Characterization of carp (Cyprinus carpio) skin gelatin extracted using different pretreatments method

Accepted Manuscript Characterization of carp (Cyprinus carpio) skin gelatin extracted using different pretreatments method

Tkaczewska Joanna, Morawska Małgorzata, Kulawik Piotr, Marzena Zając PII:

S0268-005X(17)31762-9

DOI:

10.1016/j.foodhyd.2018.02.048

Reference:

FOOHYD 4307

To appear in:

Food Hydrocolloids

Received Date:

17 October 2017

Revised Date:

19 February 2018

Accepted Date:

27 February 2018

Please cite this article as: Tkaczewska Joanna, Morawska Małgorzata, Kulawik Piotr, Marzena Zając, Characterization of carp (Cyprinus carpio) skin gelatin extracted using different pretreatments method, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.02.048

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ACCEPTED MANUSCRIPT 1

Characterization of carp (Cyprinus carpio) skin gelatin extracted using different

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pretreatments method

3

Tkaczewska Joanna1, Morawska Małgorzata2, Kulawik Piotr1, Marzena Zając1

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1Department

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Cracow, Balicka 122, 30-149 Cracow, Poland

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2Department

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University School of Physical Education in Krakow, al. Jana Pawła II 78, 31-571 Cracow,

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Poland

of Animal Products Technology, Food Technology, University of Agriculture in

of Sports Medicine & Human Nutrition, Institute of Human Physiology,

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Corresponding author:

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Dr. Joanna Tkaczewska

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Ul. Balicka 122

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30-149 Kraków

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Phone: +48 508 984 411

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e-mail adress: [email protected] 1

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Abstract

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The influence of pre-treatment method on the characteristics and gel properties of gelatine

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from Cyprinus carpio L. skin was studied. Gelatine was extracted from the carp skin using

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three methods: (I) NaCl pre-treatment, (II) NaOH and ethanol pre-treatment and (III) NaOH,

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H2SO4, C6H8O7 pre-treatment. The chemical composition and functional properties of gelatine

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were investigated. The gelatines had high protein (75.86 - 82.44%) and relatively high fat

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contents (3.99 - 10.02%). Electrophoresis showed that gelatines I and III contained α- and β-

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chains as the predominant components, while in gelatine I the distinctive bands corresponding

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to the main components of collagen were not observed. Amino acids analysis revealed high

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proline and hydroxyproline content in all the gelatines.

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The gel strength of gelatine III was higher than gelatines I and II. Gelatine I remained in a

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liquid state during the experiment. Moreover, the pre-treatment of carp skin significantly

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affected the colour, pH as well as foam-forming, amino-acids composition, water-binding and

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fat-binding properties. Gelatine III was the only gelatine with properties sufficient for

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industrial applications. The results indicate that C. carpio L. skin by-products can be utilised

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to extract gelatine with potential industrial application as an alternative source to the

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mammalian gelatine.

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Key words: gelatine, pre-treatment method, fish skin, carp, by-products utilisation

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1. Introduction

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Gelatine is a multifunctional material used commonly by the food industry during the

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production of jellies, desserts, ice-creams and meat products, as well as non-food related

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industries such as the pharmaceutical, medical and photographic ones (Boran & Regenstein,

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2010). Gelatine is obtained from collagen by means of hydrolytic degradation. Collagen is

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build from three polypeptide chains, which together form a special helical structure. During

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the gelling process, the gelatine chains undergo a conformational changes and regenerate the

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collagen helical structure forming as thermoreversible helical networks (Bigi, 2004). Global

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demand for gelatine is constantly growing. Worldwide gelatine production in 2015 achieved

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412.7 thousand tonnes, while in 2018 it is estimated to achieve 450 thousand tonnes. In 2016

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the value of the world gelatine production industry was estimated at USD 4.52 billion (TMR,

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2017). Gelatine is mostly produced from pork skin and cattle bones. Fish gelatine is an

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effective alternative source to mammalian gelatine. Its advantages include the consumer’s

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safety, since it is not related to issues connected with cattle gelatine, such as bovine

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spongiform encephalopathy (BSE), and it can be used to produce kosher and halal food

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products (Karim & Bhat, 2009).

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Global aquaculture production increases at a constant rate and in 2015 reached 106

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million tonnes in live weight. It is estimated that in 2025, 60% of world fish production will

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be supplied by freshwater species such as carp, catfish and tilapia (Xiaowei, 2017). Carp (C.

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carpio L) is one of the most commonly cultivated fish species on earth, mainly due to its high

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growth rate and feed utilisation efficiency (Tokur, Ozkütük, Atici, Ozyurt & Ozyurt, 2006).

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On the other hand, the global carp production, which in 2015 reached over 4.3 million tonnes,

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is currently decreasing (FAO, 2017). The main cause for this decline is that carp is mainly

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available for the consumer as a whole fish and cannot compete with highly processed and 3

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much more convenient fish products made of different fish species. To increase the range of

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available carp products, many processors search for alternatives and the carp processing

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industry is developing rapidly (Tokur et al., 2006). One of such alternatives is the production

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of skinless fillets. Such processing however, generates a high amount of by-products, mainly

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skins, which are an excellent source of collagen (Wasswa, Tang & Gu, 2007), that could be

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used to produce gelatine (Jayathilakan, Sultana, Radhakrishna & Bawa, 2012). Therefore, it is

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important to develop the most efficient methods for acquisition of fish gelatine in order to

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maintain efficient utilisation of by-products from fish processing as replacements for

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mammalian sources (Jellouli et al., 2011).

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Pre-treatment of the raw material is one of the essential processes of gelatine extraction,

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affecting the properties of the final product. Gelatine can be extracted from fish skin using

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alkaline or acid pretreatment, a combination of both (Niu et al. 2013, Silva, Bandeira &

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Pinto, 2014) or using chloride salts (Kołodziejska et al. 2008, Monsur, Jaswir, Salleh &

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Alkahtani, 2014) followed by thermal hydrolysis. Fish collagen contains low levels of non

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reducible intra and inter-chain crosslinks, which allows mild acid pretreatment to be sufficient

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for gelatine extraction. The alkali pretreatment on the other hand can be used for removal of

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non-collagen proteins and non-protein compounds and to improve swelling by organic acid

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pretreatment (Monsur, Jaswir, Salleh & Alkahtani, 2014). Because of this the combination of

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both acid and alkali pretreatment provides usually higher yields and better quality of acquired

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gelatine, than if those pretreatment methods are used on their own. This resulted in wider

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popularity of the combined alkali/acid pretreatment methods in recent years (Niu et al. 2013).

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The quality and yield of gelatine extracted using pretreatment with chloride salts, depends on

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the type of salts used. Sodium and potassium chlorides facilitates extraction of α- and β-chain

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polymers, while the application of MgCl2 resulted in lower content of dimers of α-chains,

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trimmers, and greater content polymers. This results in more difficult extraction of gelatine 4

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when using magnesium than sodium and potassium salts

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Montero, 2005a). All the above-mentioned processes suggest that the quality of fish gelatine

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is affected by the pretreatment method used.

