30 March 2016
plays a significant role as functional components that are essential to human health. In industry or local seafood
Available online 9 April 2016
shops, processing of seafood generates a huge quantity (50–80%) of nonedible by-products, which are discarded
Keywords:
as waste or underutilised in several parts of the world. These seafood processing by-products are rich sources of
Seafood various novel and valuable biomolecules such as collagen and gelatin. In this review, scope of seafood by-
By-products products has been explored to recover the realistic collagen. The sustainable valorisation of seafood by-
Collagen products may lead towards the development of healthy and functional food ingredients/products. Furthermore,
Collagen hydrolysates the significant challenges towards the development of collagen-based functional food ingredients are also
Bioactive peptides discussed.
Functional food ingredients Industrial relevance: With the increasing amount of seafood processing by-products worldwide, the recent trend
towards the utilisation of collagen and their derived biomaterials to develop the various functional food and bev-
erages is gaining momentum. Seafood processing by-products are a rich source of bioactive collagen molecules
with potential nutraceutical/functional properties. Seafood processing industries are constantly trying to maxi-
mum utilisation of seafood by-products.
This review article puts forward an alternative use of seafood processing by-products which may help to acceler-
ate their business with significant benefits. The collagen-based novel functional food ingredient contains a nutri-
tional benefit, such as essential and non-essential amino acid to improve the quality of different food products. It
can also be used as natural antioxidants and texturing agents that will reduce the utilisation of chemical preser-
vatives and may be able to fulfil the consumer demands for safe and green food products.
In addition, the current status, challenges and the future directions in the development of seafood derived colla-
gen peptides as functional ingredients is also discussed. This review suggests a biological solution for the disposal
of the seafood processing by-products that creates an environmental pollution issues. This article reveals the op-
portunity to utilise the seafood by-products for the development of high value-added collagen-based functional
food ingredients.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
According to food and agriculture organisation (FAO), the term sea-
food includes a group of diverse edible animals consisting of freshwater
fish, molluscan, shellfish, crustaceans, saltwater finfish, and other forms
of aquatic animal life. Marine animals (such as frogs and turtles) and ed-
ible seaweeds, which are served as food are also considered as seafood
all over the world (FAO, 2015; Suresh, Nidheesh, & Pal, 2015). The sea-
food (by capture fisheries and aquaculture) and its derived products are
the most traded food commodities for global food security and food
⁎ Corresponding author.
E-mail addresses: gauravpal00@gmail.com (G.K. Pal), drsureshpv@cftri.res.in,
drsureshpv@hotmail.com (P.V. Suresh).
trade (Shahidi & Ambigaipalan, 2015; FAO, 2014, 2015; Menon & Lele,
2015; Watson et al., 2015).
According to FAO, more than 158 million tons (MT) of seafood were
landed by capture fisheries or produced by aquaculture in 2012 of
which about 136 MT (81%) were used for direct human consumption
(FAO, 2014). A large quantity of seafood harvested is processed in a dif-
ferent way throughout the world owing to its highly perishable nature,
export potential, and demand for processed and ready-to-eat seafood
products (Suresh & Prabhu, 2013). In general, seafood processing re-
covers only 20–50% as edible portions and the remaining parts (80–
50%) are discarded as “nonedible” by-products /co-products/leftover
raw materials, with an average of 20 MT globally (Suresh & Prabhu,
2013; Pangestuti & Kim, 2014). These seafood by-products are rich
sources of various valuable components including collagen and gelatin,

http://dx.doi.org/10.1016/j.ifset.2016.03.015
1466-8564/© 2016 Elsevier Ltd. All rights reserved.
202 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
protein and peptides, oil and lipids, chitin, vitamins, minerals, enzymes,
pigments, and flavours (Suresh & Prabhu, 2013; Pangestuti & Kim, 2014;
Menon & Lele, 2015). However, a large quantity of non-edible by-
products generated by the industrial processing of seafood is wasted
or underutilised in many parts of the world. Disposal of seafood by-
products may be a costly process in some countries (e.g., in Australia
up to US$150 per tons) (Yan & Chen, 2015). With recent developments
in the biotechnology, there is a vast scope to make better utilisation of
seafood by-products as viable sources for nutraceuticals and other func-
tional ingredients for food and nutrition. The importance of seafood de-
rived functional foods, and functional ingredients have been well
documented to improve/enhance the quality of life and reduce health
care costs (Plaza, Cifuentes, & Ibáñez, 2008; Shahidi, 2009).
This review focuses on the recovery of seafood-derived bioactive
collagen and collagen peptides/hydrolysates reported from seafood
(both finfish and shellfish) and their processing by-products. Current
methods for the extraction, characterisation and purification of
seafood-derived bioactive collagen and their peptides will be outlined.
Furthermore, most relevant existing knowledge of the collagen-based
functional food and realistic challenges towards the development of
collagen-based functional food ingredients are briefly discussed.
2. Need of functional food ingredients from seafood by-products
The sense of happiness and good health maintenance are top prior-
ities for people all over the world. Food and its components may play a
dynamic role to achieve these needs. The development and utilisation of
seafood by-products as functional food ingredients have been accelerat-
ed with growing health benefits knowledge of seafood diet/products
(Pangestuti & Kim, 2014). In recent years, the consumer interest in the
relationship between seafood diet and health has increased significant-
ly. Seafood by-products can be consumed as a portion of food for human
health maintenance (Menon & Lele, 2015). In developed countries, diets
are highly caloric, rich in the saturated fats and sugars. However, the
consumption of complex carbohydrates and dietetic fibre is very low.
As well as, a decrease in physical exercise has given rise in an increase
of obesity, heart diseases, diabetes and hypertension problems (Plaza
et al., 2008). The seafood derived functional food ingredients may create
a positive contribution to health and well-being. These days consumers
preferred the foods that have a significant potential to improve health,
increase longevity, reduce the risk, or delay of the diseases (Harnedy
& FitzGerald, 2012). Consumption of seafood helps to protect against
these lifestyle diseases that pose health challenges globally. The clinical
trials evidence support the health benefits of seafood and their products
consumption is mainly derived from epidemiological studies (Hu et al.,
2002; Lavie, Milani, Mehra, & Ventura, 2009; Mozaffarian & Rimm,
2006).
Seafood by-product represents a leading valuable resource of bioac-
tive materials. The seafood derived products have significant potential
for various applications in food and health sectors such as bioactive
compounds, nutraceuticals, natural food additives, biodegradable pack-
aging, medicinal drugs, and as encapsulation materials (Menon & Lele,
2015). Bioactive substances such as collagen, gelatin, chitosan and
their derived products from seafood by-products have been used in var-
ious biotechnological, nutritional, pharmaceutical, biomedical and other
applications including food, cosmetics, drug delivery, and tissue engi-
neering (Pal, Nidheesh, & Suresh, 2015; Suresh et al., 2015; Nidheesh,
Kumar, & Suresh, 2015; Nidheesh, Pal, & Suresh, 2015). Seafood derived
collagens are suitable and outstanding as ingredients of functional food
due to their nutritive (presence of essential and non-essential amino
acids) and functional properties. In food legislations, there is no restric-
tion on the amount of collagen usage in food items. To complete the ad-
equate nutritionally, and well-balanced diets, functional foods with
seafood collagen can be consumed to fulfil the collagen need (Bilek &
Bayram, 2015; Pal et al., 2015). The antioxidant activity of collagen
can improve the oxidative stability of food products by preventing
oxidation processes. Meat shelf-life can be extended with the applica-
tion of a surface coating of collagen (Antoniewski & Barringer, 2010).
Globally, the major ongoing research is addressed to improve the man-
agement of seafood by-products in terms of searching new bioactive
collagen-based compounds and developing the new technologies that
allow a more cost-effective utilisation (Pangestuti & Kim, 2014;
Tahergorabi & Jaczynski, 2014).
3. Seafood by-products as potential source of collagen
There is a huge controversy to define the seafood ‘non-edible’ mate-
rials leftover after processing. Offal, viscera, waste and by-product terms
are frequently and interchangeably used in the seafood industry and sci-
entific literature to describe the same resources. Therefore, to overcome
the negative connotation and misleading, the seafood materials cannot
be used for any application and should be disposed of is called as ‘offal’
or ‘waste’. However, by-products and co-products term suggest that
there may be some high-value components to be recovered if treated
properly (Tahergorabi & Jaczynski, 2014).
Collagen is the most abundant structural protein found in skin and
bones of all animals and constitutes ~30% of the total protein content.
