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Fish Processing By-Products as a Potential Source of Gelatin: A Review
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Fish Processing By-Products as a Potential Source

of Gelatin: A Review
Panayotis D. Karayannakidis & Anastasios Zotos
To cite this article: Panayotis D. Karayannakidis & Anastasios Zotos (2016) Fish Processing
By-Products as a Potential Source of Gelatin: A Review, Journal of Aquatic Food Product
Technology, 25:1, 65-92, DOI: 10.1080/10498850.2013.827767
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Download by: [Panayotis Karayannakidis] Date: 21 January 2016, At: 23:26

JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY
2016, VOL. 25, NO. 1, 65–92
http://dx.doi.org/10.1080/10498850.2013.827767
Fish Processing By-Products as a Potential Source of Gelatin: A

Review
Panayotis D. Karayannakidis and Anastasios Zotos
Technology and Quality Control of Fish and Fish Products Laboratory, Department of Food Technology, School of
Food Technology and Nutrition, Alexander Technological Educational Institute of Thessaloniki, Thessaloniki, Greece
ABSTRACT KEYWORDS
The current practice of fish processing generates large amounts of by- Fish processing by-products;
products, which can account for up to three-quarters of the total fish fish gelatin; gelatin
Downloaded by [Panayotis Karayannakidis] at 23:26 21 January 2016
weight. Despite the presence of several valuable components in the fish manufacture; fish skins; fish
scales; fish bones
processing discards, the latter are usually dumped into landfills or at sea,
having potentially harmful environmental effects or end up as low com-
mercial value products (e.g., white fish meal). Still, fish processing by-
products can be considered as an alternative raw material for the prepara-
tion of high-protein ingredients, especially for the production of food grade
gelatin due to the presence of large amounts of collagen in fish skins,
scales, and bones. Although fish gelatin is an alternative to the commer-
cially available mammalian gelatins, its production on a large commercial
scale has been hampered, mainly, due to the inferior quality characteristics
compared to its mammalian counterparts. This review article summarizes
and highlights the potential utilization of by-products generated during fish
processing for gelatin extraction. Furthermore, several technical challenges
and directions of ongoing research are discussed.
Introduction
The current practice of fish processing generates large amounts of by-products, which can account
for up to three-quarters of the total fish weight (Shahidi, 1994; Zhou and Regenstein, 2004; Rustad
et al., 2011). Despite the presence of several valuable components, fish processing by-products are
usually dumped in landfills or into the oceans having potentially harmful environmental effects or
end up as low commercial value products—such as fish meal, silage, and fertilizer (Shahidi, 1994;
Gómez-Guillén et al., 2002; Muyonga et al., 2004a; Rustad et al., 2011).
Typically, fish processing by-products consist of viscera, heads, trim, skins, scales, and bones, as
well as fish that are damaged or unsuitable for human consumption or further processing, and
bycatch (Rustad, 2003; Rustad et al., 2011). Since protein is the major component in most fish on a
dry mass basis, fish processing by-products can be considered as an alternative raw material for the
preparation of high-protein ingredients, especially for the production of food grade gelatin, due to
the large amounts of collagen present in fish skins, bones (Gómez-Guillén et al., 2002; Muyonga
et al., 2004a), scales (Ikoma et al., 2003), and fins (Nagai and Suzuki, 2000).
Gelatin, which is obtained by the thermal denaturation of collagen, has been widely used in the
food industry as a means to improve the gelation, water binding, foaming, and emulsifying proper-
ties of food products as well as their elasticity and viscosity (Jongjareonrak et al., 2006;
Kittiphattanabawon et al., 2010). Furthermore, its use for encapsulation and edible film formation
makes it of interest to the pharmaceutical, biomaterial-based packaging, and photographic industries
CONTACT Panayotis D. Karayannakidis karayannakidis@yahoo.gr; pkar@food.teithe.gr P.O. Box 141, GR-57400,

Thessaloniki, Greece
© 2016 Taylor & Francis
66 P. D. KARAYANNAKIDIS AND A. ZOTOS
(Gómez-Guillén et al, 2002; Jongjareonrak et al., 2006). Besides the numerous industrial applications,
gelatin has also been reported to maintain joint and bone health, prevent osteoporosis, promote hair
growth, and improve nail strength and growth (Moskowitz, 2000; Derzavis and Mulinos, 1961;
Silvestrini, 1988). Nevertheless, gelatin production from the traditional mammalian sources (pig
skins, cattle hides, and pig and cattle bones) presents several problems for some religions—such as
Judaism, Hinduism, and Islam—which do not allow the consumption of pork- or non-religiously
slaughtered cow-related products (Pranoto et al., 2007; Choi and Regenstein, 2000; Zhou and
Regenstein, 2005). Moreover, since the recent history of livestock disease outbreaks, a growing
market demand for alternative sources of gelatin has emerged (Muyonga et al., 2004a; Lim et al.,
2001).
Although fish processing by-products are considered an alternative source for the production of
gelatin, their use on a large commercial scale has been limited, mainly due to the inferior quality
characteristics of the resulting gelatins with respect to their mammalian counterparts (Fernández-
Díaz et al, 2001; Karim and Bhat, 2009). The objective of this review article is to summarize the
existing knowledge about gelatin extraction from fish processing by-products, providing sufficient
information on the studies that have been performed thus far, including the functional characteriza-
tion of gelatin and potential use of various compounds for improving its functional properties,
known as coenhancers. In addition, several technological challenges and prospects for expanding its
industrial production, as well as research suggestions are discussed.
Classification of fish processing by-products as a collagen/gelatin source

Recent reports indicate that the annual world output of gelatin is estimated to be around 326,000
tons, of which pig skin gelatin accounts for 46%, bovine hide- and bone-derived gelatins account for
29.4 and 23.1%, respectively, while the remaining part (1.5%) is produced from other sources (Karim
and Bhat, 2009). However, because of the recent outbreaks of bovine spongiform encephalopathy
(BSE) and foot-and-mouth disease (FMD), as well as the several religious restrictions, alternative
sources for gelatin production have gained momentum (Avena-Bustillos et al., 2006).
Fish processing by-products have received considerable attention in recent years as an alternative
source of gelatin (Cheow et al., 2007), and researchers in many countries are currently involved in
exploring the suitability of various fish processing by-products from their locale for potential
conversion to gelatin. The list of fish species that can be used for gelatin manufacture seems endless;
however, the appropriateness of fish processing by-products as a whole for producing gelatin is still
under consideration, since the procedures employed for gelatin manufacture depend on the type of
the by-product (e.g., scales, skins, heads). A classification of the different by-products generated
during fish processing based on the current research interests and trends for gelatin extraction is
presented below.
Skins
Due to the high demand for fish fillets, most fish are currently commercialized as fresh or frozen
skinless fillets. The increase of filleting means that more by-products in the form of skins, scales,
heads, fins, viscera, and bones are produced (Jamilah and Harvinder, 2002). Although most of the
by-products generated during fish processing contain significant amounts of collagen, researchers in
this field have largely focused on gelatin extraction from fish skins. To date, various fish skins have
been studied for gelatin manufacture, as shown in Table 1.
Bones
Fish bones, which are another potential collagen source, have not been extensively investigated.
According to the study of Muyonga et al. (2004a), there is great potential in using both fish skins and
JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 67
Table 1. Various fish skins studied for gelatin extraction.

Skin source Species Reference
Alaska pollock Theragra chalcogramma Zhou and Regenstein (2004, 2005)
Megrim Lepidorhombus boscii Montero and Gómez-Guillén (2000)
Bigeye snapper Priacanthus macracanthus Jongjareonrak et al. (2006)
Brownstripe red snapper Lutjanus vitta
Yellowfin tuna Thunnus albacares Cho et al. (2005)
Pranoto et al. (2011)
Brownbanded bamboo shark Chiloscyllium punctatum Kittiphattanabawon et al. (2010)
Blacktip shark Carcharhinus limbatus
Dover sole Solea vulgaris Gómez-Guillén et al. (2005)
Giménez et al. (2005)
Skate Raja kenojei Cho et al. (2006)
Sin croaker Johnius dussumieri Cheow et al. (2007)
Shortfin scad Decapterus macrosoma
Black tilapia Oreochromis mossambicus Jamilah and Harvinder (2002)
Red tilapia Oreochromis nilotica
Horse mackerel Trachurus trachurus Badii and Howell (2006)
Cod Gadus morhua Gómez-Guillén et al. (2002)

Hake Merluccius merluccius
Squid Disidicus gigas
Nile perch Lates niloticus Muyonga et al. (2004a, 2004b)
Flounder Platichthys flesus Fernández-Díaz et al. (2003)
Saithe Pollachius virens Eysturskard et al. (2009)
Atlantic salmon Salmo salar Arnesen and Gildberg (2007)
Alaska pink salmon Oncorhynchus gorbuscha Chiou et al. (2008)
Rohu Labeo rohita Ninan et al. (2011)
Common carp Cyprinus carpio
Brown stingray Dasyatis annotatus Pranoto et al. (2011)
Red snapper Lutjanus altifrontalis
White cheek shark Carcharias dussmieri
Channel catfish Ictalurus punctaus Liu et al. (2008a, 2008b)
Amur sturgeon Acipenser schrenckii Nikoo et al. (2011)
Grass carp Catenopharyngodon idella Kasankala et al. (2007)
bones of Nile perch (Lates niloticus) for gelatin extraction. It has been estimated that fish skins along
with bones comprise approximately 30% of the by-products generated during fish processing (Zhou
and Regenstein, 2004; Muyonga et al., 2004a), which could serve as an additional source for gelatin
production.
Scales
Another potential source of collagen is fish scales, which are usually being removed prior to filleting
and are generated in great quantities by the fish processing industries. Some collagens that have been
isolated from the scales of various fish species include rohu (Labeo rohita), catla (Catla catla),
sardine (Sardinops melanostictus), red sea bream (Pagrus major), Japanese sea bass (Lateolabrax
japonicus; Pati et al., 2010; Nagai et al., 2004), silver carp (Hypophthalmichthys militrix; Wang and
Regenstein, 2009), lizardfish (Saurida spp.; Wangtueai et al., 2010), and bighead carp
(Hypophthalmichthys nobilis; Liu et al., 2012). According to the study of Nagai et al. (2004), the
yields of collagen from sardine, red sea bream, and Japanese sea bass were very high on a dry weight
basis, while the denaturation temperatures of the resulting collagens were somewhat lower than that
of commercial porcine collagen. In a recent study, Liu et al. (2012) showed that the melting
temperature of fish scale collagen was similar to that of fish skin collagen of the same fish species
(bighead carp). However, the melting temperature of the aforementioned collagen-containing tissues
was lower than that of calf skin collagen, owing to the greater imino acid content of the latter
compared to both fish scale and skin collagen. The above studies suggest the potential of using fish
scales for the production of collagen on a larger commercial scale.
Heads
Although fish heads can also be used for extracting gelatin, there is little information regarding the
quality assessment of gelatin preparations from fish heads. Arnesen and Gildberg (2006) reported
that gelatin extracted from cod (Gadus morhua) heads showed similar gelling properties with those
of gelatin extracted from cod skins. In the study of Kołodziejska et al. (2008), about 70% of the total
collagen was extracted from cod heads using a three-stage process. The aforementioned studies
suggest that fish heads can serve as an additional source of collagen.
Viscera
Fish viscera is another by-product generated during fish processing. However, the effective use of
viscera for the production of gelatin is minimal. From fish viscera, only swim-bladders have been
studied as a potential collagen source (Eastoe, 1957; Hickman et al., 2000). In particular, isinglass,
which derives from swim-bladders of Chinese sturgeon, has been used for the isolation of collagen.
In general, the by-products generated during fish processing have great potential for the produc-
tion of highly functional ingredients, such as edible gelatin. Therefore, utilization of all by-products
for gelatin extraction would serve to increase both economic value and better utilize our natural
resources.
Collagen and gelatin