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The properties of the gelatine are also affected by the species of fish skin from which the

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gelatine is acquired. Gelatine produced from the skin of cold-water fish can have a lower

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industrial value than gelatine produced from the skin of fish living in moderate or warm water

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temperatures and warm-blooded mammals. This is due to the lower thermal stability of

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collagen, which is affected by the proline and hydroxyproline content in the protein

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macromolecule. The higher the content of those amino acids, the higher the thermal stability

100

(Giménez, Gómez-Guillén &

of the protein (Wang et al., 2008).

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C. carpio L lives in warm waters, therefore the gelatine produced from its skin has an

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indulgently high melting point of about 28 °C and has better gelling properties than gelatine

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produced from cold-water fish (Duan, Zhang, Xing, Konno & Xu, 2011). Although the pre-

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treatment condition have been known to affect the properties of the gelatine from the skin of

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some fish species (Gómez-Guillén et al., 2002; Sinthusamran, Benjakul & Kishimura, 2014),

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no information regarding gelatine extraction from C. carpio L skin under varying pre-

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treatment conditions has yet been reported. Therefore, since there is a dynamic development

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of the carp processing industry, and the gelatine acquired from the skin of this warm-water

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fish has desirable properties, the aim of this study was to determine the effect of carp skin pre-

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treatment on the quality and functional properties of the produced gelatine.

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2. Materials and methods

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2.1 Raw material

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Fish skins from common carp (Cyprinus carpio L) were obtained from Sona Sp. z o.o.

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(Koziegłowy, Poland), where they were treated as a by-product from fillet processing. The 5

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skins for gelatine production were washed at 10 ºC and the residues of the attached flesh were

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carefully removed. Afterwards, the skins were washed again, blended and stored frozen at

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-22 oC until used for gelatine extraction. The frozen skins were thawed at 4 oC and subjected

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to the pre-treatment.

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2.2 Pre-Treatment and gelatine extraction

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Three different pre-treatment methods for gelatine extraction were chosen based on the

121

review of the pre-treatment methods used in the available literature. The methods were chosen

122

based on their simplicity and cost efficiency in order to make them the most suitable for the

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industrial application. The chosen pre-treatment methods are listed in Table 1.

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Table 1. Conditions of pre-treatment methods chosen in the experiment Samples

Pre-treatment conditions

I

2.6% NaCl, 40 min, 16°C

References Kołodziejska, Skierka, Sadowska, Kołodziejski & Niecikowska (2008)

0.1 N NaOH – 6 h , Food grade ethanol 12 h, II

Duan, et al. (2011) 4°C

III

0.2% NaOH 2 h, 0.2% H2SO 4 2 h, 1.0% C6H8O7 2 h, 21°C

Grossman & Bergman (1992)

125 126

2.2.1 Method I: Production of gelatine using sodium chloride pre-treatment

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This method was based on the method described by Kołodziejska, Skierka, Sadowska,

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Kołodziejski and Niecikowska (2008) with modifications. The batch of partially thawed skins

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(0 oC) was treated with 2.6% sodium chloride solution. The skins to NaCl ratio was 1:6 (w/v).

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The process was carried for 10 min at the temperature not exceeding 16 oC, with intensive 6

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stirring on a magnetic stirrer (WIGO MS 11H, Pruszków, Poland). After the extraction the

132

mixture was left for 10 min for sedimentation. Next, the upper layer of the solution was

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collected together with the fat layer gathered on the surface and discarded. The remaining part

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of the solution was poured through cloth with a mesh diameter of 72 µm and centrifuged

135

(BioSan LMC-3000, Riga, Latvia) for 5 min at 1000 g, and the supernatant was removed. The

136

above procedure was carried out twice. Afterwards, the remaining raw material was mixed

137

with tap water using 6:1 (v/w) ratio, stirred for 10 min at the temperature not exceeding 18 oC

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and centrifuged for 5 min at 1000 g, and the supernatant was removed. This step was repeated

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3 times. Then, the material was added to warm (approx. 45 oC) distilled water using the ratio

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of 1:3 (w/v). The extraction of gelatine was carried out for 60 min with constant stirring at 45

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± 1.5 oC. After the extraction was finished, the gelatine solution was separated from the

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insoluble material through filtration, using a double cloth with a mesh diameter of 72 µm.

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Finally, the solution was again filtered through qualitative medium filter paper and dried

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using LYO-QUEST-55ECO lyophilisator (Telstar, Terrassa, Spain). The extractions were

145

performed in triplicate.

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2.2.2 Method II: Production of gelatine using alkali pre-treatment

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This method was based on the method described by Duan et al. (2011) with

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modifications. The skins were mixed with 0.1 M NaOH for 6 h with continuous stirring at a

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sample/alkali solution ratio of 1:3 (w/v) to remove non-collagenous proteins. The alkali

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solution was changed every 3 h. Next, the samples were washed with cold distilled water,

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until neutral pH of the washing water was obtained. The skins were then soaked using food

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grade ethanol (95.6%) with a solid/solvent ration of 1:2 (w/v), left overnight to remove fat and

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washed with cold distilled water repeatedly. All the procedures were carried out at 4 °C. The

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gelatine was extracted from the pre-treated skins using a solid/distilled water ratio of 1:3 7

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(w/v) for 4 h at 45 ± 1.5 °C. Afterwards, the gelatine was collected by filtration and

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lyophilised (LYO-QUEST-55ECO, Telstar) as described for Method I. The extractions were

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performed in triplicate.

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2.2.3 Method III: Production of gelatine using diluted alkali and organic and inorganic

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acids pre-treatment

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This method was based on the method described by Grossman & Bergman (1992) with

161

modifications. The skins were soaked in 0.2% NaOH for 2 h at a sample/alkali solution ratio

162

of 1:6 (w/v). Then the alkali-treated skins were washed with distilled water at 10 ºC until

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reaching the pH of approx. 7, and soaked in 0.2% H2SO4 for 2 h at a sample/acid solution

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ratio of 1:6 (w/v). Next, the mineral acid-treated skins were washed with distilled water at 10

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ºC until the washings had a pH of about 7, and soaked in 1.0% aqueous citric acid for 2 h at a

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sample/citric acid solution ratio of 1:6 (w/v). After that, the citric acid-treated skins were

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again washed with distilled water at 10 ºC until the washings had a pH of about 7, and

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subjected to a final wash with distilled water to remove any residual salts. The pre-treated

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skins were placed in a vessel containing distilled water and extracted at a temperature of 45 ±

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1.5 °C. Following overnight extraction, the mixture was filtered and then lyophilised for

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complete removal of moisture, as described for Method I. The extractions were performed in

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triplicate.

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2.3 Proximate composition and yield of the extracted gelatine

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Moisture, lipid, ash and protein were determined using the AOAC (2007) in raw skins,

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skins after pretreatment and in gelatines. A conversion factor of 5.4 was used for calculating

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the protein content in gelatin from the Kjeldahl nitrogen content according to Muyonga, Cole

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and Duodu (2004). 8

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The yield (%) was calculated as (freeze-dried extract (g) / wet fish skins (g)) × 100.

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Hydroxyproline was determined after hydrolysis of the material in 6 M HCl for 6 h at

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105 °C, using the colorimetric method recommended by Bergman and Loxley (1963). To

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convert the amount of hydroxyproline to collagen, a factor of 11.42 was used (Sato, Ohashi,

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Ohtsuki & Kawabata, 1991).