Collagen is formed as a unique triple helix by three almost identical
polypeptide amino acids chains. Collagen is the product of an almost
continuous repeating of the Gly-X-Y-sequence, where X is mostly pro-
line and Y is hydroxyproline. Presently, genetically distinct 29 types of
collagen (type I-XXIX) with right-handed triple helical conformation
have been identified in animal tissue that differs considerably in its
amino acid composition, sequence, structural and functional properties,
more likely associated with specific genetic variants (Pal et al., 2015;
Gómez-Guillén, Giménez, López-Caballero, & Montero, 2011). Collagen
and hydrolysed form of collagen (gelatin) are presently used in various
sectors such as food, cosmetics, pharmaceutical, tissue engineering and
biomedical (Fig. 2) (Gómez-Guillén et al., 2011; Liang et al., 2014a;
Menon & Lele, 2015; Pangestuti & Kim, 2014; Pal et al., 2015). Currently,
the primary sources of commercial collagen are the skin, bones and
hides of land-based animals (bovine and porcine). In recent decades,
the production of land-based animal's collagen has been decreased in
part due to the concerns about outbreaks of bovine spongiform enceph-
alopathy (BSE), foot mouth disease (FMD) and other prions disease. In
addition, use of mammalian collagen is a hurdle in the development of
kosher and halal products due to some religious factors (Wang et al.,
2014a). Seafood by-products contain a remarkable amount of skin,
scale, fins, bone, swim bladder, which have been recognised as a poten-
tial source of collagen and other biomaterials (Suresh & Prabhu, 2013;
Pal et al., 2015) (Fig. 1). The production of collagen from seafood by-
product is expected to attract the interest of the industry as a natural al-
ternative source (Pal et al., 2015; Regenstein & Zhou, 2007;
Gómez-Guillén et al., 2011). Seafood derived collagens are grouped
into three types: fish collagens, collagens of invertebrate origin (jelly-
fish, sponges, mollusks), and marine mammal collagens (Ehrlich, 2015).
Further, collagen is the raw material for the preparation of gelatin.
Gelatin is a heterogeneous mixture of water-soluble high molecular
weight protein that does not have existence in nature. Gelatin is the par-
tially hydrolysed form of collagen. They are derived from the parent
protein collagen. For the production of gelatin, the polypeptide chains
of collagen triple helix structure cross-linking are disrupted using a min-
imal level of polypeptide bond breakage. Heat denaturation efficiently
converts the high-value collagen into gelatin. Gelatin is an important in-
dustrial biopolymer possesses the functional characteristics and gelling
properties including encapsulating, colloid stabilisation, crystallisation,
thickening, whipping, water holding, film formation, texture enhancer
and emulsification that make it useful for their potential application in
food, pharmaceutical, and other allied sectors (Regenstein & Zhou,
2007; Karim & Bhat, 2009; Kim, Mendis & Shahidi, 2007; Pangestuti &
Kim, 2014). Seafood derived gelatin releases a kind of aroma, flavour
and shows higher digestibility compare to animal gelatin. The functional
 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215 203
Huesos

Aletas

Escamas

Vejiga natatoria
Piel
Fig. 1. Major seafood processing by-products sources for collagen extraction.

and biochemical properties of seafood derived gelatin are similar to an-
imal gelatin (Regenstein & Zhou, 2007; Karim & Bhat, 2009;
Gómez-Guillén et al., 2011; Pangestuti & Kim, 2014).
4. Methods used for collagen extraction
Collagen extraction from seafood sources consists of two main steps,
viz. pre-treatment of raw materials and extraction of collagen. Raw ma-
terials have been prepared by various steps such as cleaning, size reduc-
tion, and pre-treatment, subsequent to extraction. The cleaning and size
reduction of raw material is performed to facilitate the contaminant re-
moval and maximum collagen extraction. Before pre-treatments, the
raw materials are separated from the group of different by-product ma-
terials such as skin, scale, bone, and swim bladder. The pre-treatment of
seafood by-products might be applied before extraction of the collagen
to remove impurities as well as to increase the quality of the final ex-
tracted collagen. Commonly, the raw material used for collagen prepa-
ration contains non-collagenous proteins, lipids, pigments, and so on.
Calcium or other inorganic materials were also found in bone and
scale of the seafood species (Regenstein & Zhou, 2007). Alkaline pre-
treatment with 0.1 M NaOH is a widely used method to remove the
noncollagenous proteins and pigments from the seafood by-products
(Pal et al., 2015; Benjakul, Nalinanon, & Shahidi, 2012). The raw mate-
rials can be effectively demineralised to remove calcium or other inor-
ganic materials using ethylenediaminetetraacetic acid (EDTA)
(Benjakul et al., 2012; Regenstein & Zhou, 2007). Finally, the seafood
raw materials devoid of non-collagenous proteins and minerals can be
defatted with butyl alcohol treatment. Flow chart for the preparation
and pre-treatment of seafood raw materials are shown in Fig. 3.
There are a number of methods reported for collagen extraction.
Based on the difference in extraction processes, collagen can be divided
into salt-soluble collagen (SSC), acid soluble collagen (ASC), pepsin sol-
uble collagen (PSC) and ultrasound assist collagen (UAC) (Fig. 4). The
extraction yield and properties of collagen are directly affected by the
collagen extraction method. Therefore, all procedures must be per-
formed at low temperature (~4 °C) to avoid the degradation of collagen

Fig. 2. Potential applications of the seafood derived collagen in various sectors.

204 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
Fig. 3. Outline of a process for pre-treatment of seafood processing by-products.
(Regenstein & Zhou, 2007). The conditions used by several research
groups for the extraction of collagen using salt, acid, enzyme and ultra-
sound from different sources are summarised in Table 1.
4.1. Salt solubilisation extraction
Collagen from seafood sources can be extracted using NaCl solution,
and the resultant collagen was referred as salt-solubilised collagen
(SSC). However, the salt solubilisation method has been rarely used
for collagen extraction. Extraction of SSC from skin and cartilage of
Amur sturgeon (Acipenser schrenckii) have reported using 0.45 M NaCl
at the ratio of 1:100 (w/v) for 24 h with continuous gentle stirring
(Liang et al., 2014b; Wang et al., 2014b).
4.2. Acid solubilisation extraction
The acid solubilisation method has been extensively used for the col-
lagen extraction from different seafood by-products. The collagen from
seafood by-products can be extracted using an acidic solution (most
commonly 0.5 M acetic acid), and the resultant collagen was referred
as acid-soluble collagen (ASC). In acidic conditions, collagen molecules
have the dominance of positive charge. Therefore, the enhanced
Fig. 4. Methods used for extraction of seafood derived collagen and their partial
purification.
repulsion among tropocollagen can be achieved which may be lead to
the increased solubilisation. Extraction of collagen from different sea-
food species has been successfully achieved using acetic acid (Liang
et al., 2014a, 2014b; Pal et al., 2015; Wang et al., 2014a, 2014b,
2014c). In addition to acetic acid, collagen can also be isolated with
other organic acids such as chloroacetic acid, citric acid, lactic acid or in-
organic acid such as hydrochloric acid (Benjakul et al., 2012; Regenstein
& Zhou, 2007).
Various researchers reported the isolation/extraction of collagen
using acetic acid from seafood derived by-products such as skin (Pal
et al., 2015; Wang et al., 2014a, 2014b, 2014c), scales (Chuaychan,
Benjakul, & Kishimura, 2015; Matmaroh, Benjakul, Prodpran,
Encarnacion, & Kishimura, 2011), swim bladder (Wu et al., 2015), carti-
lage (Liang et al., 2014a, 2014b), bone (Duan, Zhang, Du, Yao, & Konno,
2009), fins (Mahboob, 2015), skeletal and head bone (Jeevithan, Wu,
Nanping, Lan, & Bao, 2014) etc. Some authors reported that yield of ex-
tracted collagen depends on the several factors such as species, age and
parameters used for the extraction include acid concentration, the ratio
of acid solution and raw materials, extraction temperature and time
(Regenstein & Zhou, 2007; Pal et al., 2015). The seafood derived collagen
extraction was usually achieved at 4 °C for 24–48 h. Although an in-
crease in concentration and proportion of acid, as well as extraction
temperature and time, can offer a higher yield of collagen. It may not
be desirable due to collagen degradation (Regenstein & Zhou, 2007;
Pal et al., 2015). Benjakul et al. (2012) stated that several sequential ex-
tractions of collagen give better ASC yield as compared to extending the
extraction time.