Collagen, which is the parent compound of gelatin, is the most abundant protein in animal tissues,
representing nearly 30% of the total proteins (Pati et al., 2010). It has been shown that collagen exists
in different genetic forms, and, to date, some 27 different types of collagen have been identified, with
type I collagen occuring widely, primarily in connective tissue (skin, bones and tendons), whereas
type II collagen occurs practically exclusively in cartilage tissue (Morales et al., 2000; Schrieber and
Gareis, 2007). Type I collagen has been isolated from various marine sources including squid skin
(Kołodziejska et al., 1999), fish scales (Ikoma et al., 2003; Nagai et al., 2004), swim-bladder (Piez and
Gross, 1960; Eastoe, 1957; Bama et al., 2010), and fish skins (Sadowska et al., 2003; Senaratne et al.,
2006); while types I and V collagen have been isolated from the muscle of various cephalopods
(Morales et al., 2000) as well as from the intramuscular connective tissue of fish such as carp,
lizardfish, lamprey, Japanese eel, sturgeon, spotted shark, sardine, tiger puffer, and rainbow trout
(Sato et al., 1988, 1989, 1991, 1997) and from the fins, scales, bones, skins, and swim bladder of
bighead carp (Hypophthalmichthys nobilis; Liu et al. 2012). Unlike type I and V collagens, Sato et al.
(1989) reported that type III collagen was not contained in detectable amounts in the aforemen-
tioned fish species. Similar findings were reported in the study of Morales et al. (2000), where only
type I and V collagens were isolated from various cephalopods. According to Schrieber and Gareis
(2007), the amount of type III collagen is strongly dependent on the age of the animal. Hence, skin
from young animals can contain up to 50% of type III collagen, but in the course of time this amount
is reduced to the range of 5–10%. The differences in type I and V collagens isolated from carp
(Cyprinus carpio) muscle have been demonstrated by the differences in the electrophoretic patterns
obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the different
precipitation properties using NaCl at acidic and neutral pH values as well as their amino acid
composition. Specifically, when compared to type I collagen, type V collagen from carp muscle was
found to be rich in Glu, Hyd-Lys, and Ile, while it was characterized by a low Ala content (Sato et al.,
1988). Regarding the other collagen types, these are present in very low amounts and are mostly
organ-specific (Scrieber and Gareis, 2007).
Unlike the spherical globular proteins, collagen is a fibrous protein consisting of three polypeptide
α-chains wound together in a triple helix (Schreiber and Gareis, 2007; Senaratne et al., 2006). A
characteristic of this structure is the presence of the repeated amino acid sequence of Gly-X-Y, where
X and Y can be any amino acid, but most frequently are proline (Pro) and hydroxyproline (Hyp),
respectively (Burjanadze, 2000). It is believed that these two amino acids form the hydrogen bonds
that stabilize the triple helical structure of collagen (Norziah et al., 2009; Wasswa et al., 2007). Upon
heating, at about 40°C, the hydrogen bonds that stabilize the triple helical structure of collagen
rupture (Balian and Bowes, 1977), leading to the formation of warm-water soluble collagen—i.e.,
gelatin (Schrieber and Gareis, 2007). Many studies in this field have shown that the loss of the triple
helical conformation of collagen leads to the formation of two main components—the α-chains (α1,
α2, and α3), with molecular weights ranging from 91–95 kDa, and the β-chain, with a molecular
weight approximately twice that of the α-chain (Balian and Bowes, 1977; Bama et al., 2010; Nagai
et al., 2004; Senaratne et al., 2006). Furthermore, another component, namely the γ-chain, with a
higher molecular weight (˜ 300 kDa) than the β-chain, has been isolated and is composed of three
covalently crosslinked α-chains (Balian and Bowes, 1977).
As mentioned, collagen is the parent compound of gelatin, which is present in large amounts in
both terrestrial and aquatic animal tissues. Although gelatin is considered a protein from a chemical
point of view, gelatin preparations are not totally pure, in the sense that they contain significant
amounts of moisture and salts and small amounts of fat as well as other proteins (Schreiber and
Gareis, 2007). However, it has been demonstrated in experimental studies (Jongjareonrak et al., 2006;
Cheow et al., 2007; Rahman et al., 2008) that its chemical composition largely depends on the source
of the collagen-containing material as well as the pretreatment and extraction conditions used. The
proximate composition of commercial bovine and porcine gelatins and some gelatins extracted from
various fish skins is shown in Table 2.
Due to its protein nature, gelatin consists of amino acids, which basically define its functional properties
(Muyonga et al., 2004a; Fernández-Díaz et al., 2001). It has been reported that all the amino acids
commonly found in proteins occur in gelatin, with the exception of tryptophan and cystine (Eastoe and
Leach, 1977). Furthermore, the amino acid composition of collagen, and therefore of gelatin, is low in
methionine and tyrosine (Jamilah and Harvinder, 2002), while glycine is the most abundant amino acid
(Ikoma et al., 2003), accounting for approximately 33% of the total amino acids (Eastoe and Leach, 1977).
From the amino acids present in the gelatin molecule, special emphasis has been given to the imino
acids, Pro and Hyp, whose content varies significantly among fish species. As previously mentioned,
Pro and Hyp are responsible for the unique secondary structure of collagen and the stabilization of the
triple helical conformation (Schreiber and Gareis, 2007). Typically, cold-water fish species tend to have
a low Pro and Hyp content compared to warm-water fish species, and this results in gelatin gels of
inferior quality (e.g., lower gel strength, setting, and melting points) compared to both mammalian
gelatins and gelatins extracted from warm-water fish species (Muyonga et al., 2004a; Jongjareonrak
et al., 2006). Therefore, knowledge of a gelatin’s amino acid composition is of significance because it is
directly related to the aforementioned properties. Another factor that significantly affects the physical
properties of gelatin is the relative content of α-, β-, and γ-chains, as well as the content of higher
molecular weight aggregates and lower molecular weight protein fragments (Gómez-Guillén et al.,
2002; Fernández-Díaz et al., 2003). Table 3 shows the amino acid composition of pig skin gelatin and
several gelatins extracted from various fish skins from different experimental studies.
Table 2. Chemical composition (g/100 g gelatin) of commercial bovine and porcine gelatin and gelatin extracted from various fish
skins.
Gelatin source
Component Bovinea Porcinea Skateb Bigeye snapperc Brownstripe red snapperc Nile perchd Yellowfin tunaa
Protein 87.6 ± 0.3 85.6 ± 0.1 92.31 ± 0.33 87.9 ± 0.8 88.6 ± 0.7 88.8 ± 3.1 78.1 ± 0.2
Moisture 9.7 ± 0.3 12.3 ± 0.1 4.52 ± 0.18 8.2 ± 0.7 7.6 ± 0.2 10.4 ± 0.9 8.3 ± 0.6
Ash 0.9 ± 0.2 0.4 ± 0.5 1.42 ± 0.19 3.2 ± 0.2 1.9 ± 0.1 1.7 ± 0.4 7.8 ± 0.2
Fat 1.2 ± 0.1 1.3 ± 0.2 0.35 ± 0.09 0.6 ± 0.0 0.8 ± 0.1 0.0 ± 0.0 5.6 ± 0.1
Values are presented as mean ± standard deviation.
a
Rahman et al. (2008). bCho et al. (2006). cJongjareonrak et al. (2006). dMuyonga et al. (2004a).
Table 3. Amino acid composition (number of residues/1,000 residues) of pig skin gelatin and gelatins extracted from various fish
skins.
Gelatin source
Pig skin Brownbanded Blacktip Dover Bigeye Brownstripe
Amino acid gelatina bamboo sharkb sharkb solec Megrimd Code Pikee Carpe snapperf red snapperf
Aspartic acid/ 45.8 40 40 48 49 52 54 47 61 56
asparagine
Glutamic acid/ 72.1 76 76 74 70 75 81 74 103 105
glutamine
Serine 34.7 41 29 40 47 69 41 43 38 39
Glycine 330 322 321 345 353 345 328 317 193 204
Histidine 4 7 7 8 8 7.5 7.4 4.5 12 9
Arginine 49 51 54 53 47 51 45 53 92 94
Threonine 17.9 22 20 20 22 25 25 27 32 31
Alanine 111.7 106 120 119 119 107 114 120 103 108
Proline 131.9 113 110 129 117 102 129 124 134 141
Tyrosine 2.6 2 2 3 3 3.5 1.8 3.2 6 5
Valine 25.9 24 25 21 16 19 18 19 21 17
Methionine 3.6 12 15 14 14 13 12 12 17 15
Cysteine — 1 1 — — <1 <1 <1 — —
Isoleucine 9.5 17 18 8 7 11 9.2 12 10 9
Leucine 24 22 23 22 21 23 20 25 27 25
Phenylalanine 13.6 13 13 16 14 13 14 14 21 20
Lysine 26.6 28 27 27 26 25 22 27 38 38
Tryptophan — — — — — — — — — —
Hydroxylproline 90.7 95 91 52 60 53 70 73 91 84
Hydroxylysine 6.4 6 5 1 6 6 7.9 4.5 − —
Imino acids 222.6 208 201 181 177 155 199 193 225 225
Sum 1,000 998 997 1,000 999 1,000 999.3 999.2 999 1,000
a
Eastoe and Leach (1977). bKittiphattanabawon et al. (2010). cGiménez et al. (2005). Sarabia et al. (2000). ePiez and Gross (1960).
f
Jongjareonrak et al. (2006).
Gelatin manufacture
Gelatin is the water soluble product prepared by processes which involve the destruction of the
tertiary, secondary, and partially the primary structure of native collagen present in animal tissues
(Ledward, 1986). These changes in the structure of native collagen are usually brought about by
chemical means (acidic or alkaline pretreatment) and to a lesser extent by enzymatic modification.
The aforementioned processes, which lead to the chemical or biochemical denaturation and hydro-
lysis of collagen, are also known as the conditioning process in the gelatin industry, which lead to the
formation of warm-water soluble collagen (Schreiber and Gareis, 2007). In general, the various
processes for gelatin manufacture from different animal sources include several processing steps and
can be divided into three categories, which are described below. Furthermore, it is important to
point out that the differences among the various manufacturing processes are mostly centered on the
pretreatment and extraction processing steps.
Conventional processes
Production of gelatin from the traditional mammalian sources (pig skins, cattle hides, and pig and
cattle bones) is a prolonged process, which includes several pretreatment steps. Initially, the raw
material is degreased and demineralized, because both fat and minerals affect the quality of the final
product, as well as the efficiency of the extraction process (Hinterwaldner, 1977). Following
degreasing and demineralization, two different pretreatments are employed, based on the source
of the raw material and the final application of the gelatin (Gelatine Manufacturers of Europe
[GME], 2012). For the extraction of gelatin from cattle connective tissue, which is highly inter-
connected, an alkaline pretreatment is applied for up to 20 weeks (Ledward, 1986; GME, 2012). In
contrast to cattle hide, materials like pig skin are treated with an acid solution for 1 day due to the
lower degrees of collagen cross-linkage. From the above, it is obvious that the degree of collagen
cross-linking is a key factor in deciding the conditioning process required, prior to gelatin extraction
(Giménez et al., 2005). After the acidic or alkaline pretreatment of the collagenous material, a
neutralization step follows, which is necessary for adjusting the final pH, most often to neutral or
acidic pH values. The collagen-containing materials are then treated with hot water to extract gelatin,
and the resulting solution (gelatin liquor) is concentrated in vacuum evaporators, sterilized by flash-
heating at 140–142°C, and finally dried using excellent hygienic conditions (Hinterwaldner, 1977;
GME, 2012). The gelatin obtained by the acidic pretreatment is called type A gelatin with an
isoelectric point ranging from 6–9, whereas the gelatin obtained from the alkaline-treated raw
material is called type B gelatin with an isoelectric point of about pH 5 (Hinterwaldner, 1977;
Zhou and Regenstein, 2005; Jongjareonrak et al., 2006). While pigskins are processed for the
production of type A gelatin and cattle hides for type B gelatin, ossein, which is the gelatin producing
substance of bones after demineralization, can be processed into both types of gelatin
(Hinterwaldner, 1977). A schematic presentation of the two processes commonly employed for the
extraction of gelatin from terrestrial animals is shown in Figure 1.
Modified processes
Several alternative manufacturing protocols have been developed in recent years for the production
of gelatin from fish processing by-products and terrestrial animal sources, including some patented
methods (Grossman and Bergman, 1992; Nasrallah et al., 1993; Holzer et al, 1996). Although most of
the protocols developed are based on the concepts of the conventional processes, there are variations
in several factors that are considered of paramount importance in the pretreatment step and the
extraction process itself. These factors include: (a) temperature of pretreatment solution, (b) con-
centration of acid or alkali in the pretreatment solution, (c) pretreatment time, (d) temperature of
water as an extraction medium, and (e) extraction time. It is well established that all of the
aforementioned factors need to be optimized for two reasons—i.e., high extraction yields and high
gel strength. This is clearly demonstrated in the studies of Cho et al. (2005) and Zhou and Regenstein
(2004) on the optimization of extraction conditions of gelatin from yellowfin tuna (Thunnus
albacares) and Alaska pollock (Theragra calcogramma) skins, respectively. It is worth mentioning
that many researchers have studied gelatin extraction from fish processing by-products using
different types of acids or alkalis, beyond the traditionally used mineral acids (acidic pretreatment)
and calcium hydroxide (alkaline pretreatment), which can be also considered as a factor (type of acid
or alkali) affecting gelatin extraction. Typical examples of alternative types of acids and alkalis used
in the pretreatment step for gelatin manufacture include lactic acid (Giménez et al., 2005), acetic acid
(Gómez-Guillén et al., 2002; Montero and Gómez-Guillén, 2000), and sodium hydroxide (Cho et al.,
2005). Table 4 summarizes some of the different procedures employed for gelatin production from
various fish skins.
It must be pointed out that in contrast to fish skins, bones, scales, and heads have different
preparatory steps, prior to the conditioning process and subsequent gelatin extraction, due to
differences in their characteristics. Fish skins are usually treated with an alkaline solution to remove
noncollagenous materials and pigments (Nagarajan et al., 2013; Anand et al., 2013; Nalinanon et al.,
2008; Kittiphattanabawon et al., 2010), while a decalcification step has been reported for the
extraction of gelatin from fish bones and scales (Sha et al., 2013; Pati et al., 2010, Muyonga et al.,
2004a; Ikoma et al., 2003; Liu et al., 2009; Wang and Regenstein, 2009; Liu et al., 2012).
Decalcification has been carried out by acidulation in the case of mackerel (Scomber scombrus)
and blue whiting (Micromesistius poutassou) bones (Khiari et al., 2013) and is commonly employed
in the production of gelatin from the bones of terrestrial animals (Schreiber and Gareis, 2007);
whereas fish scale decalcification has been done using compounds such as ethylenediaminetetraa-
cetic acid (EDTA; Pati et al., 2010; Nagai et al., 2004; Ikoma et al., 2003; Wang and Regenstein, 2009;
Liu et al., 2012), hydrochloric acid (Sha et al., 2013; Wang and Regenstein, 2009), and citric acid
Pig skin Cattle hide
Degreasing Degreasing
Demineralization Demineralization
Acidic pre-treatment Alkaline pre-treatment