183

184

2.4 pH value The pH value of 6.67% (w/v) gelatine solution in distilled water was determined

185

according to

186

prepared by mixing 7.5 g of the extracted gelatine and 105 mL of warm distilled water

187

(60 °C) in a bloom bottle. As soon as the temperature of the solution equilibrated to room

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temperature, the pH of the gelatine solution was measured using an Elmetron CP-411 pH

189

meter (Zabrze, Poland).

190

Tongnuanchan, Benjakul and Prodpran (2012). The gelatine solution was

2.5 Colour measurement

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The colour of the gelatine gels (6.67% w/v) was measured by a CR 200 Minolta

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Chromameter (Osaka, Japan) and calculated using the CIE system. L∗, a∗ and b∗ parameters

193

indicating lightness/brightness, redness/greenness and yellowness/blueness, respectively. The

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colorimeter was calibrated with a white standard before analysis.

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2.6 Determination of the bloom strength and Texture Profile Analysis of the gelatine

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gels

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The gelatine (7.5 g) was weighed into a Bloom jar and mixed with 105 mL distilled

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water. The solution (6.67%) was stirred with a glass rod, covered and allowed to stand at

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room temperature for 3 h. After this time, the mixture was heated in a beaker containing water 9

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to approx. 65 °C (but not exceeding) on a magnetic heater stirrer for 20 min to dissolve the

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gelatine completely. The jar was covered and allowed to cool for 15 min at room temperature

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(22 °C). The Bloom jars were kept in a 10 °C water bath overnight (17 h), and immediately

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tested using a TA-XT2 texturometer (Stable Micro Systems, Godalming, UK) by penetration

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with a standard 0.5 radius cylinder (P/O.5R) probe. After a trigger force of 4 g was attained,

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the probe proceeded to penetrate into the gel to a depth of 4 mm. At this depth the maximum

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force reading was obtained and translated as the 'Bloom Strength' (g) of the gel. For TPA

207

analysis a P/100 probe was used. The gel was subjected to two cycle compression to 40% of

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its original height (15 mm). The detailed pretest speed : 1. 0 mm/s, test speed 5mm/s, trigger

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force 0.05 N (Sow &Young, 2015). The TPA analysis have been made in temperature 4°C.

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The analysis was done in triplicate.

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2.7 Micro- and macroelements analysis

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The micro- and macroelements content, with the exception on mercury, was analysed

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using ICP-OES according to the method described by Kulawik et al. (2015) with

214

modifications. Half a g of the sample was mineralised with 30% HCl and concentrated HNO3

215

(Suprapur, Merck KGaA, Darmstadt, Germany). Mineralisation was carried out in an Anton

216

Paar microwave oven (Graz, Austria) at 1400 W, after which the samples were subjected to

217

ICP-OES analysis using the Perkin-Elmer ICP-OES 7300 Dual View apparatus (Perkin-

218

Elmer, Waltham, USA). Wavelengths, detection limits and recovery rates for individual

219

elements are shown in Table 2. The recovery rates were determined using the NCS DC733448

220

certified material.

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Table 2. Wavelengths and detection limits for individual elements measured by ICP Element

Wavelength [nm]

Detection limit

10

Recovery rate [%]

ACCEPTED MANUSCRIPT [mg/l] Fe

238.204

0.0046

106.1

Zn

206.200

0.0059

106.3

Ni

231.604

0.0150

109.0

Cd

327.393

0.0097

121.4

Mn

257.610

0.0014

87.9

Cr

267.716

0.0071

91,3

Ca

317.933

0.0100

98.2

Mg

285.208

0.0016

96.5

Na

589.592

0.0690

95.0

P

213.617

0.0760

98.8

K

766.490

0.0090

89.9

Pb

220.355

0.0420

100.4

Cu

327.393

0.0097

98.3

222 223

The mercury content was analysed using an Advanced Mercury Analyser AMA-254

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(Spectro-Lab, Łomianki, Poland). The sample weight was 30 mg and the absorbance was

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measured at 254 nm (Costley et al., 2000). The detection limit for mercury was 0.01 ng.

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2.8 Electrophoretic analysis

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One g of sample was homogenised with 20 ml distilled water. Homogenates were diluted

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(1:1) with a denaturing buffer (0.125 M Tris, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol,

229

pH 6.8) and heated for 90 s in a boiling water bath. The extracts were centrifuged for 3000 g

230

for 3 min (Centrifuge MPW-352R) and the clear supernatant was collected. SDS-PAGE was

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carried out according to the method by Laemmli (1970) using a 12% w/v gel concentration.

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2.9 Amino acids analysis 11

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The amino acids composition was determined by means of RP-HPLC using the Waters

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ACCQ-Tag Ultra Derivatization kit (186003836, Waters, Milford, MA, USA). Thirty mg of

235

sample was hydrolysed using 4 mL of 6M HCl and 15 µL of phenol at 110 oC for 24 h. The

236

sample was sealed in nitrogen atmosphere during the process of hydrolysis. The acquired

237

hydrolysate was filtered using a syringe filter with a pore diameter of 45 µm and dried under a

238

constant stream of nitrogen. Such prepared samples were diluted accordingly and derivatised

239

by mixing 10 µL of sample with 70 µL of boron buffer (pH in the range of 8.2 - 9.0) and 20

240

µL of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) in a concentration of 3 mg

241

of ACQ/mL of acetonitrile. The standards were prepared in the same manner as the samples.

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The separation was carried out using the Dionex Ultimate 3000 HPLC system (Thermo

243

Scientific, Waltham, MA, USA) equipped with an LPG-3400 SD four channel gradient pump,

244

WPS 3000 TSL autosampler and VWD 3400RS four channel UV/VIS detector. Analysis was

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performed using a Nova-Pak C18, 4 µm (150 x 3.9 mm) column (Waters, USA). The elution

246

procedure used acetate-phosphate buffer (Eluent A) and 60:40 acetonitrile/water (Eluent B)

247

according to the procedure recommend by Waters (USA). The separation temperature was set

248

at 37 oC Detection was carried out at 240 nm wavelength. The quantitative analysis was

249

performed using 1-point calibration using the analytical standards (100 pmol for each

250

concentration)

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2.10 Functional properties

252

The functional properties of gelatine were measured by a partially modified method of

253

Cho et al. (2004). To measure foam formation ability, half a g sample was placed in 50 ml

254

distilled water and left for 15 min for swelling. The sample solution was dissolved at 60 °C

255

and the foam was prepared by homogenising the mixture at 10,000 rpm for 5 min (Unidrive X

256

1000, CAT Scientific, Paso Robles, CA, USA). The homogenised solution was poured into a 12

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250 mL measuring cylinder. The foam formation ability was calculated as the volume ratio of

258

foam to liquid. The foam stability was calculated as the ratio of the initial volume of foam to

259

the volume of foam after 30 min.

260

For measuring water-holding capacity and fat-binding capacity, 50 ml of distilled water

261

or 10 ml of sunflower oil were added to 1 g of gelatine andkept at room temperature for 1 h.