 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215 205

Table 1 2015; Jeevithan, Wu, Nanping, Lan, & Bao, 2014; Veeruraj,
Extraction conditions and yield of seafood derived collagens at low temperature (4 °C). Arumugam, & Balasubramanian, 2013; Tamilmozhi, Veeruraj, &
Raw materials Method Time Yield References Arumugam, 2013).
(h) (%) The extraction of collagen with the aid of limited amount of enzyme
Amur sturgeon cartilage SSC 24 × 5 2.18 Liang et al. (2014a, 2014b) (pepsin) is a potential method for several reasons: a) pepsin can hydro-
ASC 24 × 2 27.04 lyse the noncollagenous proteins, and the noncollagenous proteins hy-
PSC 48 55.92 drolysate can be easily removed using the salt precipitation and
Amur sturgeon Skin SSC 24 × 6 4.55 Wang et al. (2014a, 2014b,
dialysis to improve the purity of collagen; b) hydrolyse the telopeptides
ASC 24 × 2 37.42 2014c)
PSC 48 52.80 of the collagen to make it readily solubilise in the acid solution that may
Catla Skin ASC 72 63.40 Pal et al. (2015) be resulting in improvement of the extraction efficiency; c) pepsin di-
PSC 48 69.53 gestion may reduce the antigenicity caused by telopeptide in the colla-
Rohu Skin ASC 72 46.13 gen that serve a significant problem in food and pharmaceutical
PSC 48 64.94
Seabass scales ASC 48 0.38 Chuaychan et al. (2015)
applications (Duan, Zhang, Du, Yao, & Konno, 2009; Matmaroh,
PSC 48 1.06 Benjakul, Prodpran, Encarnacion, & Kishimura, 2011; Benjakul et al.,
Spotted golden ASC 48 0.46 Matmaroh et al. (2011) 2012; Wang et al., 2014a, 2014b, 2014c).
goatfish scale PSC 48 1.20
Carp skin ASC 72 41.3 Duan et al. (2009)
4.4. Ultrasound-assisted extraction
Carp scales ASC 96 1.35
Carp bone ASC 72 1.06
Catla skin ASC 24 5.8 ⁎Mahboob (2015) Ultrasonic treatments (20 kHz with amplitude of 20–80%) have used
PSC 48 7.2 to improve the yield of collagen from seafood by-products by few re-
Catla scales ASC 24 3.9 search groups (Kim, Kim, Park, & Lee, 2013; Ran & Wang, 2014; Yang,
PSC 48 5.6
Guo, Yu, & Li, 2013). In an ultrasound-assisted extraction process, pre-
Catla fins ASC 24 6.7
PSC 48 8.8 treated seafood by-product samples were soaked in acetic acid for
Mrigala skin ASC 24 4.7 12 h. Subsequently, the collagen was extracted using an ultrasonic pro-
PSC 48 6.5 cessor for ultrasonic treatment. It was reported that the yield of collagen
Mrigala scales ASC 24 3.2
depends on the amplitudes and duration of ultrasonic treatment. Kim,
PSC 48 5.1
Mrigala fins ASC 24 5.7
Kim, Park, and Lee (2013) reported improved yields of collagen from
PSC 48 7.7 sea bass skin as compared with the traditional extraction process. Fur-
Silvertip shark skeletal PSC 96 – Jeevithan et al. (2014) ther, it was reported that there were no changes in the main compo-
and head bone nents of collagen after the ultrasonic treatment. Therefore, the
Marine eel-fish skin PSC 72 80 Veeruraj et al. (2013)
ultrasonic-assisted method could offer promising practical applications
Sailfish skin ASC 72 5.76 Tamilmozhi et al. (2013)
PSC 72 2.11 in the industrial production of collagen (Kim et al., 2013; Ran & Wang,
Grass Carp skin ASC 72 25.5 Wang et al. (2014a, 2014b, 2014; Yang, Guo, Yu, & Li, 2013). More details of ultrasound-assisted ex-
PSC 72 19.8 2014c) traction are described in Section 9.
Grass Carp scale ASC 72 16.7
PSC 72 16.1
Grass Carp bone ASC 72 0.7
5. Recovery of seafood derived collagen
PSC 72 3.5
Seabass skins UAC 24 17.97 Kim et al. (2013) After extraction process, the collagen is usually recovered using the
Cattle tendon UAC 48 2.4 Ran and Wang (2014) salt precipitation, centrifugation, dialysis, and freeze-drying. Generally,
UPAC 45 2.7
collagen solution is precipitated using 2.6 M NaCl in the presence of
Yak bovine tendons UAC 24 31.04 Yang, Guo, Yu, and Li (2013)
tris(hydroxymethyl)aminomethane at pH 7.5. The NaCl concentrations
ASC = Acid soluble collagen; PSC = Pepsin soluble collagen; UAC = Ultrasound assist col-
used for collagen precipitation can be adjusted to maximise the collagen
lagen extraction; UPAC = Ultrasound-pepsin assist collagen extraction.
⁎ Wet weight basis. recovery and removal of impurities. Centrifugation (usually 10,000–
20,000 rpm; 4 °C; 60 min) is used to collect the precipitated collagen.
The resultant precipitate is dissolved in a minimal volume of acetic
4.3. Enzymatic solubilisation extraction acid with prior to dialysis against distilled water. The dialysate is finally
freeze-dried, and the obtain collagen powder was stored at 4 °C (Pal
Traditionally, collagen is extracted using an acidic (acetic acid) et al., 2015; Benjakul et al., 2012).
solution without the aid of an enzyme. However, the collagens
from the by-products of seafood species were not entirely dissolved 6. Physiochemical characteristics of seafood derived collagen
in acetic acid solution (Liang et al., 2014a, 2014b; Wang et al., 2014a,
2014b, 2014c; Pal et al., 2015). Due to the lower yield of extracted Collagens from different seafood sources have varying properties,
collagen obtained from the acidic process, the enzymatic extraction especially thermal denaturation susceptibility and cleavage by animal,
process has been developed to maximise the collagen yield, and re- plant and microbial proteases. It may affect the seafood derived colla-
sultant extracted collagen is called as enzymatically extracted colla- gens properties that directly affected collagen applications in food and
gen. Various enzymes such as trypsin, pepsin, and collagenase have other allied sectors (Pal et al., 2015). Collagen from the scale and bone
been used to maximise the yield of collagen (Benjakul et al., 2012; of seafood species showed the almost similar characteristics. Most of
Regenstein & Zhou, 2007). Pepsin is most widely used enzyme in the collagens reported from seafood by-products are types I collagen
the extraction of collagen from seafood derived materials. Pepsin and type V also reported in minor quantity based on its electrophoretic
can be used either in combination with an acetic acid or alone after mobility (Benjakul et al., 2012). Duan et al. (2009) stated that the mo-
initial extracting of collagen within acetic acid. The resultant pepsin lecular weight of collagen had some relations with the thermal stability.
extracted collagen was called as pepsin soluble collagen (PSC). The The collagen with higher molecular weight may have greater thermal
several researchers showed that use of limited pepsin to extract col- stability. The imino acid content also plays a significant role in thermo-
lagen from seafood by-products increased the yield of collagen stability of collagen. However, seafood derived collagens have the lower
(Liang et al., 2014a, 2014b; Wang et al., 2014a, 2014b, 2014c; Pal imino acid content. Therefore, these seafood by-product derived colla-
et al., 2015; Mahboob, 2015; Chuaychan, Benjakul, & Kishimura, gens are less stable compared to mammalian collagen. One of the
206 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
primary and simplest ways to analyse the extracted seafood derived col-
lagen is the wave scan of the collagen sample from 200 to 400 nm (Pal
et al., 2015; Duan et al., 2009). However, triple helical collagen has the
maximum absorbance peak at 230–235 nm (Pal et al., 2015). Most sea-
food by-product derived collagens exhibited a maximum absorbance
peak at 230–240 nm with a little absorption peak at 280 nm. The absor-
bance at 280 nm is mainly because of tryptophan, tyrosine, and phenylal-
anine. The FTIR spectra of extracted collagen from the by-products of
seafood species exhibited a characteristic peak of amide I, II, III and
Amide A, B. The presence of these amide bonds confirming that collagen
has well maintained triple helical structures. These are the major spectral
peak locations of collagen. Thermal stability of collagen mainly linked
with pyrrolidine rings of both proline and hydroxyproline and partially
with hydrogen bonding of hydroxyl group of hydroxyproline (Pal et al.,
2015; Matmaroh et al., 2011). Collagen containing small concentrations
of both imino acids have denatured at a lower temperature compare to
those with the larger concentrations. The high content of imino acid need-
ed for stabilisation of collagen (Benjakul et al., 2012; Pal et al., 2015). Fur-
thermore, the thermal stability of collagen not only depends on the imino
acid content but is also directly correlated with body temperatures of fish
species and their habitat temperatures (Matmaroh et al., 2011; Duan
et al., 2009; Minh Thuy, Okazaki, & Osako, 2014).