(1 day) (up to 20 weeks)
Extraction Extraction
Evaporation Evaporation
Sterilization Sterilization
Drying Drying
Type A gelatin Type B gelatin
Figure 1. Schematic presentation of gelatin manufacture from pig skins and cattle hides.
(Wang and Regenstein, 2009). It must also be noted that the decalcification step in the case of fish
bones and scales may last several days, making the gelatin manufacturing process time-consuming
compared to the processes employed for fish skins. Still, total processing time for the production of
gelatin from fish bones and scales is much shorter compared to the time required for the production
of some mammalian gelatins, especially those deriving from cattle, where the alkaline pretreatment
may be applied for up to 20 weeks (Ledward, 1986). Regarding fish heads, Liu et al. (2009) reported
that a hydrolysis step using an alkaline protease was necessary to obtain the head bones from
channel catfish (Ictalurus punctatus), which were then decalcified using hydrochloric acid. In
another study, Arnesen and Gildberg (2006) investigated the extraction of muscle proteins and
gelatin from cod (Gadus morhua) heads. It was found that comminution of fish heads and
subsequent successive extractions to recover muscle proteins by pH adjustments enabled the
separation of head bones from skins and residual tissue due to differences in density, thus allowing
the extraction of gelatin from head bones. Table 5 summarizes some of the procedures employed for
the production of gelatin from fish bones, scales, and heads.
Table 4. Different procedures employed for gelatin extraction from various fish skins.
Gelatin source Pretreatment Extraction Reference
Nile perch (Lates niloticus) Skins were pretreated with 0.01 M Sequencial extraction at 50, 60 Muyonga et al. (2004a)
H2SO4 for 16 h (1:2 w/v) at room and 70°C followed by boiling
temperature. for 5 h each
Yellowfin tuna (Thunnus Skins were pretreated with 1.89% Extraction with hot water (˜ 58° Cho et al. (2005)
albacares) (w/v) NaOH at 10°C for 2.87 days. C) for 4.72 h
Megrim (Lepidorhombus Skins were pretreated with 0.05 N Distilled water at 45°C, Montero and Gómez-
boscii) Hake (Merluccius acetic acid (1:10 w/v) at room overnight Guillén (2000); Sarabia
merluccius) Dover sole temperature for 3 h after having et al. (2000); Gómez-
(Solea vulgaris) Cod (Gadus been soaked with 0.8 M NaCl and Guillén et al. (2002)
morhua) 0.2 M NaOH at 5°C for 30 min (1:6
w/v).
Black tilapia (Oreochromis Skins were soaked in 0.2% (w/v) Distilled water at 45°C for 12 h Jamilah and Harvinder
mossambicus) Red tilapia NaOH for 40 min followed by (2002)
(Oreochromis nilotica) soaking in 0.2% (w/v) H2SO4 and 1%
(w/v) citric acid.
Atlantic cod (Gadus morhua) Skins are pretreated with 0.3% (w/v) Extraction with water at 45°C Gudmundsson and
Sin croaker (Johnius H2SO4 and 0.7% (w/v) citric acid. overnight Hafsteinsson (1997);
dussumieri) Shortfin scad Cheow et al. (2007)
(Decapterus macrosoma)
Alaska pollock (Theragra Soaking in 0.1 M Ca(OH)2 and 0.05 M Extraction with water at 50°C Zhou and Regenstein
chalcogramma) acetic acid (1:6 w/v) for 60 min at for 3 h (2004, 2005)
2–4°C
Bigeye snapper (Priacanthus Skins were stirred with 0.025 M Extraction with water at 45°C Nalinanon et al. (2008)
macracanthus) NaOH (1:10 w/v) at room for 12 h
temperature for 2 h followed by
soaking with 0.2 M acetic acid (1:10
w/v) in the presence of bigeye
snapper pepsin or porcine pepsin for
2 days at 4°C. Soybean trypsin
inhibitor was added to mixture for
30 min.
Harp seal (Phoca Skins were pretreated with 0.65% Extraction with hot water (60°C) Arnesen and Gildberg
groendlandica) (w/v) H2SO4 for 20 h at 10–15°C. for 7 h (2002)
Atlantic salmon (Salmo salar) Skins were pretreated with 0.12 M Two-step extraction with hot Arnesen and Gildberg
H2SO4 for 30 min and 0.005 M citric water at 56°C and 65°C for 2 h (2007)
acid for another 30 min. each
Cod (Gadus morhua) Skins were pretreated with 0.65% Extraction with hot water (60°C) Arnesen and Gildberg
(w/v) H2SO4 for 20 h at 10–15°C. for 7 h (2006)
Alaska pollock (Theragra Skins were stirred with 0.2 M NaOH Extraction with hot water (45°C) Avena-Bustillos et al.
chalcogramma) Pink (1:6 w/v) and pretreated with 0.2 N overnight (2006)
salmon (Onchorynchus H2SO4 and finally 0.7% (w/v) citric
gorbuscha) acid for 40 min.
Squid (Dosidicus gigas) Skins were pretreated with 0.05 N Distilled water at 80°C, Gómez-Guillén et al.
acetic acid (1:10 w/v) at room overnight (2002); Giménez et al.
temperature for 3 h after having (2009)
been soaked with 0.8 M NaCl and
0.2 M NaOH at 5°C for 30 min (1:6
w/v).
Dover sole (Solea vulgaris) Skins were pretreated with 0.05 N Extraction with water (45°C) Gómez-Guillén et al.
acetic acid (1:10 w/v) at room overnight aided with (2005)
temperature for 3 h after having application of high pressure
been soaked with 0.8 M NaCl and (250–400 MPa) for various
0.2 M NaOH at 5°C for 30 min (1:6 times
w/v).
Bigeye snapper (Priacanthus Skins were soaked in 0.2 M NaOH Extraction with water (45°C) for Jongjareonrak et al.
macracanthus) Brownstripe (1:10 w/v) at 4°C and pretreated 12 h (2006)
red snapper (Lutjanus vitta) with 0.05 M acetic acid (1:10 w/v) for
3 h at room temperature.
(Continued )
Table 4. (Continued).
Gelatin source Pretreatment Extraction Reference
Dover sole (Solea vulgaris) Skins were pretreated with various Extraction with water (45°C) Giménez et al. (2005)
concentrations of lactic acid (1:10 w/ overnight
v) at room temperature for 3 h after
having been soaked with 0.8 M NaCl
and 0.2 M NaOH at 5°C for 30 min
(1:6 w/v).
Horse mackerel (Trachurus Skins were stirred with 0.2% (w/v) Extraction with water (45°C) Badii and Howell
trachurus) H2SO4 for 10 h at 4°C followed by overnight (2006)
stirring with 0.7% (w/v) citric acid for
18h at 4°C
Grass carp Skins were pretreated with 1.19% Extraction with hot water (52.6° Kasankala et al. (2007)
(Catenopharyngodon idella) (w/v) HCl for 24 h at 7°C C) for 5.1 h
Channel catfish (Ictalurus Skins were pretreated with 0.1% (w/ Extraction with hot water (43.2° Liu et al. (2008b)
punctaus) v) Ca(OH)2 for 68.8 h C) for 5.73 h
Channel catfish (Ictalurus Skin were soaked with 50 mM acetic Extraction with distilled water Liu et al. (2008a)
punctaus) acid (1:8 w/v) at 15°C for 18 h (45°C) for 7 h
Amur sturgeon (Ascipenser Skins were soaked in 0.2 M H3PO4 Extraction with distilled water Nikoo et al. (2011)
schrenckii) (1:10 w/v) at 4°C for 24 h (50°C, 1:5 w/v) for 6 h
Carp (Cyprinus carpio) Skins were soaked in 10% (v/v) butyl Extraction with distilled water Duan et al. (2011)
alcohol (1:10 w/v) at 4°C (1:15 w/v) for 4 h at 60, 70 and
80°C
Enzymatic processes
Gelatin extraction has been carried out mostly using acidic and alkaline pretreatments followed by
successive extractions with hot water. However, an alternative approach for the production of gelatin
from collagen-containing materials is proposed by the patented enzymatic method of Rowlands and
Burrows (2000). The advantages of this method are that it does not use chemical compounds in the
pretreatment step (lack of residual chemicals in gelatin) and the resulting gelatin gels show high gel
strength. However, this method has not been applied for gelatin production from fish processing
by-products. Kawahara and Tanihata (2005) have developed a patented enzymatic process for the
production of gelatin from fish skins, which is preferably applied to white meat fish skins, such as
those of Alaska pollock and Pacific cod. The resulting fish gelatin has been reported to be devoid of
fishy odor (after treating the gelatin liquor with activated carbon), have a white appearance, and give
a clear and colorless solution when dissolved in water.
Other factors affecting gelatin extraction efficiency and gelatin functionality