262

The gelatine solutions were mixed with a vortex mixer for 5 s every 15 min. Next, the

263

solutions were centrifuged at 450 xg for 20 min. The upper phase was removed and the

264

centrifuge tube was drained for 30 min on a filter paper after tilting to a 45° angle. The water-

265

and fat-holding capacities were calculated as the weight of the contents of the tube after

266

draining divided by the weight of the dried gelatine, and expressed as the wt % of the dried

267

gelatine.

268

2.11 Rheological properties

269

The gelatins were prepared in the same way as for the Bloom strength analysis. The

270

solutions were kept in the water bath (65°C) until analyzed. The measurements were made

271

using RS6000 rheometer (Haake, Germany). A plate-plate sensor was used. The lane

272

parameter was as follows: diameter 60 mm. The gap size was selected on the basis of the

273

preliminary studies, and adjusted to 0.5 mm. Rheological studies relied on the

274

measurement of the values of the complex modulus G* as a function of frequency, within

275

the range of 0.1 to 10 Hz at 5°C to 30°C. DMTA investigation were performed in

276

temperature range: for cooling from 30°C to 6°C and for heating from 6°C to 30°C and

277

ware conducted for the strain amplitude γ = 0.005 and frequency f=1Hz. The first step was

278

to determine the area of linear viscoelasticity (Ferry, 1980; Tschoegl, 1989). This test

279

relies, in the case of frequency domain measurements, on measurements of the absolute

280

value of complex modulus as |G*()| function of deformation amplitude at constant 13

ACCEPTED MANUSCRIPT 281

frequency. Linear viscoelasticity area determines the range of deformation amplitude (o),

282

for which G*(o) values are parallel to axis of abscissa (o axis). Analyzed for all the

283

systems, the measurements of linear viscoelasticity range were done at extreme values of

284

frequency. It allowed to determine the value of o = 0.01, common for all the systems,

285

which was used in the following investigations. For each gelatine G’(f) and G”(f)

286

measurements were done in triplicate.

287

2.12 Statistical analysis

288

All analyses were performed in triplicate and the data was subjected to a statistical

289

analysis using STATISTICA 12 software (Dell Software, Tulsa, OK, USA). The normality of

290

the results was established using the Shapiro–Wilk test. One-way analyses of variances

291

(ANOVA) were performed to compare the results between the groups. The significance of the

292

differences between means (P < 0.05 or P < 0.01), was established using the Tukey post-hoc

293

test. The results are presented as average ± standard deviations.

294 295

3. Results and discussion

296

3.1 Yield and proximate composition of the extracted gelatine and skin after

297

pretreatment

298

The yield of the extracted gelatine depended significantly on the skin pretreatment

299

method. The highest yield, 12.00% of the skins wet weight, was obtained using the method

300

III, while the yield obtained by method II was 10.47%. The lowest yield, 5.21% was obtained

301

using method I. According to Gómez-Guillén et al. (2002), and Zhou & Regenstein (2005)

302

gelatine production yield from fish skin depends on the fish species and pretrement method

303

used. For example, the yields acquired from the skin of sole, megrim, cod and hake were 8.3, 14

ACCEPTED MANUSCRIPT 304

7.4, 7.2 and 6.5%, respectively. Grossman & Bergman (1992) report that the yield of gelatine

305

production from tilapia, while using pre-treatment method III, was 15%, while Muyonga et al.

306

(2004) achieved a 12.3% yield while treating the skins of Nile perch with diluted sulphuric

307

acid, using a low ratio of material to solution (approx. 1:2 w/v) and four step extraction of

308

gelatine. Much lower yields, ranging from 5.39 - 7.81%, were reported from tilapia skins by

309

Jamilah and Harvinder (2002), who used pre-treatment method III.

310

Cho, Jahncke, Chin and Eun (2006) performed the optimisation of gelatine production

311

from the skin of skate, and found the best yield of 17% when the skins were pre-treated with

312

1.5% solution (w/v) of Ca(OH)2 and extracted with water (1:3 w/v) at pH 6 and 50 oC for 4 h.

313

The washing of salmon skins with NaCl solution resulted in a 12.6% yield of gelatine

314

production. When the same method was applied to the skins of smoked salmon and

315

marinated, salted herring, the yield was 25 and 3.5%, respectively (Kołodziejska et al., 2008).

316

In this study, the yield of skins subjected to the same pretreatment as described by

317

Kołodziejska et al. (2008), was 5.21% which indicates that not only the pretreatment method

318

but also the fish species influences the gelatine formation yield. The proximate composition of the extracted gelatines and skin after pre-treatment is

319 320

summarised in Table 3.

321

Table 3. Chemical composition and pH of raw material, skins after pre-treatment and

322

gelatines (Mean value ± standard deviation)

Content [%]

Skin Dry weight

Skin I

Skin II

Skin III

49.97±0.66 36.81c±0.82 22.61a±1.03 29.78b±1.4

Gelatine I

Gelatine II

Gelatine III

91.63b± 0.4 94.53c±0.27

87.95a±0.06

Total protein

18.21±0.43 15.64c±0.63 6.58b±0.44

3.67a±0.05 75.86a±0.52 82.44c±0.49

77.87b±0.34

Collagen*

16.48c±0.46 14.03b±0.82 5.39a±0.02

1.47±0.56

70.99a±0.40 82.36c±1.52

77.01b±1.27

Lipids

28.50±0.23 17.99a±0.82 15.43a±1.9 25.17b±1.80 10.02b±0.2

15

9.82b±0.03

3.99a±0.2

ACCEPTED MANUSCRIPT Ash

0.26±0.02

0.11a±0.03

0.13a±0.01

0.00

1.91c±0.03

0.92b±0.01

0.30a±0.01

Carbohydrates

2.92±1.36

3.08c±0.98

0.47a±0.15

0.94b±0.56

3.84b±0.42

1.35a±0.44

5.78c±0.15

pH

-

-

-

-

6.94b±0.01

9.28c±0.02

3.18a±0.01

323 324

*The conversion factor for calculating the content of skin collagen from hydroxyproline was

325

11.34 according to Sato et al. (1991)

326 327 328 329

Different letters (a, b, c) indicate significant differences at p <0.05

330

treatment method. The highest protein content was found in gelatine pre-treated with weak

331

alkali (gelatine II), and the lowest when the skins were washed with sodium chloride (gelatine

332

I). According to Jongjareonrak, Benjakul, Visessanguan and Tanaka (2006), the crude protein

333

content reported for fish skin gelatine from different fish species is in the range of 87 - 89%.

334

The total protein content in the residual skin treated by method III was lower (3.67%) than

335

that in the residual skin after pretreatment II ( 6.58%) and I (15.64%). Similar trend could be

336

observed in collagen content in residual skins. The highest collagen content was observed in

337

skins after subjecting to pretreatment I (14.03%), followed by skins subjected to pretreatment

338

II (5.39%) and the lowest in skins subjected to pretreatment III (1.47%). Skins treated with

339

weak alkali and weak acid solutions had the most swelled structure of all the studied raw

340

materials, which eased the extraction process. Skins treated with high ionic strength and low

341

pH acid could facilitate the swelling process caused by more repulsive force among the

342

collagen molecules compared with other method. With the loosen structure of swollen

343

collagen, warm water could penetrate into the skin matrix effectively, acid pretreatment more

344

likely provided a greater swelling power for destabilization of acid labile cross-links at the

345

telopeptide region and amide bonds of the triple helical structure of collagen as well as non-

The chemical composition of the studied gelatines differed significantly depending on the pre-

16

ACCEPTED MANUSCRIPT 346

covalent intra- and inter-molecular bonds, compared with other preatretment method [Ahmad

347

& Benjakul, 2011]. The high protein and collagen content in skin after pre-treatment I suggest

348

that the extraction processes was not effective. Despite achieving relatively high levels of

349

hydroxyproline (calculate to collagen as 70.99%) in gelatine I, the process yield is not

350

satisfactory.