Some factors have been known to influence the collagen properties.
pH and salt concentration were reported to affect water binding capac-
ity, viscosity, and emulsifying properties of collagen from seafood mus-
cle and skin connective tissues (Benjakul et al., 2012). The solubility
characteristics of collagens with changes in pH and NaCl concentrations
may play a crucial role in their extraction and application in various sec-
tors (Pal et al., 2015; Duan et al., 2009; Matmaroh et al., 2011; Veeruraj,
Arumugam, & Balasubramanian, 2013; Tamilmozhi, Veeruraj, &
Arumugam, 2013). Pal et al. (2015) reported that maximum solubility
of collagen from Indian major carp's catla and rohu were observed in
the presence of 0.4 M NaCl concentrations. Freeze-drying may cause
the reduction in solubility and emulsifying capacity (Benjakul et al.,
2012).
7. Development of the seafood derived collagen peptides and
hydrolysate
Seafood sources have gained much attention as potential bioactive
collagen hydrolysate/peptides. It is mostly due to the abundance of
underutilised seafood processing species and by-products as raw mate-
rials (Suresh & Prabhu, 2013; Samaranayaka & Li-Chan, 2011; Kim &
Wijesekara, 2010). Seafood by-product derived collagen peptides, and
essential amino acids are specific protein fragments that play a signifi-
cant role to maintain health, immune systems and to prevent the car-
diovascular, nervous diseases. These collagen peptides are inactive
within the parent collagen polypeptide sequences. They become active
upon release from the parent sequences. They usually contain 3–20
amino acid residues, and their activities are based on their amino acid
Fig. 5. Chemical and biological methods used for collagen peptide generations.
sequence and composition (Menon & Lele, 2015; Pangestuti & Kim,
2014). Several bioactive collagen peptides can be released from differ-
ent seafood derived collagen and gelatin during degradation/hydrolysis
using exogenous and/or endogenous enzymes, food processing or dur-
ing microbial fermentation, as well as during gastrointestinal digestion
of seafood derived proteins (Suresh & Prabhu, 2013; Samaranayaka &
Li-Chan, 2011; Kim & Wijesekara, 2010; Liu, Nikoo, Boran, Zhou, &
Regenstein, 2015). However, commercially available collagen derived
hydrolysates/peptides are produced mainly by chemical (acid-alkaline
hydrolysis) and enzymatic hydrolysis of collagen or gelatin (Fig. 5).
7.1. Chemical methods
Chemical methods of collagen hydrolysis are classified into two
groups: acid and alkali hydrolysis methods. Acid-alkaline hydrolysis
methods are cost effective and simple operative. They have short hydro-
lysis time and are applicable to industrial processes. However, the uses
of strong acids or strong base make the hydrolysis process ecologically
unacceptable and reduce the nutritional qualities of the products
(Anal, Noomhorm, & Vongsawasdi, 2013).
Acid hydrolysis process is usually carried out with hydrochloric acid
(HCl) under high-temperature conditions (at 110–120 °C) and over a
long period (18–48 h). 6 M concentration of HCl is frequently used to in-
creases the rate of peptide bond cleavage. Hydrolysate product pre-
pared by this process has a highly acceptable sensory profile (Anal,
Noomhorm, & Vongsawasdi, 2013). Alkaline hydrolysis process uses a
strong alkali (sodium hydroxide or potassium hydroxide) in water at
high temperatures (130–180 °C). However, this process may destroy
some of the amino acids such as serine, cysteine, histidine and threonine
(Anal et al., 2013).
7.2. Biological methods
Bioactive collagen peptides/hydrolysate can be released from hydro-
lysis of seafood derived collagen using exogenous and/or endogenous
enzymes, food processing or during microbial fermentation, as well as
during gastrointestinal digestion (Suresh & Prabhu, 2013;
Samaranayaka & Li-Chan, 2011; Kim & Wijesekara, 2010; Anal et al.,
2013; Liu, Nikoo, Boran, Zhou, & Regenstein, 2015). However, most
commonly used commercial method is an enzymatic hydrolysis
(Samaranayaka & Li-Chan, 2011; Anal et al., 2013).
Enzymatic hydrolysis is widely used to improve the functional and
nutritional properties of seafood derived proteins (Liu et al., 2015;
Kim & Wijesekara, 2010). Enzymatic hydrolysis has been the leading
process for the production of bioactive collagen hydrolysate/peptides
from seafood derived collagen (Samaranayaka & Li-Chan, 2011; Kim &
Wijesekara, 2010). During enzymatic hydrolysis of seafood derived col-
lagen and gelatin, the α and β polypeptides chains are gradually degrad-
ed into smaller peptides (molecular weight range of 1–4 kDa) with a
higher degree of hydrolysis (Liu et al., 2015).
 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
Commercial proteolytic enzymes from microbes such as food-grade
(alcalase, flavourzyme, protamex, etc.) and non-food grade (collagenase,
proteinase K, metalloproteases, serine-protease), plants (papain, brome-
lain, ficin) and animal (α-chymotrypsin, neutrase, trypsin, etc.) have
been widely used for the hydrolysis process of seafood derived collagen
to produce collagen peptides/hydrolysate (Kim & Wijesekara, 2010; Liu
et al., 2015; Samaranayaka & Li-Chan, 2011; Mora, Reig & Toldra, 2014;
Lafarga & Hayes, 2014). Use of exogenous enzymes was preferred due
to better control of hydrolysis and shorter time required to obtain the op-
timum degree of hydrolysis with more consistent molecular weight pro-
files of collagen peptides (Samaranayaka & Li-Chan, 2011). For the
preparation of seafood derived collagen peptides with desirable function-
al and bioactive properties, careful choice of a suitable enzyme and
physiochemical conditions of enzymatic reaction such as protein solution
pH, temperature, hydrolysis time and enzyme concentration used should
be optimise for the maximum activity (Kim & Wijesekaraa, 2010;
Samaranayaka & Li-Chan, 2011). The molecular weight of bioactive pep-
tide is one of the most important factor to produce bioactive peptides
with desired functional properties. A suitable method is required to obtain
the bioactive seafood derived collagen peptides with a desired molecular
size and functional properties (Kim & Wijesekaraa, 2010; Liu et al., 2015;
Samaranayaka & Li-Chan, 2011). The molecular weight distribution of the
collagen hydrolysates/peptides depends on collagen sources, enzyme
types, and hydrolysis conditions (Liu et al., 2015).
8. Separation and purification of collagen hydrolysate/peptides
A large number of bioactive peptides are generated during the enzy-
matic hydrolysis of collagen, but only a few have a positive health pro-
moting and functional properties. When the desirable and optimum
Fig. 6. Isolation, characterisation and biological activity assays of collagen peptides from seafood processing by-products.
207
degree of hydrolysis is over, the hydrolysed product was submitted to
partial purification and fractionation through various filtration/chro-
matographic methods (Mora, Reig & Toldra, 2014). Collagen hydrolysis
reaction with specific commercial proteases is usually carried out in
batch-fed or continuous reactors. After hydrolysis, it is necessary to frac-
tionate and purify the mixture of peptides for bioactive peptides isola-
tion. The most common methods used for peptide purification include
ultrafiltration, gel filtration chromatography, reversed-phase chroma-
tography, ion exchange chromatography, and size exclusion chroma-
tography (Fig. 6). Ultrafiltration method is one of the most preferred
method for efficient recovery and purification of seafood derived colla-
gen hydrolysis products (Cheung, Ng, & Wong, 2015). Membrane filtra-
tion method can be used to concentrate and separate according to the
different molecular weights of collagen hydrolysate or peptides. The
molecular weight distribution of the desired peptide can be controlled
using an appropriate ultrafiltration membrane (Kim & Wijesekara,
2010). Recently, membrane bioreactor equipped with ultrafiltration
membranes is emerging technology for the development of seafood de-
rived bioactive peptides/compounds. It is considered as a potential
method to utilise the seafood proteins as a value-added functional
food ingredients/nutraceuticals (Kim & Wijesekara, 2010).