Thus far, the factors that have been shown to affect gelatin extraction efficiency, as well as the
functional properties of the resulting gelatin, are mostly related to the pretreatment and extraction
processing steps of the gelatin manufacture process and the different degrees of cross-linkage of the
collagen molecules, which appear to be species dependent. However, the quality of the raw material
and the different methods of preserving it along with several modifications in the temperature of the
drying process following gelatin extraction have also been shown to affect the aforementioned
parameters.
It is well established that fish, and therefore, their by-products are among the most perishable of
foodstuffs (Garthwaite, 1997; Rustad, 2003). The high perishability of fish is due in part to the
proteases present in situ, which are active over a wide temperature and pH range, as well as the
highly unsaturated lipids that are prone to oxidative deterioration. Intarasirisawat et al. (2007)
showed that bigeye snapper (Priacanthus macracanthus) skins contained a heat-activated serine
proteinase, most likely a collagenase, which affected the yield of gelatin extracted from the skins.
Nalinanon et al. (2008) reported that the addition of an appropriate protease inhibitor (soybean
trypsin inhibitor) in combination with a pepsin-aided process for the production of gelatin from the
Table 5. Procedures employed for gelatin manufacture from fish bones, scales, and heads.
Gelatin source Pretreatments Extraction Reference
Red snapper (L. Bones were washed with water and treated Extraction with hot water (45°C) at a Shakila et al.
campechanus) Brown with 0.2% (w/w) NaOH (1:6, w/v) for 45 min. 1:1 (w/v) ratio for 24 h (2012)
spotted grouper (E. Alkaline-treated bones were washed with
chlorostigma) water. Swelling and decalcification was
performed with 0.2% (w/w) H2SO4 (1:6, w/v)
for 45 min. Bones were then washed with
water and treated twice with 0.1% (w/w)
citric acid (1:6, w/v) for 45 min.
Catfish (Clarias Bones were treated with 3.35% HCl at 4°C for Extraction with distilled water (1:8, w/ Sanaei et al.
gariepinus) 14.5 h to dimineralize. Acid-treated bones v) at ˜ 67.2°C for 5.2 h (2013)
were neutralized by washing with water.
Atlantic mackerel Bones were treated with 0.1 N NaOH (1:3, w/ Extraction with distilled water (1:3, w/ Khiari et al.
(Scomber scombrus) v) for 30 min. This step was repeated 3 times. v) at 45°C for 18 h (2013)
Blue whiting Mixing with 0.1 M phosphate buffer and heat
(Merluccius poutassou) treatment for 5 min to inactivate
endogenous enzymes. Bones were then
subjected to hydrolysis at 50°C for 4 h with

Flavourzyme or Alcalase (enzyme/substrate,
0.1%) and hydrolysis was terminated by heat
treatment. Filtration to separate bones from
protein hydrolysates. The collected bones
were demineralized with 0.25 N HCl (1:3, w/
v) for 18 h and washed with water for 15
min.
Pangasius catfish Bones were degreased with warm water (35° Extraction with distilled water (1:8, w/ Mahmoodani
(Pangasius sutchi) C) and demineralized with 2.74% HCl for ˜ 21 w) at 74.7°C for 5.26 h et al. (2014)
h. Acid-treated bone was rinsed with water
and then neutralized with NaOH.
Tiger-toothed croaker Bones were soaked in 0.2% (w/v) NaOH, Extraction with distilled water (1:3, w/ Koli et al.
(Otolithes rubber) Pink 0.2% (w/v) sulfuric acid, and 1.0% (w/v) citric v) at 45°C for 12 h (2012)
perch (Nemipterus acid with a ratio of 1:7 (w/v). Each soaking
japonicus) was carried out for 40 min. After each
soaking, bones were neutralized with tap
water.
Lizard fish (Saurida spp.) Scales were treated with 0.51% (w/v) NaOH Extraction with distilled water (1:2, w/ Wangtueai
(1:2, w/v) for 3.10 h at 30°C and neutralized v) at 78.5°C for ˜ 3 h and
by washing with water. Noomhorm
(2009)
Grass carp Scales were washed with 10% (w/v) NaCl Extraction with distilled water at 60°C Zhang et al.
(Ctenopharyngodon solution for 24 h and then washed with for 6 h (2011)
idella) distilled water. Demineralization was
achieved with 0.4 M HCl (1:15, w/v) for 90
min. Acid-treated scales were washed with
water 3 times, dried, and minced. Minced
scales were washed with water (1:10, w/v)
and pH was adjusted at 7.0 followed by the
addition of protease A 2G. Hydrolysis was
carried out at ˜ 30.7°C for ˜ 5.5 h with an
enzyme concentration of 0.22% (w/w)
followed by successive washings with water
for 15 min each.
Asian silver carp Washing with water and drying for 1 day. Successive extractions with distilled Wang and
(Hypophthalmichthys Fish scales were then treated with 0.2 M water (1:20, w/v) at 60°C for 1 h, 65°C Regenstein
molitrix) EDTA (1:10, w/v) for 2 h to remove Ca salts for 1.5 h, and 70°C for 2 h (2009)
and repeatedly washed with water until pH
was neutral.
(Continued )
Table 5. (Continued).
Gelatin source Pretreatments Extraction Reference
Mackerel (Scomber Mince head was treated with 0.1 N NaOH Extraction with distilled water (1:3, w/ Khiari et al.
scombrus) (1:3, w/v) to remove pigments and v) at 45°C overnight (2011)
noncollagenous proteins for 30 min. This
step was repeated 3 times. Alkaline-treated
minced head was neutralized with water
followed by pretreatments with various
organic acids (acetic, citric lactic, malic, or
tartaric) at a ratio of 1:3 (w/v) for 4 h. Heads
were washed with tap water before
extraction.
Channel catfish (Ictalurus Head bones were obtained through Sequential extractions with water Liu et al.
punctatus) hydrolysis at 50°C for 140 min with an (1:4, w/v) at 75°C, pH 4.0 for 4 h; 82° (2009)
alkaline protease at pH 9.0. After hydrolysis C, pH 2.5 for 2 h; and 90°C, pH 3.0 for
the heads were dried and crushed to obtain 3h
dry bone powder. The powder was subjected
to decalcification with 0.4 M HCl (1:5, w/v)
for 7.5 h at 20°C and washed with 0.1 N