351

The studied gelatines contained high fat content; however, gelatine III contained significantly

352

lower fat content than gelatines I and II. The high level of fat in the produced gelatines is due

353

to the high levels of fat in the carp skins and it is difficult to eliminate lipids during the

354

extraction procedure. The ash content of the studied gelatines varied from 0.30 - 1.91%.

355

Gelatine of good quality should not contain more than 0.50% of ash (Ockerman & Hansen,

356

1999), and only gelatine III meets these criteria. On the other hand, all the produced gelatines

357

contained lower ash content than the maximum ash content recommended for carp skin

358

(2.6%) (Jones, 1977) and the limit given for edible gelatine (2%) (GME, 2005). Relatively

359

low ash content both in gelatines and in residual skins indicates that inorganic salts that might

360

have been created during the pretreatment phase were washed out into the pretreatment

361

solution.

362

All the extracted gelatines were high-protein products with relatively high fat and ash

363

content. Comparing the residue levels in the raw material and the final product, it can be

364

concluded that the efficiency of residues removal through pre-treatment methods used in this

365

study was low, wherein the highest quality gelatine was produced using method III.

366

Wangtueai and Noomhorm (2009) used an alkali pre-treatment method to acquire gelatine

367

from Saurida spp. scales with water, protein, fat and ash content of 10.5, 86.9, 0 and 2.3%,

368

respectively. The gelatine acquired from skate skin using the alkali pre-treatment contained

369

92% of protein, 1.4% of ash, 0.35% of lipids and 4.5% of water (S.-H. Cho et al., 2006). The

370

carp skin gelatine acquired by Ninan, Joseph and Aliyamveettil (2014), using the same pre17

ACCEPTED MANUSCRIPT 371

treatment method as in method III, contained 8.5% of moisture, 90.4% of protein and 1.2% of

372

ash. Based on the results, it can be concluded that the pre-treatment method has significant

373

effect on the chemical composition of the produced gelatine.

374 375

3.2 pH value

376

The pH of the gelatine solution reflects the chemical treatment used during the

377

extraction stage (da Trindade Alfaro, Balbinot, Weber, Tonial & Machado-Lunkes, 2015).

378

The pH of the studied gelatines differed significantly (Table 3). These differences probably

379

result from the type of reagents used, with gelatine III having acidic, gelatine I neutral and

380

gelatine II alkali pH. The pH of the acquired gelatine is an important parameter given that the

381

functional properties of the produced gelatine are dependent on the pH (Karim & Baht, 2009).

382

Due to this, the possible use of the produced gelatine relies on the pre-treatment method used.

383 384

385

3.3 Colour measurement Table 4. Color of analysed gelatines (Mean value ± standard deviation) L*

a*

b*

Gelatine I

71.14c±0.40

0.30b±0.03

6.72a±0.16

Gelatine II

68.25a±0.70

0.86c±0.09

17.92c±0.17

Gelatine III

69.25b±0.59

-1.06a±0.06

11.66b±0.35

Different letters (a. b. c.) indicate significant differences at p <0.05

386

There were significant differences in all the colour parameters of the produced gelatines

387

(Table 4). The lowest lightness was observed in gelatine II, and the highest in gelatine I. The

388

lowest redness was observed in gelatine III, which means its colour was the closest to the

389

yellow-green colour. Gelatine II was a darker yellow than the other gelatines, showing the 18

ACCEPTED MANUSCRIPT 390

highest b* and lowest L*. The dark colour of gelatine is commonly caused by inorganic,

391

protein and mucosubstance contaminants introduced or not removed during its manufacture

392

(Avena‐Bustillos et al., 2006). All the gelatines were slightly yellow in colour, as

393

characterised by the values of the b* parameter. According to Ninan, Jose and Abubacker

394

(2011), the colour of the gelatine is dependent on the raw material used; however, since

395

gelatines produced in this study were acquired from the same batch of raw material, the

396

colour of the gelatine is also significantly affected by the pre-treatment method used.

397

3.4 Determination of the bloom strength and Texture Profile Analysis of the gelatine

398

gels

399

Gel strength is the most important attribute of gelatine and determines the quality of the

400

produced gelatine. The bloom strength of the carp gelatine was highly diverse and ranged

401

from 5.68 Bloom (gelatine I) to 267.08 Bloom (gelatine III) (Table 5). Gelatine I, during the

402

whole experiment, remained in a state of liquid solution which might be due to the presence

403

of mostly low molecular weight protein (Fig 1). The low hydroxyproline content of fish skin

404

gelatine is a main reason for the low gel strength or no gelling since the hydrogen bonds

405

between water molecules and the free hydroxyl groups of amino acids in gelatine are essential

406

for high gel strength (Arnesen & Gildberg, 2002). Although the hydroxyproline content in

407

gelatine I is relatively high, in this case this amino acid is not bound in the form of β, α1 and

408

α2 collagen chains but is a part of low molecular weight proteins (Fig. 1) which resulted in no

409

gelling properties. Dowgiałło (2013) used the same pre-treatment method to produce gelatine

410

from salmon skins and also reported no gelling properties of obtained gelatine (56 Bloom). It

411

is possible that the residues of this compound prevent gel formation. The addition of NaCl to

412

gelatine increases the ionic strength of the solution which can result in a reduction of

413

electrostatic bridges of α-chain due to the screening effect of the short range electrostatic 19

ACCEPTED MANUSCRIPT 414

interactions (Sow & Yang, 2015). Another possible explanation may be the lower content of

415

high molecular weight compounds in gelatine I than in gelatine II and III. Lack of gel-forming

416

properties of gelatine I discards the use of pre-treatment method I for the industrial

417

application.

418

Wangtueai & Noomhorm (2009) acquired gelatine extracted from lizardfish scales with a

419

gel strength of 252 g, using method II for pre-treatment, which is much higher than the gel

420

strength of gelatine II acquired in this study. The reported gel strength of gelatine extracted

421

from tilapia using pre-treatment method III ranged from 128 - 263 g (Grossman & Bergman,

422

1992; Jamilah & Harvinder, 2002) and the Bloom value of gelatine produced from carp skin

423

using the same pre-treatment was also lower (181 g) than that acquired in this study (Ninan et

424

al., 2011). A higher content of amino acids with free hydroxyl groups (hydroxyproline, serine.

425

threonine and tyrosine) may contribute to more hydrogen bonds, which in turn increases the

426

gel strength.