Reversed-phase-high-performance liquid chromatography (RP-
HPLC) has been extensively used in the characterisation of seafood de-
rived collagen peptides. However, liquid chromatography (LC)-mass
spectrometry (MS) has simplified and advanced the identification of
peptides separated by HPLC and is now one of the preferred character-
isation methods (Lafarga & Hayes, 2014). Electrospray ionisation (ESI)
and matrix-assisted laser desorption/ionisation (MALDI) mass spectros-
copies are the most common methods used for peptide and protein
identification. However, these methods can be economically prohibitive
208 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
as scale-up costs are expensive (Lafarga & Hayes, 2014). Individual col-
lagen peptide fractions can be identified using the combined techniques
of mass spectrometry and protein.
9. Emerging methodologies in the isolation/extraction and
purification of seafood collagen and collagen derivatives
Utilisation of conventional methods for isolation/extraction and
purification of bioactive polymers/molecules are often restricted by
several problems that are difficult to overcome. In recent years, iso-
lation and purification methodologies have been continuously
established towards reducing solvent/reagent consumption, short-
ening processing times, and increasing isolation efficiency, with an
objective of reducing expenses and environmental hazards (Huang,
Hsu, Yang, & Wang, 2013). Many approaches are already under ac-
tive investigation, including ultrasonic extraction, supercritical
fluid extraction, microwave-assisted extraction, and high-pressure
extraction (Mustafa & Turner, 2011). Most of these methods require
some degree of heating, which could easily lead to thermo-sensitive
ingredients for losing their biological activities or transforming/
degrading into other substances (Huang, Hsu, Yang, & Wang,
2013). In addition, many of these methods are not suitable for use
with heat-sensitive seafood origin bioactive collagen and collagen
peptides. Few works reported by some research group Gomez-
Guillen, Gimenez, & Montero (2005) and Kim, Kim, Kim, Park, &
Lee (2012), on this direction briefly described below.
9.1. Ultrasound-assisted extraction
Ultrasounds are the high-frequency sound waves (20 kHz) that
exceed the frequency hearing capacity of the human ear. Ultrasound
waves principally act by generating bubble cavitation in biological
medium and efficient alternative to traditional methods (Kadam,
Tiwari, & O′Donnell, 2013; Kadam, Tiwari, Alvarez, & O′Donnell,
2015). Ultrasound is a novel, simple, green, safe, rapid and econom-
ically viable technology that is suitable for scale up, extraction, pro-
tein digestion, production and drug delivery of seafood derived
bioactive peptides (Galanakis, 2012; Kadam, Tiwari, & O'Donnell,
2013). It facilitates the extraction of heat sensitive compounds with
minimal damage and higher yield. Equipment costs are lower than
other emerging methodologies. However the sound wave amplitude
controlling with distance is a difficult task (Kadam et al., 2013). The
applications of ultrasound for the extraction, production and deliv-
ery of bioactive seafood based proteins and peptides were recently
reviewed (Kadam et al., 2013; Kadam, Tiwari, Álvarez, & O'Donnell,
2015). Ultrasound-assisted encapsulation of peptide-based drugs
with biodegradable polymers (eg. chitosan, gelatin) can improve sta-
bility and bioavailability. Moreover, in sonophoresis applications,
low-frequency ultrasound can be used to transport high molecular
weight peptide drugs (Kadam et al., 2015). Ultrasound extensively
reported for extraction of bioactive compounds from natural prod-
ucts (Kadam et al., 2013; Kadam et al., 2015).
Kim, Kim, Kim, Park, and Lee (2012) were investigated the effects of
ultrasonic treatment on acid soluble collagen extraction yield from
seabass (Lateolabrax japonicas) skins. It was reported that the yield of
collagen increased rapidly at higher amplitudes of ultrasonic treatment
(Kim et al., 2012). The ultrasonic method of collagen extraction requires
a less amount of acid and a short time for extraction (Kim et al., 2013).
However, these studies confirmed the structural stability of extracted
collagen on the basis of SDS-PAGE analysis. The digestion patterns of
collagen with pepsin enzyme could not be able to confirm the stability
of the collagen. Therefore, ultrasonic treatment may be lead to the struc-
tural damage of extracted collagen. It may also be converted in the de-
nature form of collagen.
9.2. Pressurised liquid extraction
Pressurised liquid extraction is also known as high-pressure solvent
extraction, pressurised fluid extraction, accelerated solvent extraction,
and enhanced solvent extraction. This procedure is based on the use
of solvents at high temperatures and pressure (50–200 °C and 3.5–
20 MPa). During the extraction process, extracted solvents are main-
tained in the liquid form. Sometimes, water is used as an extraction sol-
vent; then this method is called as subcritical water extraction,
superheated water extraction, pressurised hot-water extraction
(Kadam et al., 2013; Herrero, del Pilar Sánchez-Camargo, Cifuentes, &
Ibáñez, 2015; Marcet, Álvarez, Paredes, & Díaz, 2016). This is a faster
process that requires a low amount of solvent compared to traditional
extraction methods. Pressurised liquid applications for the extraction,
identification and delivery of food proteins and bioactive peptides
reviewed recently (Herrero et al., 2015; Marcet et al., 2016).
Gomez-Guillen et al. (2005) reported that extraction of gelatin from
the fish skin by high pressure is a useful alternative to conventional pro-
cedures. This method was able to drastically reducing the longest part
process duration and also producing a high-quality gelatin. However
there in no report so far the extraction of the collagen using these
technologies. It may be due to the high temperature and pressure
employed in these technologies. The higher temperature used during
these extraction process could lead to the transformation of collagen
into gelatin.
10. Biological potential of seafood derived collagen
The seafood derived collagen has various applications in food, phar-
maceutical, biomedical, leather, biomaterials and cosmetic industries.
Further, seafood derived collagen is the most favourable biomaterial
for tissue engineering purpose due to its excellent biocompatibility, bio-
degradability, high cell adhesion properties and weak antigenicity
(Bilek & Bayram, 2015; Pal et al., 2015; Yamada, Yamamoto, Ikeda,
Yanagiguchi, & Hayashi, 2014). In food, seafood derived collagen is
used as a functional as well as nutritional ingredients towards the devel-
opment of health promotion foods. In recent years, the demand for food
enriched with seafood derived collagen is growing due to the improve-
ment in the nutritive and functional properties of the foodstuffs and
their health benefits (Bilek & Bayram, 2015). Collagen is required for
the health development. In the older human body, synthesis of collagen
will decrease. The best alternative to gain collagen is through collagen
containing food products. Seafood derived collagen supplements are
proposed to complete the collagen requirement of the body. Therefore,
seafood collagens have been used in a variety of foodstuffs and bever-
ages products. Seafood-derived collagen was suggested for applications
in functional food, drink and beverage, dietary supplements, and con-
fectionery (Antoniewski & Barringer, 2010; Bilek & Bayram, 2015;
Hashim, Ridzwan, Bakar, & Hashim, 2015; Regenstein & Zhou, 2007).
Seafood collagen-containing drinks are a trend in the global market
(Bilek & Bayram, 2015; Regenstein & Zhou, 2007).
Collagen can be used to produce edible sausage casing. Seafood de-
rived collagens are also used as food additives to improve the rheologi-
cal properties of sausages. Seafood derived collagen films have the
ability of water and oxygen barrier that helps to protect against the mi-
gration of oxygen, moisture and providing structural integrity to food
items. Collagen films or coatings could function as carriers of active sub-
stances. Moreover, edible seafood derived collagen surface coating can
be used to extend the shelf life of meat food products. Collagen coatings
are used as a barrier in meat products to reduce purge, aroma deteriora-
tion, colour deterioration, spoilage, improve sensory scores, and can act
as an antioxidant (Antoniewski & Barringer, 2010; Gómez-Guillén et al.,
2011; Olsen, Toppe, & Karunasagar, 2014).