NaOH until pH was 10–11. It was then
treated with 0.9% (w/v) Ca(OH)2 for 144 h
under agitation and washed with water.
aforementioned fish skins could yield gelatin gels of higher gel strength compared to those prepared
by the conventional process. Furthermore, if the collagen-containing material has a high lipid
content, then soaps may be formed which can contaminate the resulting gelatin (Benjakul et al.,
2012). Therefore, knowledge of autolytic activities and lipid variations according to species, season,
and fishing ground are necessary and have to be taken into account by gelatin manufacturers to
extract gelatin with desirable properties from fish processing by-products.
Another factor of significant importance is the preservation method of the raw material, which is
necessary especially when processing large amounts of by-products. Fernández-Díaz et al. (2003)
conducted a comparison study regarding the rheological properties of gelatin extracted from fresh
flounder (Platichthys flesus) skins and gelatins extracted from previously frozen fish skins stored at
−12 or −20°C of the same species. It was found that gelatins from frozen stored fish skins showed
lower gel strength values compared to that prepared from fresh fish skins, but gelatins from frozen
stored fish skins showed significantly higher melting points. These differences were attributed to the
low recovery of β- and γ-chains, as well as the higher molecular weight components obtained due to
frozen storage of fish skins. However, the above study implies that frozen storage of fish skins at
temperatures below −20°C may lead to higher recoveries of the aforementioned components and
yield gelatin gels of higher gel strength compared to those frozen at higher temperatures. Still,
further studies are necessary to clarify these relationships.
To date, the best preservation method for storing the collagenous materials for gelatin extraction
seems to be drying. Giménez et al. (2005) evaluated the functional properties of gelatin extracted
from dried Dover sole (Solea vulgaris) skins stored at room temperature for 160 days. Although the
melting and gelling points of extracted gelatins decreased for all the drying methods employed as
storage time increased, gel strength values did not show noticeable changes throughout the storage
period studied. Similar results have been reported by Liu et al. (2008a) on extracting gelatin from
dried channel catfish (Ictalurus punctaus) skins.
A recent study by Kwak et al. (2009) has also demonstrated that the different drying methods
(freeze drying, hot-air drying, and spray drying) following gelatin extraction from shortfin mako
shark (Isurus oxyrinchus) cartilage significantly affect its functional properties. It was found that
freeze-dried gelatin preparations formed stronger gels, while gelatins dried at high temperatures
formed weaker gels. Therefore, selection of drying temperature appears to be another critical factor
in gelatin manufacture, since it significantly affects its functional properties.
Functional properties of gelatin

The functional properties of gelatin can be divided into two categories. The first category encom-
passes those that are associated with the gelling properties (gel strength, viscosity, and setting and
melting points), while the second category includes those properties that are related to the surface
behavior of gelatin (foaming, emulsification, film formation, etc.) (Schrieber and Gareis, 2007).
Although both categories are considered important in assessing the functional properties of gelatin,
the focus on the evaluation of the different methods for gelatin extraction has mainly led to the
assessment of the former category.
Gelling properties
Gelatin gelation
During heating of gelatin solutions, the molecules assume a random coil conformation. Subsequent
cooling below a critical temperature causes the native structure to begin to reform, namely the
random coils undergo a coil to helix transition (Tosh et al., 2003). In particular, gel nuclei are formed
from helical interactions within and between polymer molecules, whereas the rate of nucleation is
affected by the concentration of polymers and the degree of cooling below the maximum gelation
temperature. The formation of a gel network is achieved by involvement of polymer chains in more
than one junction zone, where triple helices are forming the junction zones of the network.
Following the formation of triple helices, a packing into fibrils, similar to but not as organized as
native tropocollagen fibrils, is observed, which constitutes a dynamic process where bonds are
continually breaking and reforming within the gel due to weak polymer–polymer interactions
(Tosh et al., 2003, Karim and Bhat, 2009). Unlike the chemical gels, where the bonds responsible
for linking the subunits are covalent chemical bonds, gelatin gels are physical gels formed by physical
interactions. Such interactions include van der Waals forces and hydrogen bonds (Jones 2002; Karim
and Bhat, 2009). During aging, the gel gradually assumes a more stable conformation, the final
structure of the gel being highly dependent on the gel setting temperature (Djabourov, 1988).
Bloom strength
The principal gelling property that determines the value of commercial gelatins is gel strength
(Wainewright, 1977). According to Schrieber and Gareis (2007), gel strength is highly dependent
on the proportion of fractions with a molecular weight of ˜ 100 kDa, the size of one α-chain (Haug
et al., 2004), and it is considered to be proportional to the α-chain content of gelatin (Liu et al.,
2008a). However, a recent study by Eysturskard et al. (2010) showed that the gel strength of
mammalian gelatins was positively correlated with the fractions of α- and β-chains as well as the
high molecular weight protein fragments (>200 kDa) and negatively correlated with the low
molecular weight proteins (<100 kDa). In addition, it was found that in cold-water fish gelatin,
the dynamic storage modulus was positively correlated with the fractions of β-chains and the high
molecular weight protein fragments and negatively correlated with the low molecular weight
proteins and the α-chains. Gel strength, also known as Bloom value in the gelatin industry, is
defined as the weight in grams required for a 12.7 mm diameter cylindical plunger to penetrate
4 mm into a previously prepared gelatin gel (6.67% w/w final concentration) matured at 10°C for
16–18 h (Montero and Gómez-Guillén, 2000; Cho et al., 2005; Jongjareonrak et al., 2006; Schrieber
and Gareis, 2007). The Bloom values of most commercial gelatins range from 80–320 g
(Wainewright, 1977). Generally, gelatins with a Bloom value <150 g are characterized as low
Bloom, while those with a Bloom value ranging from 150–220 g and >220 g are characterized as
medium and high Bloom, respectively (Badii and Howell, 2006; Anonymous, 2013). In general,
gelatins with Bloom values of 250–260 g are the most desirable products (Holzer, 1996). High gel
strength values have been reported for gelatin from skins of warm-water fish species—such as tilapia
(Zhou et al., 2006), grass carp (Kasankala et al., 2007), sole, and megrim (Gómez-Guillén et al.,
2002). Gelatin from cold-water species does not form a gel at room temperature or produces very
soft and unstable gels (Giménez et al., 2005) and may remain liquid at 10°C (Bloom test conditions),
which could restrict its use in the food sector. Bloom values of 70 to 110 g were obtained from
gelatin extracted from cod, salmon, Alaska pollock, and hake (Arnesen and Gildberg, 2007; Zhou
et al., 2006; Gómez-Guillén et al., 2002). The aforementioned differences in gel strength have been
attributed to the variability of Pro and Hyp content in collagen from various species and the varying
temperature of the animal’s living habitat (Jongjareonrak et al., 2006). It is also worth mentioning
that because of sample limitations and lack of gelling at 10°C, some of the research is done using
non-standard conditions, and therefore, a condition-specific gel strength is obtained rather than a
Bloom strength.
Viscosity
Viscosity is the second most commercially important physical property of gelatin (Wainewright,
1977), and viscosity measurements are usually conducted above the gelation temperatures of gelatin
preparations. Avena-Bustillos et al. (2006) studied the viscosity of gelatin preparations from mam-
mals and fish at initial testing temperatures of 35 and 25°C, respectively, considering their differ-
ences in gelling temperature. The viscosity of a concentrated gelatin solution depends on the
hydrodynamic interactions amongst gelatin molecules. Flow behaviors of gelatin solutions from
farmed Amur sturgeon (Acipenser schrenckii) as a function of concentration (1, 3 and 5% w/v) and
temperature (10, 30, 45, and 60°C) indicated a clear non-Newtonian pseudoplastic behavior at 10°C
and 5% (w/v) gelatin solution (Nikoo et al., 2011). However, Wulansari et al. (1998) reported, using a
limed hide (225 Bloom) gelatin at different concentrations (0.5–3.5% w/v), that the resulting gelatin
solutions showed a Newtonian behavior at 50°C. Furthermore, Busnel and Ross-Murphy (1988) have
shown that very dilute gelatin solutions from limed bone ossein exhibit a thixotropic behavior.
Viscosities for commercial gelatins range from 2 to 7 cP or up to 13 cP for specialized preparations,
which stabilize the foam in confectionery products—such as gelatin gums, chewable sweets, marsh-
mallows, and nougat (Anonymous, 2013). It has been demonstrated that low viscosity gelatin
solutions yield brittle gels, while high viscosity gelatin solutions yield rigid and extensible gels
(Wainewright, 1977). Haug et al. (2004) reported that the intrinsic viscosity of fish gelatins (0.2%
w/v) from cold-water species determined at 30°C was comparable to that of mammalian gelatins,
implying that fish gelatins behave like the mammalian with respect to their molecular weight and
hydrodynamic volume. Zhou and Regenstein (2004) also studied the gelling properties and viscosity
of gelatin solution (3.3% w/v) extracted from Alaska pollock skins at 25°C, as affected by extraction
parameters and predicted values from an equation obtained using response surface methodology, as
reported in Table 6.
Setting and melting points

The setting and melting points of gelatin are also considered important indices of the quality of
gelatin preparations. Being thermo-reversible, gelatin gels will start melting when the temperature
is increased above a specific point, the melting point, which is usually lower than the temperature
of the human body. The aforementioned phenomenon governs the melt-in-mouth properties of
gelatin gels and is manipulated by the food and pharmaceutical industries. Regarding gelatins from
fish species, setting temperatures are in the range of 8–25°C, while the range of melting temperatures
is 11–28°C. Although there is a standardized procedure for the determination of gelatin’s melting
point (Wainewright, 1977), this method is rarely employed (Jamilah and Harvinder, 2002; Ninan
et al., 2011) due to several factors affecting its accuracy (maturing time, pH, salt content, and
concentration dependence; (Wainewright, 1977). The aforementioned method is based on the
determination of the temperature at which gelatin gels soften sufficiently to allow carbon tetra-
chloride drops with a red oil soluble dye to sink through them (Wainewright, 1977). Furthermore,
there appears to be no standardized procedure for the determination of gelatin’s setting point.
Methods that are currently employed for the determination of melting and setting points of gelatins
Table 6. Gelling properties of gelatins extracted from various fish species.