427

The above-mentioned differences in gel strength could be explained by differences in the

428

manufacturing process used and the intrinsic properties of collagen which varies among fish

429

species. According to Mohtar, Perera and Quek (2010), the gel strength of fish gelatine ranges

430

from 0 - 426 g, while gel strength of pork and bovine gelatine usually ranges from 200 - 300

431

g. The gel strength of gelatine acquired from warm-water fish is usually close to the gel

432

strength exhibited by gelatine from warm-blooded animals, in contrast to cold-water fish

433

gelatine. The gelling strength of commercial gelatines ranges from 100 - 300 g, but gelatines

434

with Bloom values of 250 - 260 are the most desirable (Karim & Baht, 2009). Only gelatine

435

produced using pre-treatment method III showed sufficient gel strength to be used in

436

industrial applications.

437 438

The texture parameters of gelatin II and gelatin III are presented in table 5. Gelatine I was not analysed as it did not have any gelling properties. 20

ACCEPTED MANUSCRIPT 439

Table 5. TPA parameters and gel strength of analysed gelatines (Mean value ± standard

440

deviation) Chewiness [g]

Cohesiveness

Hardness [g]

Springiness

Gelatin I

-

-

-

-

Gelatin II

226.48a ± 20.90

0.99a ± 0.04

212.95a ± 16.80

0.94a ± 0.01

Gelatin III

429.13b ± 26.46

0.95a ± 0.06

366.31b ± 43.26

0.90b ± 0.01

Bloom value 5.68a±1.02

158.70a±3.70

267.08a± 14.9

441

Different letters (a. b. c.) indicate significant differences at p <0.05

442

Hardness is a parameter referring to the strength of the gel structure during compression. It is

443

a force that is necessary to reach certain deformation. Cohesiveness is a measurement of the

444

degree of difficulty in breaking down the gel's internal structure. Springiness (also called

445

“elasticity”) is the rate of achieving the original state. Chewiness is the energy needed to

446

masticate a solid food to a state ready for swallowing, in TPA tests it is calculated based on

447

hardness, springiness and cohesiveness values (Breene, 1975). The gelatin is considered better

448

if those qualities are high. There were significant differences between those two products. The

449

results of the gel strength analysis were confirmed by TPA tests. Gelatin III was harder, more

450

chewy, but the cohesiveness was lower. The springiness parameter was comparable in both

451

analysed gelatins. Similarly Sow and Young

452

springiness between fish gelatins with sodium chloride or sucrose. The hardness values for

453

both gelatin II and III are much lower compared to other fish gelatins obtained by various

454

authors (Sow&Young, 2007; Wangtueai & Noomhorm, 2009; Boran, Lawless and

455

Regenstein, 2010; Yang et al., 2007).

456

457

3.5 Micro- and macroelements analysis 21

(2015) did not detect any difference in

ACCEPTED MANUSCRIPT 458

The statistical analysis of mineral composition showed significant differences between the produced gelatines (Table 6).

460

Table 6 . Mineral composition of carp skin and gelatines

461 462 463

[mg/kg dry matter]

Heavy metals

Microelements [mg/kg dry matter]

Macroelements [g/kg dry matter]

459

Skin

Gelatine I

Gelatine II

Gelatine III

Na

0.348±0.017

1.121c ±0.030

0.617b±0.06

0.342a±0.170

Mg

0.060±0.005

0.047a±0.002

0.211c±0.000

0.190b±0.030

K

0.518±0.064

0.140a±0.007

0.145a±0.017

0.198b±0.014

Ca

0.380±0.126

0.456a±0.038

1.030b±0.042

1.587c±0.058

P

0.556±0.047

0.246c±0.008

0.212b±0.003

0.146a±0.003

Cr

0.658±0.225

0.491a±0.171

0.581ab±0.051

0.848b±0.101

Mn

0.626±0.240

0.352a±0.08

0.324a±0.062

0.611b±0.075

Fe

12.109±2.971

5.850a±1.306

6.980a±1.130

14.798b±1.108

Ni

0.177±0.073

0.162a±0.021

0.140a±0.013

0.563b±0.056

Zn

31.837±0.168

16.897b±3.309

23.497c±0.815

8.452a±0.545

Cu

0.851±0.168

0.753a±0.199

0.897a±0.082

0.647a±0.065

Cd

n/d

n/d

n/d

n/d

Pb

n/d

n/d

n/d

n/d

Hg

0.024±0.004

0.030b±0.008

0.014ab±0.012

0.004a±0.000

Results expressed as maean value ± standard deviation Different letters (a. b. c.) indicate significant differences at p <0.05. n/d – not detected 22

ACCEPTED MANUSCRIPT 464 465

The major mineral present in gelatine II and III was calcium with levels of 1.030 g/kg dw

466

and 1.587 g/kg dw respectively. Gelatine I contained the lowest levels of calcium but the

467

sodium content in this gelatine was almost twice as high as in other gelatines, which is

468

probably due to the residue of sodium chloride which was used during the pre-treatment

469

phase. Gelatine II had the highest magnesium content, with levels close to magnesium levels

470

of pork gelatine (0.214 g/kg dw) (Savadkoohi, Hoogenkamp, Shamsi & Farahnaky, 2014).

471

According to Benjakul, Oungbho, Visessanguan, Thiansilakul and Roytrakul (2009), fish

472

gelatines contain low levels of magnesium. The authors reported magnesium levels of 170 -

473

570 mg/kg of product in fish gelatine from Priacanthus tayenus and Priacanthus

474

macracanthus. Gelatine III contained significantly higher levels of Ca, K, Cr, Fe, Mn and Ni,

475

which is surprising since treating the fish skin with mineral acids should increase the removal

476

of minerals from the gelatine (Akagündüz et al., 2014).

477

The content of all the microelements in gelatine III differed significantly from the other

478

studied gelatines. The microelements content in gelatine I and II were Zn > Fe > Cu > Cr

479

>Mn > Ni, while gelatine IIIcontained more Zn than Fe and more Cr than Cu. Metallic

480

compounds are easily dissolved in rainwater getting into water and soil systems and finally

481

reaching the water environment. The metals also accumulate in bottom sediments where

482

numerous invertebrates live. These invertebrates are one of the mains constituents of the

483

carps’ diet (Protasowicki, 1991).

484

Neither cadmium nor lead was detected in any of the studied gelatine samples. Mercury

485

content in all the studied samples was low ranging from 0.004 - 0.030 mg/kg dw, with the

486

lowest content found in gelatine III. Castro-González and Méndez-Armenta (2008) reported

487

that carp muscle contains 0.016 mg of Cd/kg, 0.11 - 0.28 mg of Hg/kg, 0.21 - 0.43 mg of

488

Pb/kg and 0.16 - 0.17 mg of As/kg. These results are much higher than the results acquired in 23

ACCEPTED MANUSCRIPT 489

this study, however carp from Polish aquacultures are not associated with high heavy metal

490

contamination (Tkaczewska & Migdal, 2012)

491

The results show that carp skins do not accumulate high levels of heavy metals and the

492

gelatine produced from those skins is a product which does not contain high levels of those

493

contaminants.

494

495 496

3.6 Electrophoretic analysis The approximate molecular weight distribution of the gelatines from the carp achieved by the three different methods is compared and presented in Figure 1.

497 498

Fig 1. Electrophoretic analysis of the gelatines.