Different types of seafood derived collagen-based biomaterials such
as gels, scaffolds, sponges, films, membranes, and composites is fre-
quently used in medical and pharmaceutical industries as a carrier for
 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
proteins, genes, and drugs (Lee, Singla, & Lee, 2001; Ehrlich, 2015). Sea-
food collagen is promising drug carrier for cancer treatment (Kim &
Mendis, 2006). The main clinical and experimental applications of colla-
gen include wound healing, treatment and prophylaxis of bone and soft
tissue infections. Collagen is used as efficient drug carriers for controlled
release of drug concentration at target sites. In addition, collagen film
may be used as gene delivery agents promoting cartilage and bone for-
mation. It has also been reported that collagen plays a role in the forma-
tion of tissues, organs and cell expression (Ehrlich, 2015; Kim & Mendis,
2006; Lee, Singla, & Lee, 2001). Collagen also contributes to the promo-
tion of cell growth and differentiation and the regulation of various cell
functions (Yamada, Yamamoto, Ikeda, Yanagiguchi, & Hayashi, 2014).
11. Biological potential of seafood derived collagen hydrolysates/
peptides
Research findings are indicating an array of the biological activities
for seafood derived collagen hydrolysates/peptides (Fig. 7). They have
several biological activities such as antihypertensive, immunomodula-
tory, neuroactive, antimicrobial, mineral and hormonal regulating prop-
erties (Liang et al., 2014a, 2014b; Gómez-Guillén et al., 2011; Wang
et al., 2013; Chi et al., 2014; Guo, Hong & Yi, 2015a; Zhuang et al.,
2009a, 2009b; Zhuang, Zhao & Li, 2009b). Clinical investigations suggest
that ingestion of collagen hydrolysates reduces pain in osteoarthritis
suffering patients. Hydrolysed collagen has also been involved in carti-
lage matrix synthesis (Kim & Mendis, 2006).
11.1. Antioxidant activity
Oxidation is a vital process, particularly in vertebrates and humans.
This process leads to the formation of free radicals. The reactive oxygen
species (ROS) formation is an unavoidable consequence of the body's
normal use of oxygen during respiration. It includes superoxide anion
Fig. 7. Biological activities of the seafood derived collagen peptides.
209
radicals, hydroxyl radicals, and non-free radical species (hydrogen perox-
ide and singlet oxygen radicals) (Najafian & Babji, 2012). Oxygen radicals
are a reactive molecule that can react with every cellular component and
give rise to functional and morphological disturbances in cells. It is evi-
dent that oxygen radicals are significant pathological mediators in various
human diseases and ageing processes, such as skin ageing, osteoarthritis,
osteoporosis, rheumatoid arthritis, hair and nail damage and others
(Najafian & Babji, 2012; Liang et al., 2014a, 2014b). Antioxidants are
widely used to reduce the risk associated with oxidation reaction.
Food deterioration has been linked to the oxidation of lipids and the
formation of undesirable secondary lipid peroxidation products. Antiox-
idants are widely used in food to reduce the deterioration due to oxida-
tion of lipids. Butylated hydroxyanisole (BHA), tert-butyl hydroquinone
(TBHQ), butylated hydroxytoluene (BHT), and propyl gallate (PG) are
the mostly used synthetic antioxidants in food and pharmaceutical in-
dustries. However, the use of synthetic antioxidants is limited due to po-
tential health issues. Hence, there is an increasing attention to the
replacement of synthetic antioxidants with natural antioxidants
(Najafian & Babji, 2012). Recently, the antioxidant activity of seafood
derived collagen peptides has been widely demonstrated in different
oxidative systems (Gómez-Guillén et al., 2011). The collagen peptides
isolated from Jellyfish (Rhopilema esculentum), exhibited the antioxi-
dant activity in a linoleic acid emulsion system (Zhuang et al., 2009a,
2009b; Liang et al., 2014a, 2014b). Collagen peptides obtained by hydro-
lysis of acid soluble collagen from the scales of croceine croaker
(Pseudosciaena crocea) showed strong scavenging activities on hydrox-
yl, DPPH, superoxide and ABTS radicals scavenging activity. These colla-
gen peptides were also effective in lipid peroxidation model system
(Wang et al., 2013). Acid and pepsin soluble collagens derived from
the scale and swim bladder of Indian major carp catla and rohu have
the antioxidant activities (unpublished Data). Numbers of studies
have shown that seafood derived collagen hydrolysate/peptides act as
potential antioxidants (Table 2).
210 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215

11.2. Metal chelating ability
Dietary mineral (calcium, zinc and iron) deficiencies can lead to nu-
merous diseases that have adverse effects on health (Guo et al., 2015a,
2015b, 2015c). Iron and calcium deficiency are the most concerning de-
ficiency problems in the world. Iron deficiency can induce microcytic
hypochromic anaemia, impaired physical activity and endurance in
adults and cognitive impairment in children. An adequate amount of
calcium was required for the development of a child's bones and
teeth. Insufficient calcium intake may increase the risk of osteoporosis
(Guo, Harnedy, Zhang, et al., 2015c; Huang, Wu, Yang, Li, & Kuo,
2015). For health considerations and dietary preferences, nutritional
scientists encourage consumers to choose metal elements enriched
foods rich in their daily nutritional supplement diets. Seafood derived
collagen peptides could be easier to absorb directly into human body
due to lower molecular weight. These collagen peptides showed the sig-
nificant capacity in incorporating with divalent ions (calcium, iron
ions). The chelating complex chelated between collagen peptides, and
calcium ion can promote calcium absorption in the human body, there-
fore, improve the bioavailability of divalent ions (Guo et al., 2015a,
2015b, 2015c). Mineral chelating collagen peptides have been
recognised as potential functional food ingredients to improve mineral
bioavailability (Guo, Harnedy, O'Keeffe, et al., 2015b; Guo, Harnedy,
Zhang, et al., 2015c). Guo et al. (2015a, 2015b, 2015c) reported the cal-
cium chelation ability of collagen peptides derived fynodontidae fish
scales and its potential as a calcium supplement. Collagen peptides iso-
lated from Jellyfish (R. esculentum) exhibited the copper-chelating abil-
ity (Zhuang et al., 2009a; Zhuang et al., 2009b). Collagen hydrolysates
from squid skin have the ferrous chelating ability in a dose-dependent
manner (Nakchum & Kim, 2016). Huang, Wu, Yang, Li, & Kuo, (2015) re-
ported a high iron (II)-binding activity of Chanos chanos collagen pep-
tides as compared to other fish collagen peptides. Collagen peptides
from Alaska pollock skin collagen showed the iron and copper chelating
activity (Guo, Harnedy, Zhang, et al., 2015c). Therefore, it is suggested
that collagen peptides can be used as functional food ingredients in
the management of mineral deficiencies.
11.3. Tyrosinase inhibitory activity
Melanin biosynthesis proceeds through chains of complex enzymat-
ic and oxidative reactions. A copper-containing compound, tyrosinase is
a key enzyme in melanin synthesis and widely distributed in microor-
ganisms, plants, and animals. It is catalysing the hydroxylation of tyro-
sine to 3, 4-dihydroxyphenylalanine (DOPA) and the oxidation of
DOPA to dopaquinone (Zhuang et al., 2009a, 2009b). Although, melanin
plays an essential role to protect skin against ultraviolet injury,
abnormal pigmentations, and age spots (Zhuang et al., 2009a, 2009b).
Moreover, fruits and vegetables browning are related to tyrosinase in
plant tissues. Therefore, tyrosinase inhibitors have recently attracted
more and more attention (Zhuang et al., 2009a, 2009b). However,
many potent tyrosinase inhibitors (sulfite and kojic acid) have side ef-
fects such as high cells toxicity and low oxygen and water stability. Sea-
food derived collagen peptides were reported as a natural inhibitor of
tyrosinase activity (Nakchum & Kim, 2016; Zhuang et al., 2009a,
2009b). Jellyfish umbrella collagen hydrolysate was able to inhibit ty-
rosinase activity due to their Cu2 +-chelating ability (Zhuang et al.,
2009a, 2009b).
11.4. Neuroprotective activity
Oxidative stress plays a crucial role in neurodegenerative diseases.
Oxidative stress resulting from an imbalance of antioxidant homeostasis
leads to the generation of ROS, which causes extensive damage to pro-
teins, lipids, and DNA (Cai et al., 2014; Nidheesh, Salim, Rajini &
Suresh, 2016). Seafood derived peptides can be used to suppress the de-
velopment of neurodegenerative diseases such as Alzheimer's disease,
Parkinson's disease, and multiple sclerosis. Direct interaction of
absorbed marine/seafood derived collagen peptides with a variety of
cellular and molecular targets, enzyme/ion channels brought the neuro-
protective activities (Cheung et al., 2015; Cai et al., 2014). It was demon-
strated that neuroprotective effect of marine collagen peptides can
facilitate long-term learning and memory in male rats by reducing oxi-
dative damage and acetylcholinesterase activity in the brain (Pei et al.,
2010; Xu, Dong, Zhao, & Xu, 2015). It is suggested that dietary interven-
tion may prevent such neurodegenerative disease. Therefore, undoubt-
edly collagen peptides could be a suitable candidate for functional food
ingredients or drugs to manage/reduce memory deficits associated with
ageing (Pei et al., 2010).