Gel setting Gel melting
Fish species Viscosity Bloom strength (g) temperature (°C) temperature (°C) Reference
Alaska pollock 120 cP at 60°C 98 at 10°C 217 at 2° NR 16.1–21.2a Zhou et al. (2006)
skin C
Alaska pollock 6.2 cP at 25°C 460 NR NR Zhou and Regenstein (2004)
skin
Alaska pollock NR ˜ 194 4.6b NR Avena-Bustillos et al. (2006)
skin
Alaskan pink NR ˜ 216 5.3b NR Avena-Bustillos et al. (2006)
salmon skin
Atlantic salmon ˜ 30 cP at 60°C 108 12b NR Arnesen and Gildberg (2007)
skin
Atlantic cod skin 31.2 cP at 60°C24.7 at 10°C 123.2 10b NR Arnesen and Gildberg (2006)
at 4°C
Atlantic cod skin NR ˜ 80 12.0–13.0d 13–15d Gómez-Guillén et al. (2002)
Atlantic cod 18–25.2 cP at 60° 4.7–13.4 at 10°C NR NR Arnesen and Gildberg (2006)
bones C 28.5–90.9 at 4°C
11.0d 13–15d
Hake skin NR 110 Gómez-Guillén et al. (2002)

Cod, haddock, or Intrinsic 42–47 NR 4.0-5.0d 12.0–13.0d Haug et al. (2004)
pollock mL/g at 30°C
Megrim NR NR 13.0d NR Sarabia et al. (2000)
Megrim 220 NR ˜ 11.0d Montero and Gómez-Guillén
(2000)
Megrim skin NR 340 17d 21.0d Gómez-Guillén et al. (2002)
Tilapia skin 38 cP at 60°C 273 at 10°C 395 at NR 24.9–25.4a Zhou et al. (2006)
2°C
Tilapia skin NR NR 15d at pH 5 16d 19.0d Sarabia et al. (2000)
at pH 8
Yellowfin Tuna NR 426 18.7d 24.3d Cho et al. (2005)
skin
Bigeye snapper NR 105.7 NR NR Jongjareonrak et al. (2006)
skin
Brownstripe red NR 219 NR NR Jongjareonrak et al. (2006)
snapper skin
Black tilapia skin 7.12 cP 181 at 10°C NR 28.9a Jamilah and Harvinder (2002)
Red tilapia skin 3.20 cP 128 at 10°C NR 22.4a Jamilah and Harvinder (2002)
Harp seal skin NR ˜ 260 at 10°C ˜ 390 NR NR Arnesen and Gildberg (2002)
at 4°C
Dover sole skin NR 350 13–19d 21–24d Gómez-Guillén et al. (2002);
Giménez et al. (2005)
Sin croaker skin NR 124.9 7.1d 24.6c and 17.7d Cheow et al. (2007)
Shortfin scad NR 176.9 9.9d 18.5c and 23.8d Cheow et al. (2007)
skin
Grass carp skin NR 267 19.5d 26.8d Kasankala et al. (2007)
Horse mackerel NR 230 ˜ 16d NR Badii and Howell (2006)
skin
Channel catfish NR 276 NR NR Liu et al. (2008b)
skin
Channel catfish NR 243–256 15–18d 23–27d Liu et al. (2008a)
skin
Lizard fish scales3.43–5.63 cP at 268 NR NR Wangtueai and Noomhorm
25°C (2009)
Grass carp scales NR 276 20.8d 26.9d Zhang et al. (2011)
Amur sturgeon 4.5 cP at 60°C 9.3 316.3 13.0d 19.6d Nikoo et al. (2011)
skin cP at 30°C
NR = not reported.
a
Determined according to Wainewright (1977). bDetermined using viscometry. cDetermined using differential scanning calorimetry,
d
Determined using dynamic mechanical analysis.
are differential scanning calorimetry (DSC; Cheow et al., 2007; Norziah et al., 2009; Rahman et al.,
2008) and dynamic mechanical analysis (DMA; Gómez-Guillén et al., 2005; Cheow et al., 2007;
Giménez et al., 2005; Sarabia et al., 2000; Cho et al., 2005). In the former, the melting and setting
points are obtained from the DSC thermogram as the peak temperature where the transition upon
heating (melting) or cooling (gelling) occurs; while in DMA, melting and setting points are
determined as the cross-over temperature where the elastic modulus (G′) is equal to the viscous
modulus (G′′) during heating and cooling, respectively (Nikoo et al., 2011). In addition, melting and
setting points have been determined using different types of viscometers with controlled temperature
programs (Arnesen and Gildberg, 2002; Pati et al., 2010; Avena-Bustillos et al., 2006) as the
temperatures at which a sharp decrease (upon heating) or increase (upon cooling) in the viscosity
is observed. It is worth mentioning that the different methods used for the determination of setting
and melting points of gelatins may provide different values. This is clearly demonstrated in the
studies of Norziah et al. (2009) and Cheow et al. (2007), where fish gelatins from the same fish
species showed different setting and melting points when determined using DSC and rheol-
ogy (DMA).
The gelling properties (Bloom strength, viscosity, and setting and melting points) of various fish
gelatins are shown in Table 6. Generally, the gelling properties, such as Bloom strength and melting
temperature of gelatin, prepared from the skins of warm-blooded animals and warm-water fish are
higher than that of gelatin from the skin of fish living in cold water, owing to the greater imino acid
content and increased degree of hydroxylation (Gilsenan and Ross-Murphy, 2000; Avena-Bustillos
et al., 2006). In addition, Badii and Howell (2006) have suggested that a higher content of hydro-
phobic amino acids (in warm-water fish species such as tilapia or horse mackerel) produces gelatin
gels of increased gel strength, in relation to gels from cold-water species, such as cod.
The melting and gelling temperatures of gelatin have been shown to correlate with the proportion
of Pro and Hyp in the original collagen (Haug et al., 2004). It has been reported that calf skin gelatin
contains approximately 94 Hyp and 138 Pro residues per 1,000 total amino acid residues, while cod
skin gelatin contains approximately 53 and 102 amino acid residues of Hyp and Pro, respectively, per
1,000 residues (Piez and Gross, 1960). However, tilapia skin gelatin contains approximately 70 and
119 residues of Hyp and Pro, respectively, per 1,000 residues and has similar properties to those of
mammalian gelatins (Sarabia et al., 2000). The difference in imino acid content between cold- and
warm-water fish species is also shown in Table 3.
Surface properties
Due to its surface-active properties, gelatin has been also used as a foaming, emulsifying, and wetting
agent in food, pharmaceutical, medical, and technical applications (Karim and Bhat, 2008). The
hydrophobic areas on the peptide chain are responsible for giving gelatin its emulsifying and
foaming properties (Cole, 2000). Factors affecting foam formation are solubility, viscosity, protein
unfolding, and aggregation (Kinsella, 1981). Regarding its applications in food, gelatin is used in
making marshmallows and premixed coffee beverages, among others (Kwak et al., 2009), thanks to
its excellent foam-stabilizing ability. Different gelatins have different foam-stabilizing abilities, and,
thus, gelatin for this purpose has to be carefully selected (Baziwane and He, 2003). Generally, gelatin
foam formation ability and stability is decreased, due to unfolding and aggregation of molecules
during the high temperatures of drying; such as the findings of Kwak et al. (2009), who reported a
decrease in foam formation ability of gelatin from shark cartilage with increased drying tempera-
tures. Spray-dried gelatin showed the highest foam stability in relation to other drying methods, such
as hot-air drying and freeze drying. Ninan et al. (2011) reported increased foam formation ability in
gelatins from rohu and common carp. Common carp produced gelatin of moderately inferior
foaming properties compared to rohu, possibly due to protein aggregation. Furthermore, Cho
et al. (2004) showed that gelatin from shark cartilage had foaming properties comparable to those
from porcine skin.
Emulsion formation is an essential property of proteins required for manufacturing foods such as
mayonnaise, where gelatins are frequently used as emulsifiers (Kwak et al., 2009). However, in the
United States egg yolk is normally used as an emulsifier for the production of mayonnaise.
Hydrophobicity is known to be closely associated with the emulsifying capacity of proteins. In
general, fish gelatin emulsions are moderately stable to creaming (Karim and Bhat, 2009). Surh et al.
(2006) determined the influence of gelatin molecular weight on emulsifying capacity and the effect of
environmental stresses (pH, salt, and thermal processing) on the stability of such emulsions.
Emulsions with monomodal particle size distributions and small mean droplet diameters could be
produced at protein concentrations of 4.0% (w/v) for low- and high-molecular weight fish gelatins,
but large droplets or flocculated droplets were present indicating a destabilized oil fraction.
Emulsions were fairly stable with high salt concentrations (250 mM sodium chloride), thermal
treatments (30 and 90°C for 30 min), and different pH values (pH 3–8), showing that fish gelatin
may have limited use as a protein emulsifier for oil-in-water emulsions.
Kwak et al. (2009) reported that the best emulsifying capacity was shown by spray-dried gelatin,
compared to hot-air dried and freeze-dried gelatin from shark cartilage. All drying methods were
shown to yield gelatins which produced stable emulsions (0.01% w/v gelatin). Ninan et al. (2011)
measured the fat binding capacity of gelatin from the skins of rohu and common carp and concluded
that the higher content of the hydrophobic amino acid tyrosine in rohu, in relation to common carp,
was responsible for the high binding capacity of rohu.
Sensory properties
Color, transparency, odor, and flavor are considered important sensory quality characteristics in
gelatin preparations (Giménez et al., 2005; Montero and Gómez-Guillén, 2000). However, one of the
main drawbacks of fish gelatin limiting its applicability for industrial use is often the dark color
(Wasswa et al., 2007). Fish gelatin color depends on the raw materials from which it is extracted and
whether one, two, or more extractions are employed; however, it does not influence other functional
properties (Ockerman and Hansen, 1999). The “L” values of bovine and sin croaker (Johnius
dussumieri) gelatins have been reported to be significantly higher than those of shortfin scad
(Decapterus macrosoma) gelatin. Bovine and sin croaker gelatins showed the brightest and whitest
appearances. Sin croaker gelatins were found to have comparable lightness to bovine gelatins, but
significantly lower “b” values (less yellow hue) than bovine and shortfin scad gelatin (Cheow et al.,
2007). Ninan et al. (2011) reported that color determination of gelatin from rohu (Labeo rohita) and
common carp (Cyprinus carpio) gave “L” values of around 90 with light green and light yellow hues.
Another disadvantage when producing gelatin from aquatic sources is the unpleasant smell often
associated with fish processing byproducts (Jamilah and Harvinder, 2002). Muyonga et al. (2004a),
using gelatin from Nile perch (Lates niloticus) skins and bones, filtered extracted gelatins through
compressed cotton wool, followed by passing through a column with activated carbon, which was
aimed at removing the fishy odor. This was confirmed by a sensory test conducted using a 20-
member panel, indicating that the activated carbon pretreament eliminated the fishy odor from fish
gelatins. The physicochemical and sensory characteristics of fish gelatin compared to commercial
porcine gelatin of similar Bloom strength have been studied by Choi and Regenstein (2000). In their
study, the flavored fish gelatin-water desserts had less undesirable off-flavor and off-odor and a more
desirable release of flavor and aroma. This was attributed to the lower melting point of fish gelatin,
suggesting that it may offer new opportunities to product developers.
Potential use of coenhancers for improving the functional properties of fish gelatin
As previously mentioned, fish gelatins, especially from cold-water fish species are of inferior quality
compared to both mammalian gelatins and gelatins extracted from warm-water fish species. These
differences concern, primarily, the gelling properties (Bloom strength, viscosity, and setting and
melting points) of extracted gelatin. This has led to the incorporation of several compounds into
gelatin preparations, known as coenhancers, with one main goal—i.e., to improve gelatin’s functional
properties.
Classification of compounds as potential coenhancers