499

S- standard; I – geltaine I; II – gelatine II, III- gelatine III

500

The maximum molecular weight of gelatines I, II and III was 50, 200 and 200 kDa,

501

respectively. The minimum molecular weights were < 5 kDa, >30 kDa and > 25 kDa, 24

ACCEPTED MANUSCRIPT 502

respectively. In addition, gelatines II and III showed the typical electrophoretic patterns of

503

type I collagen, including α-chains, β-chains and other high molecular weight aggregates.

504

High levels of α-chain polymers (dimmers and trimmers) allow better stabilisation and the

505

formation of more organised triple helical structures, which explains the higher gel strength of

506

gelatines II and III than gelatine I. Gelatines II and III contained additional bands with

507

molecular weight < 100 kDa below the α-chain, which could be the results of basic collagen

508

chains hydrolysis or the residues of non-collagenous proteins (Kołodziejska et al., 2008).

509

In the case of gelatine I, the distinct bands corresponding to the main components of

510

collagen were not observed, even at higher protein concentrations. Over the whole length of

511

the gel only a smudged band was visible. Gelatine I showed faint bands at molecular weights

512

of approximately 40 and 50 kDa, and intensive bands corresponding to molecular weight < 5

513

kDa. These results indicate advanced hydrolysis of collagen. Probably some small molecular

514

weight products of hydrolysis were also present but not stopped in the gel (Kołodziejska et al.,

515

2008; Mohtar et al., 2010). Sato et al. (1987) found that naturally present proteases might

516

hydrolyze collagen during the isolation of collagen from fish. Zhou & Regenstein (2005)

517

reported that the pretreatment of fish skin with alkali or acid can significantly inhibit protease

518

activity and decrease the enzymatic degradation of gelatin extracts. Based on those results, it

519

can be assumed that the skin pretreatment with 0.45M NaCl is too mild and does not result in

520

the inhibition of proteases which are naturally present in fish skin, resulting in higher degree

521

of collagen hydrolysis. It is also possible that pre-treatment method I is able to extract only

522

low molecular weight protein from the skin. This is the other reason discards the use of pre-

523

treatment method I for the industrial application.

524

Nikoo et al. (2014) acquired gelatine from Japanese sturgeon using pre-treatment with

525

acid in different concentrations. They reported that the changes in acid concentration during 25

ACCEPTED MANUSCRIPT 526

the pre-treatment phase did not affect the electrophoretic protein separation. On the other

527

hand, Sha et al. (2014) reported that the pre-treatment conditions affected the molecular

528

weight distribution of the produced gelatine. Research performed by Weng, Zheng and Su

529

(2014) showed bands of protein with molecular weight < 80 kDa on the electrophoregram of

530

tilapia gelatine produced using alkali environment (pH 9), while the same bands were not

531

visible when gelatine was pre-treated with acids (pH 3 and 5). These protein fractions might

532

be the result of the degradation of the internal peptide bonds during the gelatine production.

533

Low molecular weight peptides ( < 100 kDa) from fish gelatine can be acquired by sodium

534

hydroxide and acid pre-treatment, as well as by using acid pre-treatment alone (Binsi,

535

Shamasundar, Dileep, Badii & Howell, 2009; Giménez, Gómez-Guillén & Montero, 2005b).

536

Gelatine III, prepared with sulphuric and citric acid pre-treatment, contained a number of

537

components with molecular weight < 100 kDa. Zhang, Wang, Herring and Oh (2007) treated

538

catfish skins with six selected pre-treatment methods. They reported that almost all the

539

gelatines extracted by the different pre-treatment methods exhibited very weak and broad

540

bands around 120 kDa, indicating that α-chains might degrade to smaller chains during the

541

extraction. During the process of converting collagen to gelatine, the breakage of the

542

interchain chemical bonds, and to some extent of intra-chain polypeptide bonds, occurs that

543

results in the production of gelatines with a wide range of molecular weight

544

3.7 Amino acids analysis

545

The amino acids composition of the gelatines acquired with different pre-treatment methods

546

differed significantly (Table 7).

547

Table 7. The amino acid composition of carp gelatines expressed as residues per 1000 total

548

amino acid residues Amino acid

Skin

26

Gelatine I

Gelatine II Gelatine III

ACCEPTED MANUSCRIPT [g/100g skin]

[residues/1000 residues]

Aspartic acid (Asp)

1.35

6.23a

7.22b

15.74c

Serine (Ser)

1.07

13.47a

13.33a

21.55b

Glutamic acid (Glu)

2.06

14.41a

16.01a

32.35b

Histidine (His)

0.06

6.90b

2.76a

10.33c

Glycine (Gly)

5.40

302.92a

313.21a

351.70b

Arginine (Arg)

1.34

78.29b

81.44b

73.57a

Threonine (Thr)

0.56

43.45b

34.51a

33.82a

Alanine (Ala)

1.49

169.69b

175.00b

147.16a

Proline + hydroxyproline (Prol+Hyp)

1.84

175.94b

185.21c

144.86a

Tyrosine (Tyr)

0.12

4.08c

3.16b

2.56a

Valine (Val)

0.49

36.01b

33.05a

32.92a

Methionine (Met)

0.51

24.23a

23.18a

24.20a

Lysine (Lys)

0.84

43.98a

43.31a

42.16a

Isoleucine (Ile)

0.39

20.00c

17.72b

16.88a

Leucine (Leu)

0.63

35.82b

29.50a

30.05a

Phenylalanine (Phe)

0.43

24.54b

21.41a

20.15a

549

Different letters (a. b. c.) indicate significant differences at p <0.05

550

Glycine was the most abundant amino acid present, with the highest content observed in

551

gelatine III (351.70 residues / 1000 residues). Glycine content in gelatines I and II was

552

similar. Around 60% of α-chains consist of tripeptides having the general formula Gly-X-Y, 27

ACCEPTED MANUSCRIPT 553

where X is generally proline and Y is mainly hydroxyproline (Jellouli et al., 2011). The

554

gelatine acquired from grey triggerfish skin using combined pre-treatment with 0.2 M NaOH

555

and acetic acid contained lower glycine levels (289 residues / 1000 residues) than carp

556

gelatine (Jellouli et al., 2011). The content of proline and hydroxyproline in gelatines I and II

557

(175.94 and 185.21 residues per 1000 residues receptivity) was higher than the levels reported

558

in salmon skin (166 residues per 1000 residues) (Arnesen & Gildberg, 2007), but lower than

559

the content reported by Sinthusamran et al. (2014) in seabass skin gelatine (198 - 202 residues

560

per 1000 residues). The results acquired in this study are in accordance with the data by da

561

Trindade Alfaro et al. (2015), who reported that fish gelatines from warm-water fish contain

562

about 189 hydroxyproline and proline residues per 1000 amino acid residues. This suggests

563

that carp gelatine can be a good substitute for gelatine produced from warm-blooded animals.

564

The high content of imino acids in the gelatine leads to physical properties similar to gelatine

565

extracted from mammalians, with higher melting and gelation temperature.

566

Gelatines I and II had a high content of alanine with 169.69 and 175 residues per 1000

567

residues, respectively. Gelatine III contained less alanine than others gelatines (147.16

568

residues per 1000 residues). Gelatine I and II contained a higher content of arginine (78.29

569

and 81.14 residues per 1000 residues, respectively) than gelatine III (73.57 residues per 1000

570

residues).