11.5. Angiotensin I-converting enzyme inhibitory activity
Angiotensin I-converting enzyme (ACE, EC 3.4.15.1.) is a multifunc-
tional zinc-containing enzyme that converts the inactive angiotensin I
to angiotensin II. It is a part of the renin-angiotensin system, which
plays a key physiological role to maintain blood pressure homeostasis
and fluid and salt balance as well as local tissue growth, remodelling
and inflammation (Young et al., 2013; Liu, Chen, Su, & Zeng, 2011;
Zhuang, Sun & Li, 2012a). Currently, hypertension is considered as one
of the most serious chronic illnesses. It is one of the major controllable
risk factors associated with cardiovascular disease (CVD). CVD has be-
come an increasingly serious medical and public health issue (Cheung,
Nakayama, Hsu, Samaranayaka, & Li-Chan, 2009; Guo, Harnedy,

Table 2
Seafood derived antioxidant collagen peptides, sources, molecular weight, and enzyme used for hydrolysis.

Source Enzyme used Purification methods Degree of Molecular References

organism hydrolysis (%) weight

Jellyfish Trypsin, properase E Ion exchange and gel filtration chromatography 27.8 400–1200 Da Zhuang et al. (2009a, 2009b)
Croceine croaker Trypsin, pepsin Ultrafiltration, gel chromatography, RP-HPLC 25.11 1–3 kDa Wang et al. (2013)
Jellyfish umbrella Trypsin High-performance liquid chromatography (HPLC) – 1–3 kDa Zhuang, Zhao & Li (2009b)
Spanish mackerel Pepsin Gel filtration chromatography – 5–47 kDa Chi et al. (2014)
Whale shark Thermolysin Ultrafiltration 8.37 37 kDa Jeevithan, Bao, Zhang, Hong, & Wu
(2015)
Sea cucumber Trypsin – – 5–25 kDa Abedin et al. (2014)
Squid Alcalase Ultrafiltration – 1–10 kDa Nakchum & Kim (2016)
Jumbo squid fin Trypsin, chymotrypsin, peptidase – ~20–40 20–77 kDa Cuevas-Acuña, Robles-Sanchez,
Torres-Arreola,
Marquez-Rios, & Ezquerra-Brauer
(2015)
Jellyfish Protamex – – – Ding et al. (2011)
Ribbon jellyfish Trypsin, alcalase, protamex – ~25–48 677–1351 Da Barzideh, Latiff, Gan, Benjakul, &
Karim, (2014)
Alaska pollock Trypsin RP-HPLC, gel permeation 2.5–12.9 o.5–5 kDa Guo, Harnedy, Zhang, et al.
chromatography (GPC-HPLC) (2015c)
 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
Zhang, et al., 2015c; Kong et al., 2011). Although, synthetic ACE inhibi-
tors are effective as anti-hypertensive drugs, cause several adverse
side effects. Development of seafood protein-derived ACE-inhibitory
peptides is thought to be a safer alternative to commercial ACE-
inhibitory medicines (Liu, Chen, Su, & Zeng, 2011). Seafood derived col-
lagen peptides and hydrolysates generated from enzymatic hydrolysis
are a potential source of ACE inhibitors (Kong et al., 2011; Guo, Guo,
Harnedy, Zhang, et al., 2015c). Peptides generated from enzymatic hy-
drolysis of the Alaska pollock skin collagen (Guo, Harnedy, Zhang,
et al., 2015c) and giant red sea cucumber (Parastichopus californicus)
collagen hydrolysates (Liu et al., 2011) exhibited ACE-inhibitory activi-
ty. The production of collagen peptides ACE-inhibitory activity from
giant red sea cucumber collagen have optimised by central composite
rotatable design modelling (Liu et al., 2011). The systolic and diastolic
blood pressure of the renovascular hypertension rats were significantly
reduced with administration of jellyfish (Rhopilema esculentum) colla-
gen peptides, compared with model control (Zhuang, Sun, Zhang &
Liu, 2012b). These studies pointing that seafood derived collagen pep-
tides may offer protection against diabetes and hypertension by affect-
ing molecules involved in these diseases. Further study could be lead to
the development of new antihypertensive drugs based on bioactive col-
lagen peptides from seafood sources. A list of the source of collagen pep-
tides and hydrolysates and their ACE inhibitory activity were shown in
Table 3.
11.6. Antifreeze activity
Antifreeze proteins/peptides have characteristics to inhibit the
growth and recrystallisation of ice. Inhibition of ice growth reduces
the freezing point without altering the melting point. Due to ice
recrystallisation inhibition, antifreeze proteins can preserve the struc-
ture, texture and quality of frozen foods, protect crops from freeze–
thaw cycle; improve the storage of blood, oranges, and tissues;
minimising or preventing damage to cells and tissues (Cao et al.,
2016; Wang, Zhao, Chen, Zhou & Wu, 2014c). Recently, Cao et al.
(2016) isolated and characterised collagen peptides (GLLGPLGPRGLL)
of molecular weight 1162.8 Da were able to inhibit recrystallisation.
Wang et al. (2014a, 2014b, 2014c) reported the preparation and isola-
tion of specific antifreeze collagen peptides (GAIGPAGPLGP) with a mo-
lecular weight of 906 Da from shark skin. The antifreeze collagen
peptides showed a significantly cryoprotective effect on freeze-dried
Streptococcus thermophiles. It significantly improves the viability and in-
tracellular enzyme activities of S. thermophiles after freeze drying, which
specifies that it could be able to protect the cell membranes (Wang,
Chen, Wu & Wang, 2015b). It was suggested that possible protective
mechanism of antifreeze collagen peptides might be through the inter-
action with phospholipids membrane (Wang et al., 2015a, 2015b). Col-
lagen peptides which exhibit strong ice-binding activity could be
potentially developed as a new and beneficial probiotics or other proc-
essed foods (require low-temperature storage) cryoprotectant in the
food industry (Wang et al., 2014a, 2014b, 2014c; Wang et al., 2015a,
2015b). However, the limited availability of hyperactive seafood
Table 3
ACE inhibitory activity of collagen peptides and hydrolysates.
Sources Enzyme Purification
Jellyfish Alcalase Ultrafiltration, ion-exchange and gel filtration
chromatography
Marine fish Mixed proteases Ultra-filtration
– Pepsin with Papain, trypsin, papain, – –
and protease M
Squid skins Pepsin-pancreatin Reverse-phase high-performance liquid
chromatography
Jellyfish Alcalase Ultrafiltration, ion-exchange and gel filtration
chromatography
211
derived antifreeze collagen peptides has restricted their application in
food industry.
11.7. Wound healing activity
Wound healing is a systematic process that completed in three clas-
sic phases: inflammation, proliferation, and maturation (Wang et al.,
2015a, 2015b). Collagen deposition is an important event in wound
healing. Collagen peptides and hydrolysates are chemotactic and pro-
mote cellular proliferation and differentiation (Wang et al., 2015a,
2015b; Zhang, Wang, Ding, Dai, & Li, 2011). Collagen-based biomaterials
(gel, sponge, and dressing), have been reported to have beneficial bio-
logical functions on wound healing (Wang et al., 2015a, 2015b). Wang
et al. (2015a, 2015b) reported that oral administration of chum salmon
(Oncorhynchus keta) collagen peptides improves wound healing. The re-
sults of several studies confirm the significant role of collagen peptides
to accelerating the healing process and justify their possible utilisation
as a functional food ingredient or pharmaceutical agent (Zhang, Wang,
Ding, Dai, & Li, 2011; Banerjee, Suguna, & Shanthi, 2015).
11.8. Cell proliferation activity
Collagen peptides derived from tilapia (Oreochromis sp.) scale colla-
gen have the ability to stimulate fibroblast cells proliferation, collagen
synthesis and procollagen synthesis in a dose and time-dependent
manner (Chai et al., 2010). Liu et al. (2014) demonstrated that collagen
peptides have the ability to increased osteoblast proliferation and differ-
entiation and mineralised bone matrix formation. Ohara et al. (2010)
studied the effect of collagen peptides on dermal extracellular matrix
components and cell proliferation using human dermal fibroblasts cul-
ture. They suggest that cells and skin of extracellular membrane matrix
were modulated by oral ingestion of collagen peptides (Ohara et al.,
2010).