Several compounds have been studied over the last decade as potential coenhancers for improving
the functional properties of gelatin gels from both aquatic and terrestrial animal sources. The
compounds that have been used thus far can be classified into five categories, as follows:
● Cross-linking enzymes: Transglutaminase (TGase) appears to be the only cross-linking enzyme

used for improving the functional properties of gelatin gels and films (Fernández-Díaz et al.,
2001; Kołodziejska et al., 2004; Yi et al., 2006; Jongjareonrak et al., 2006; Norziah et al., 2009;
Bae et al., 2009).
● Protein and polysaccharide macromolecules: Several protein and polysaccharide macromolecules
—such as egg albumen (Badii and Howell, 2006), κ-carrageenan (Haug et al., 2004; Pranoto
et al., 2007), gellan (Pranoto et al., 2007), pectin (Liu et al., 2007), and chitosan (Gómez-Estaca
et al., 2011)—have been assessed to determine their potential to improve the functional
properties of gelatin.
● Phenolic compounds: Recently, the use of plant phenolic compounds as potential coenhancers
has attracted the interest of many researchers (Strauss and Gibson, 2004; Gómez-Guillén et al.,
2007; Rattaya et al., 2009).
● Protease inhibitors: The use of a protease inhibitor has been reported during the extraction
process of gelatin from bigeye snapper and brownstripe red snapper skins to eliminate
problems associated with gelatin hydrolysis from the proteolytic enzymes present in situ
(Nalinanon et al., 2008). Therefore, protease inhibitors can be considered, indirectly, as
coenhancers.
● Other compounds: This category encompasses all coenhancers that cannot be classified into one
of the aforementioned categories and includes several chemical compounds like salts (Sarabia
et al., 2000; Choi and Regenstein, 2000; Fernádez-Díaz et al., 2001), glycerol (Fernández-Díaz
et al., 2001), glutaraldehyde (Chiou et al., 2008), sucrose (Choi and Regenstein, 2000), glyoxal,
and formaldehyde (de Carvalho and Grosso, 2004).
Qualitative and quantitative characteristics of coenhancers

Among the various compounds that have been used thus far for improving the functional properties
of gelatin, the concentration level of the coenhancer seems to be of significant importance for
attaining maximal gel strength of gelatin gels for a given fish species. According to the study of
Norziah et al. (2009), addition of TGase in herring (Tenualosa ilisha) gelatin solutions at concentra-
tions up to 1.0 mg/g gelatin, significantly increases gel strength. However, increasing the enzyme
concentration beyond that level leads to a decrease in gel strength (Figure 2). Similar results have
been reported in the study of Jongjareonrak et al. (2006). Fernández-Díaz et al. (2001) also
demonstrated the beneficial effect of the addition of glycerol and magnesium sulfate on the gelling
properties of gelatins extracted from cod (Gadus morhua) and hake (Merluccius merluccius).
However, both compounds improved Bloom strength of fish gelatins at specific concentrations,
above which, gel strength significantly decreased.
Besides improving the functional properties of extracted gelatin, an ideal compound to be used as
a coenhancer should not have any detrimental effects on other important quality characteristics of
gelatin—such as color, transparency, flavor, and odor. Therefore, when a compound is studied as a
potential coenhancer, it is of paramount importance to evaluate the aforementioned sensorial traits
along with the gelation-related parameters of gelatin gels or films. Pranoto et al. (2007) studied the
effect of added gellan and κ-carrageenan at concentrations of 1 and 2 g/100 g gelatin granules.
Although both macromolecules significantly improved the functional properties of fish gelatin,
gelatin films with added gellan and κ-carrageenan appeared to be darker.
Figure 2. Changes in gel strength of fish gelatin as related to transglutaminase concentration. Reprinted from Norziah, M. H., Al-
Hassan, A., Khairulnizam, A. B., Mordi, M. N., and Norita, M., 2009, Food Hydrocolloids, 23: 1610–1616, with permission from
Elsevier.
Another important factor that should be considered when using chemical cross-linking agents for
improving the functional properties of gelatin preparations is their toxicity and cost (Rattaya et al.,
2009). Although formaldehyde, glyoxal, and glutaraldehyde have been shown to be potent coenhan-
cers (de Carvalho and Grosso, 2004; Chiou et al., 2008), the resulting gelatin will have limited
applicability if destined for human consumption.
Ultraviolet radiation as a means to improve the functional properties of gelatin

Besides the various compounds that have been used for improving the functional properties of
gelatin, Bhat and Karim (2009) reported that improvements in Bloom strength of fish gelatins will
occur after exposure to ultraviolet (UV) irradiation. In their study, fish gelatin preparations exposed
to UV irradiation (253.7 nm) for 30 and 60 min showed higher Bloom strength values compared to
the untreated fish gelatin (Figure 3). However, a significant decrease in the viscosity of the UV-
treated gelatins was observed, while no appreciable changes were noticed for melting points. The
beneficial effect of UVB radiation exposure (at doses up to 29.7 J·cm−2) on the functional properties
of fish gelatins from cold- (cod, haddock, pollock) and warm-water (tilapia) fish has also been shown
by Otoni et al. (2012). Interestingly, SDS-PAGE and refractive index measurements indicated the
formation of cross-links in gelatins exposed to UVB. Furthermore, it was found that the Bloom
strengths and viscosities of all gelatins significantly increased when higher doses of UV light were
used, while higher tensile strength was observed for gelatin films made from cold-water fish species
only.
Gelatin hydrolysates
Recently, the production of fish gelatin and collagen-derived peptides with bioactive properties has
attracted the interest of many researchers. Such peptides are generally obtained by enzymatic
hydrolysis using various commercial proteases. Some of the enzymes that have been used for the
production of gelatin and collagen-derived hydrolysates include papaya latex enzyme
(Kittiphattanabawon et al., 2012a, 2012b); pepsin (Lin et al., 2012); Alcalase®, Flavourzyme®, and
bromelain (Li-Chan et al., 2012; Cheung et al., 2012); Properase E and Multifect® Neutral (Zhang
et al., 2012); trypsin (Li et al., 2013); Protamex® (Cheung et al., 2012); and papain (Wang et al., 2013).
Furthermore, Wang et al. (2013) have produced collagen hydrolysates from tilapia (Oreochromis
niloticus) skins by simply retorting in an autoclave at 121°C for 3 h (thermal hydrolysis), while
Figure 3. Changes in gel strength of fish gelatin as related to UV exposure time. Reprinted from Bhat, R., and Karim, A. A., 2009,
Ultraviolet irradiation improves gel strength of fish gelatin, Food Chemistry, 113, 1160–1164, with permission from Elsevier.
enzymatic hydrolysis of fish gelatin under high pressure treatment has also been reported (Alemán
et al., 2011a). Gelatin and collagen-derived hydrolysates have been assessed in terms of their
antioxidant properties, antihypertensive activity (angiotensin-I-converting enzyme inhibitory activ-
ity), dipeptidyl-peptidase IV (DPP-IV) inhibitory activity, cryoprotective effect, and anticancer
activity among others. Below are some of the bioactive properties of various fish gelatin hydrolysates
based on several experimental studies.
Antioxidant properties
The antioxidant activity of gelatin hydrolysates from various fish species has been demonstrated in
numerous studies. Zhang et al. (2012) purified and characterized novel antioxidant peptides from
enzymatic hydrolysates of tilapia (Oreochromis niloticus) skin gelatin using Multifect® Neutral and
Properase E. In their study, it was found that the hydrolysates obtained by progressive hydrolysis
using Multifect® Neutral and Properase E exhibited the highest degree of hydrolysis and hydroxyl
radical scavenging activity. Furthermore, two peptides, formed during hydrolysis of gelatin with a
high antioxidant activity were identified, and their amino acid sequences were Glu-Gly-Leu (317.33
Da) and Tyr-Lys-Asp-Glu-Tyr (645.21 Da). Ngo et al. (2011) prepared hydrolysates from Pacific cod
(Gadus macrocephalus) skin gelatin using Alcalase®, Neutrase®, papain, trypsin, pepsin, and α-
chymotrypsin and found that papain was the most successful enzyme in producing hydrolysates
with the highest antioxidant activity. The papain-derived hydrolysate was further purified, and two
peptides were obtained with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-
Gly-Asn-Val (485.5 Da).
In another study, Kittiphattanabawon et al. (2012a) evaluated the antioxidant properties of gelatin
hydrolysates from blacktip shark (Carcharhinus limbatus) skin, prepared with different degrees of
hydrolysis (10, 20, 30, and 40%), in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis
(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) free-radical scavenging as well as hydroxyl radical
scavenging activity and oxygen radical absorbance capacity (ORAC). It was found that the antiox-
idant activity of gelatin hydrolysates increased as the degree of hydrolysis increased and that at levels
of 500 and 1,000 ppm, the peptides could inhibit the oxidation in both β-carotene linoleate and
cooked comminuted pork model systems.
Antihypertensive properties
Hypertension is one of the most common human chronic health problems and is considered as the
major risk factor for arteriosclerosis, stroke, myocardial infraction, and end-stage renal disease (Itou
et al., 2007). Angiotensin-I-converting enzyme (ACE) plays an important role in the regulation of
blood pressure and hypertension because it catalyzes the conversion of inactive angiotensin-I into
angiotensin-II, a potent vasoconstrictor (Alemán et al., 2011b). Numerous studies in this field have
shown that fish gelatin hydrolysates contain peptides that are able to inhibit the ACE and therefore
prevent hypertension. The antihypertensive properties of gelatin hydrolysates have been reported for
various fish species; some of them are blacktip shark (Kittiphattanabawon et al., 2013), Pacific cod
(Ngo et al., 2011), pangasius catfish (Mahmoodani et al., 2014), as well as squid (Lin et al., 2012;
Alemán et al., 2011b). Himaya et al. (2012) produced gelatin hydrolysates from Pacific cod skin
using different gastrointestinal proteases and purified a very active peptide, which not only exhibited
a strong ACE inhibitory activity but also strong antioxidant activity. The amino acid sequence of the
peptide was found to be Leu-Leu-Met-Leu-Asp-Asn-Asp-Leu-Pro-Pro and had a molecular weight
of 1,301 Da.
Cryoprotective properties
Antifreeze peptides, which have the ability to inhibit the growth of crystals, thus decreasing the
injury of cells and helping to retain the structure and quality of products, have also been reported to
be formed upon fish skin gelatin hydrolysis. In a recent study of Wang et al. (2011), gelatin derived
from shark skin was hydrolyzed to obtain antifreeze peptides. The resulting hydrolysate was then
investigated for its hypothermia protection activity on bacteria such as E. coli. The hypothermia
protection assay showed that the survival rate of E. coli was 80.8% when the concentration of
peptides was up to 500 μg per mL. A cryoprotective effect of gelatin hydrolysates from blacktip shark
(Carcharhinus limbatus) skin has also been observed by Kittiphattanabawon et al. (2012b) on surimi
gels from threadfin bream (Nemipterus spp.) subjected to three and six freeze-thaw cycles.
Specifically, the gelatin hydrolysate with a 10% degree of hydrolysis was able to prevent the
denaturation of surimi proteins (high Ca2+-ATPase activity and protein solubility, low surface
hydrophobicity, and a small decrease in total sulfhydryl groups), and its performance as a cryopro-
tectant was found to be equal to that of commercial cryoprotectants (sucrose/sorbitol blend, 3:1),
which are responsible for the sweet taste of surimi products.
Anticancerous activity
Recently, Alemán et al. (2011b) showed that gelatin hydrolysates produced from giant squid
(Dosidicus gigas) using esperase had a high cytotoxic effect on cancer cells, with IC50 values of
0.13 and 0.10 mg per mL for human breast carcinoma and glioma, respectively. Kittiphattanabawon
et al. (2013) have also shown that gelatin hydrolysates from blacktip shark skin with a 40% degree of
hydrolysis, effectively inhibited hydroxyl and peroxyl radical-induced DNA scission, implying that
gelatin hydrolysates might be used as functional food ingredients and antimutagenic agents.
Management of type 2 diabetes

Another important bioactive property, which has been demonstrated using gelatin hydrolysates
produced from salmon (Salmo salar) using Flavourzyme®, bromelain, and Alcalase® is the inhibition
of the DPP-IV enzyme, suggesting that gelatin hydrolysates may also be used in the treatment of type
II diabetes (Li-Chan et al., 2012). In the aforementioned study of Li-Chan et al. (2012), the gelatin
hydrolysates produced using Flavourzyme® showed the highest inhibition of DPP-IV. Furthermore,
isolation of the peptides formed upon hydrolysis of salmon skin gelatin using Flavourzyme® showed
that the peptide fraction with a molecular weight <1 kDa had the highest DPP-IV inhibitory activity.
From the above fraction, two peptides were isolated using high performance liquid chromatography
(HPLC), and their amino acid sequences were Gly-Pro-Ala-Glu (372.4 Da) and Gly-Pro-Gly-Ala
(300.4 Da).
In general, it can be concluded that besides the numerous applications of gelatin in the food,
pharmaceutical, and photographic industries, gelatin can also be used for the production of hydro-
lysates, which could serve as a potential source of functional ingredients for health promotion.
Challenges associated with gelatin extraction from fish processing by-products and
research suggestions for future work
Gelatin extraction from fish processing by-products has several technical challenges, as follows:
● To date, most researchers have largely focused on gelatin extraction, mainly from the skins of
several fish species, while there is much less information regarding gelatin extraction from
other body parts such as bones, heads and scales, which also contain gelatin (Muyonga et al.,
2004a; Kołodziejska et al., 2008; Nagai et al., 2004). The use of these other body parts can help
lead to the complete utilization of the fish processing by-products that are currently being
discarded or converted into low value products by the fish processing industry.
● Another point to be made is that extraction of gelatin is a prolonged process, which involves
several pretreatment steps. Therefore, shortening the processing time and reducing the proces-
sing steps for efficient extraction of gelatin should be addressed.
● The functional properties of gelatin depend on the raw material as well as the pretreatment of
the raw material. It is well established that the optimum conditions for extracting gelatin vary
among different fish species due to differences in the susceptibility of their collagen to
degradation during the processing steps as well as the activation of several enzymes (proteases)
present in situ (Eysturskard et al., 2009; Nalinanon et al., 2008). Therefore, optimizing the
extraction conditions and minimizing proteolysis is required to obtain gelatin of superior
quality with functional properties similar to those of commercial gelatin.
● Most investigations have focused on gelatin extraction from lean fish species (e.g., cod, pollock,
megrim, etc.; Zhou and Regenstein, 2004; Gómez-Guillén et al., 2002), and very few studies
have been carried out on gelatin extraction from fatty fish species (using skins only) with no
attempt to recover other ingredients (e.g., lipids) at the same time (Arnesen and Gildberg,
2007). Therefore, it would be of great interest to investigate the extraction and recovery of
gelatin and lipids using a single multistep procedure.
● In general, fish gelatin’s functional properties differ from those of mammalian gelatin, and,
therefore, several chemical compounds known as coenhancers (e.g., glycerol, magnesium
sulphate, transglutaminase, etc.) have been used to improve its functional properties
(Fernández-Díaz et al., 2001; Kołodziejska et al., 2004). However, recent studies have shown
that alternative compounds such as phenolics extracted from plants can successfully be used as
coenhancers (Strauss and Gibson, 2004). Although this has not been extensively studied, the
use of phenolic compounds might be a simple way to modify and improve the functional
properties of fish gelatin.
Conclusions
Fish processing by-products are a very promising and cost-effective gelatin source. However, further
studies working with fish gelatin production are required to develop low cost and high quality
products. Gelatin production from fish processing by-products on a large commercial scale will not
only satisfy the needs of the fish processing industries that currently face waste disposal problems,
but it will also fill the needs of diverse cultures that do not consume pork- or cow-related products
(e.g., kosher, halal, and Hindu market), opening new market opportunities to gelatin manufacturers.
Acknowledgments
The research project is implemented within the framework of the Action “Supporting Postdoctoral
Researchers” of the Operational Program “Education and Lifelong Learning” (Action’s Beneficiary:
General Secretariat for Research and Technology).
Funding
The research project is cofinanced by the European Social Fund (ESF) and the Greek State.
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Fish Processing By-Products as a Potential Source of Gelatin: A Review

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Fish Processing By-Products as a Potential Source

Panayotis D. Karayannakidis & Anastasios Zotos

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Fish Processing By-Products as a Potential Source of Gelatin: A

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Classification of fish processing by-products as a collagen/gelatin source

Table 1. Various fish skins studied for gelatin extraction.

Cod Gadus morhua Gómez-Guillén et al. (2002)

Collagen and gelatin

Pig skin Cattle hide

Acidic pre-treatment Alkaline pre-treatment

Type A gelatin Type B gelatin

Other factors affecting gelatin extraction efficiency and gelatin functionality

subjected to hydrolysis at 50°C for 4 h with

for 7.5 h at 20°C and washed with 0.1 N

Functional properties of gelatin

Setting and melting points

Table 6. Gelling properties of gelatins extracted from various fish species.

Hake skin NR 110 Gómez-Guillén et al. (2002)

Classification of compounds as potential coenhancers

● Cross-linking enzymes: Transglutaminase (TGase) appears to be the only cross-linking enzyme

Qualitative and quantitative characteristics of coenhancers

Ultraviolet radiation as a means to improve the functional properties of gelatin

Management of type 2 diabetes

glyoxal and formaldehyde. Food Hydrocolloid. 18: 717–726.

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