11.9. Treatment of bones and joints diseases and anti-ageing activity
A lot of evidence demonstrates that collagen hydrolysate and colla-
gen peptides may provide beneficial effects on bone tissue, including
stimulation of bone forming cells, improvement of calcium absorption
capacities, without obvious undesirable effects (Antoniewski &
Barringer, 2010; Guillerminet et al., 2012). The collagen hydrolysate
plays a significant role in the management of bone, joint disorders and
osteoarthritis. Potential utilisation of collagen peptides in the preven-
tion and treatment of osteoporosis is reported (Guillerminet et al.,
2012; Ficai et al., 2013; Liu et al., 2014). In Japan, collagen and collagen
peptide have been marketed as functional food for treatment of joint
problems and maintenance of bone integrity (Wu, Fujioka, Sugimoto,
Mu, & Ishimi, 2004). The anti-ageing effect of collagen hydrolysates
might be related to their antioxidant activities. Therefore, it is suggested
that the collagen hydrolysate and peptides exhibited anti-ageing activ-
ity and might be used as nutraceutical ingredients in functional food,
cosmetics and another health care purpose (Liu, Peng, Yang, Li, & Li,
2010).
MW References
200–600 Da Zhuang et al. (2012a, 2012b)
100–860 Da Cui-Feng et al. (2010)
Kong et al. (2011)
1000–10,000 Da Alemán, Gómez-Guillén,
& Montero (2013)
200–600 Da Zhuang, Sun & Li (2012a)
212 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
12. Hurdles and challenges: commercialisation of seafood collagen
and collagen peptides
It would be expected that research on bioactive collagen/collagen
peptides would be succeeded by the development of seafood collagen-
based functional food products for consumer health. However, it was
observed that translation of seafood derived collagen-based food prod-
ucts in health promotion has been faced many hurdles. The seafood de-
rived functional food sector seems to be one of the fastest growing food
sectors all over the world. However, the launching of novel seafood de-
rived functional foods onto the market is not an easy task and certainly
not always a success (Grasso, Brunton, Lyng, Lalor, & Monahan, 2014;
Udenigwe, 2014). The challenges that might be responsible for delay
the commercialisation of seafood collagen-based functional foods
includes:
12.1. Sustainability of the seafood by-product resources
The sufficient quantities of the seafood processing by-products
should be available without supply disruptions regularly. It should
allow the continuous manufacturing of seafood derived collagen so
that it can be used as food ingredients (Silva et al., 2014).
12.2. Consumer acceptance
Seafood collagen and collagen peptides may have the amino acid
residues (Phe and Pro) results in a bitter taste and potentially affect con-
sumer acceptability of seafood collagen-based functional food products.
It was thought that consumers may compromise taste towards the
health effects of bitter functional foods (Udenigwe, 2014). However, it
was proven that consumer does not like to compromise with taste for
health benefits. For example, Novartis withdrew a range of functional
food products most markets of European countries due to low sales
(Grasso, Brunton, Lyng, Lalor, & Monahan, 2014). Taste seems to be a
key factor for the successful commercialisation of seafood collagen-
based functional food. Still, besides the taste, some other parameters
such as health claims through clinical studies, approval from regulatory
authorities, etc. should be considered while launching a new functional
food product (Grasso et al., 2014).
12.3. Safety profile
The seafood derived collagens should be as safe. The animal derived
collagens have the concerns about the transmission of the prions dis-
eases. They should present acceptable safety and toxicological profiles.
The seafood derived collagen should be free from the pathogens/resi-
dues that potentially harmful to humans (Silva et al., 2014).
12.4. The cost of products
The cost is the major factor for commercialisation of collagen-based
functional food products. The price of the innovative functional food
product must be accessible for consumers to purchase these products
(Grasso et al., 2014).
12.5. Reproducibility and the economic viability of collagen
Seafood collagen must be designed and recover in such a way as to
guaranteeing the identity, purity, characteristics and performances.
The recovery of seafood collagen should be shown the batch-to-batch
quality and yield consistency (Silva et al., 2014).
13. Patents on seafood collagen and its derived products
Limited numbers of the patent are available in the development of
seafood processing by-product collagen as well as their applications
towards the development of functional food or nutraceutical products.
A summary of recent patents on the extraction of bioactive seafood de-
rived collagen and their applications in the food industry are described
in Table 4.
14. Challenges associated with seafood collagen and their hydroly-
sate/peptides
Collagen peptide as a functional food ingredient is a new and rapidly
growing sector. It is a scientific discipline that focuses on the seafood
sources for the production of novel collagen peptides and their func-
tional activities. The isolation of collagen from sea animals may create
an opportunity for the sustainable utilisation of seafood derived by-
products. A few major challenges such as technical, conceptual and in-
tellectual prevent a complete isolation of collagen peptides and under-
standing of their functional bioactivities at present. Therefore, it is
clear that there is an urgent need for additional knowledge on seafood
derived collagen and its hydrolysate/peptides. The exploration of func-
tional activities of collagen peptides is limited due to the several
challenges.
i. Isolation and preparation of specific collagen peptides with max-
imum yield.
ii. To deliver an effective dosage of collagen peptides for a specific
health benefit.
iii. The impact of collagen peptides on taste and aroma of the final
food product.
iv. To prevent the undesirable interactions of the collagen peptides
with other food components of the matrix.
v. Should be suitable for industrial processing.
vi. Should not contain impurities residues, toxic compounds and
chemical and biological pathogens.
vii. Collagen peptides should not be degraded when it is incorporat-
ed into foodstuffs.
viii. It should not lose the functional activities during processing.
ix. It should ensure food-grade nature of the final functional food-
stuff.
x. Safety profiles and cost aspects.
xi. Complications in methodology for quality assurance of collagen
peptides.
xii. Inadequate clinical evidence of bioefficacy.
xiii. Collagen peptides should not contain the bitter taste.
xiv. Reproducibility of seafood derived collagen peptides batches.
xv. Economically viable at industrial scales.
xvi. Collagen peptides containing food products should provide the
intended health benefit after ingestion.
15. Conclusions
The agro-food industrial by-products have attracted the interest of
various researchers, regulatory bodies, industry, and consumers. To
this direction, recent scientific findings provide opportunities for effec-
tive reuse of seafood derived by-products and waste leading to the de-
velopment of high economic importance innovative and functional
food items. Identification of the functional food ingredients and nutra-
ceutical from seafood by-products is a growing field. It is a novel ap-
proach to developing more commercial functional food and
nutraceutical ingredients. In conclusion, seafood is highly nutritious.
Therefore, consumption of adequate quantities of seafood derived colla-
gen and its hydrolysate/peptides can significantly help to improve the
health. Based on evidence of potential biological activities and health
benefits, seafood derived collagen peptides have promising applications
as a nutraceutical and natural functional food ingredients. The interac-
tion between collagen peptides and other components of the foods
needs to be understood. These interactions can affect the bioavailability
 G.K. Pal, P.V. Suresh / Innovative Food Science and Emerging Technologies 37 (2016) 201–215
Table 4
A summary of recent patents on the collagen and their applications in food industry.
Patent no. Name of patent
260,909 Collagen peptide composition and food or beverage containing the same
248,497 A method of producing collagen or fibrillar collagen
US 20130252899A1 Collagen hydrolysate for use to improve the health of human skin, hair and/or nails
CN105123983A Kind of fish collagen whole bean curd and its preparation method
CN105039479A Method for preparing fish collagen peptide complexes
CN102697136A Preparation method of collagen drink
CN101946961A Collagen beverage formula
CN101926472A Fish sausage containing trophic factors and preparation method thereof
JP2016005477A Masking method of unpleasant taste with collagen
JP2013126391A Food or beverage product containing fish-derived collagen and derivative thereof
and biological efficacy of the collagen peptides. In addition, there is a
need to understand the mechanisms by which different collagen pep-
tides/hydrolysates mediate their beneficial health effects.
Acknowledgements
Gaurav Kumar Pal thanks to Department of Science and Technology
(DST), Govt. of India, New Delhi, India for the award of a Research Fel-
lowship. Authors thank Director, CSIR-CFTRI, Mysuru for his encourage-
ment and permission to publish this work. The authors would like to
thank anonymous reviewers for the valuable comments provided to im-
prove the manuscript.
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