571

The lysine content was similar in all the tested gelatines. The aspartic acid, histidine and

572

tyrosine content was low in all the studied carp gelatines, however the differences in the

573

content of these amino acids significantly depends on the pre-treatment method used. Gelatine

574

III had the highest amount of serine and the highest gel strength. Serine has free hydroxyl

575

groups which can contribute to the gel strength by the generation of hydrogen bonds and

576

helical structures.

28

ACCEPTED MANUSCRIPT 577

Cysteine, which is not commonly present in gelatine, was not found in any of the carp

578

gelatines. Cysteine does not take part in the structure of type I collagen and its presence could

579

indicate that gelatine contains water insoluble stroma proteins, such as elastin (Nagarajan,

580

Benjakul, Prodpran, Songtipya & Kishimura, 2012). It was previously established that the

581

extraction time and temperature influences the amino acids composition of the produced

582

gelatine (Nagarajan et al., 2012). Our research shows that pre-treatment conditions can also

583

influence the amino acids composition of fish skin gelatine.

584

585

3.8 Functional properties Foam formation ability (FF) is one of the most important properties of gelatine for

586

commonly used food products. The foam formation ability and foam stability of carp

587

gelatines are shown in Fig 2.

588 589

Fig.2 . Foam formation ability and foam stability of carps gelatine.

590

Different letters (a. b. c.) indicate significant differences at p <0.05

591

The foam formation ability of gelatine III was 1.0 which meant that gelatine was not foaming

592

at all. The highest foam formation ability was exhibited by gelatine I (close to 1.6). The foam

593

formation ability differed significantly between all the studied gelatines. Although the foam 29

ACCEPTED MANUSCRIPT 594

stability (FS) of gelatine I was 1.27 which was lower than the foam stability of gelatine II

595

(1.53), these differences were statistically insignificant. Gelatine I contained mostly proteins

596

with molecular weight < 30 kDa. Smaller molecular weight peptides are able to reach the air-

597

liquid interface and undergo unfolding and rearrangement at this interface resulting in better

598

FE and FS (Liu et al., 2017). A positive correlation exists between the hydrophobicity of the

599

unfolded proteins and foaming characteristics. Additional hydrophobic residues form a large

600

hydrophobic sphere on the surface of the protein and improve the foaming capacity (Shakila,

601

Jeevithan, Varatharajakumar, Jeyasekaran & Sukumar, 2012). The lowest content of

602

hydrophobic amino acids was found in gelatine III, which resulted in a complete lack of

603

foaming properties of the gelatine. The results showed that the pre-treatment conditions

604

affected the foaming properties of the carp gelatine.

605 606 607

Fig. 3. Water-holding capacity and fat-binding capacity carps gelatine.

608

Different letters (a. b. c.) indicate significant differences at p <0.05

609

The amount of water bound by protein depends on a number of factors, such as: amino

610

acids composition of the protein, number of polar groups within the particle, availability of

611

hydrophilic spots, pH of the environment, ionic strength, temperature and protein

612

concentration (Zayas, 1997). Gelatine II showed significantly higher water-holding capacity 30

ACCEPTED MANUSCRIPT 613

(WHC) than the other carp gelatines, reaching a WHC of 160% (Fig. 3). The water binding

614

capacity of the solubilised gelatine makes it a suitable material for reducing drip loss and the

615

impairs the juiciness in frozen fish or meat products when thawed or cooked, and where

616

denatured protein has suffered a partial loss of its WHC (Koli, 2012).

617

The low fat-binding capacity of carp skin gelatines (Fig. 3) suggests the presence of a large

618

proportion of hydrophilic as compared to hydrophobic groups on the surface of the protein

619

molecules. The mechanism of fat-binding by proteins is not fully understood, but it appears to

620

be affected by protein and lipid-protein complexes content (Chavan, McKenzie & Shahidi,

621

2001). Khalid, Babiker & El Tinay (2003) suggested that the oil absorption capacity is due to

622

the non-polar side chains of the protein, as well as due to the different conformational features

623

of the proteins. Our results suggest that carp gelatine had poor fat-holding capacity, dependent

624

on the pre-treatment methods.

625

3.8 Rheological properties

626

Figure 4 shows the relationship between the absolute value of the complex relaxation modulus

627

in the function of temperature for gelatins II and III. The plots for gelatin I are not presented

628

because of the lack of the gelling ability of that product. Gelatine I was a viscous liquid in the

629

whole analysed temperature range.

31

ACCEPTED MANUSCRIPT

630 631

Figure 4. The complex modulus (G*) as a function of temperature of gelatin II and gelatin III

632

Both systems are comparable in terms of the temperature of gel-sol and sol-gel

633

transformation. The sol-gel transformation was observed at the temperature of ~14°C and the

634

gel-sol transformation was observed at ~20°C. These are much lower values than those

635

obtained by Sinthusamran et al., (2017) for a fish gelatin of an unknown origin. The gelling

636

temperature of the gelatins analysed in this study was the same as obtained for the gelatin

637

from eel skin while the melting point for the same gelatin was slightly higher compared to the

638

carp skin gelatin (Sila et al., 2017). There was a hysteresis observed on the figure which

639

means that the gel is created in lower temperature than its melting.

640

The elastic modulus (G’) and loss modulus (G”) of the gelatines II and III in the function of

641

frequency in the linear range of frequency are presented on figures 5 and 6.

32

ACCEPTED MANUSCRIPT

642 643 644 645

Figure 5. Dynamic viscoelastic storage (G’) and loss modulus (G”) as function of frequency of gelatin II (+ symbol) and gelatin III (x symbol). Measurements taken at 5°C.

646 647 648 649

Figure 6. Dynamic viscoelastic storage (G’) and loss modulus (G”) as function of frequency of gelatin II (+ symbol) and gelatin III (x symbol). Measurements taken at 10°C.

650

The typical behavior of gels was observed for both gelatins II and III where G’ values were

651

much higher compared to G” values. It means that there can be mechanical energy stored in

652

the system but only a small part of it can be dissipated. Higher G’ and G” values obtained for

33

ACCEPTED MANUSCRIPT 653

gelatin III also show that the product is harder and more brittle, that is why the cohesiveness

654

was significantly lower.

655 656

4. Conclusions

657

Carp skin gelatine is a high-protein product with relatively high fat and ash content. The

658

effectiveness of residues removal from the raw material by pre-treatment methods used in this

659

study was low. The highest quality gelatine (gelatine III) was produced using weak alkali,

660

followed by weak mineral acid and organic acid pre-treatment. Moreover, the pre-treatment

661

method significantly affected the colour, amino acids composition, gel strength and

662

electrophoretic protein separation of the produced gelatines. To obtain gelatine with high

663

quality and functional properties, the recommended pre-treatment conditions were 0.2%

664

NaOH for 2 h; 0.2% H2SO4 for 2 h; 1% C6H8O7 for 2 h; 21 °C. The results indicate that C.

665

carpio L. skins can be utilised to extract gelatine with potential application for the industry as

666

an alternative gelatine source to mammalian gelatine.

667 668

Acknowledgements

669 670

We would like to express our gratitude to the technical assistance from dr hab. inż.

671

Paweł Ptaszek and mgr Iwona Duda. This work was supported by the National Centre for

672

Research and Development, Poland [Grant no: Lider/21/0003/l-7/15/NCBR/2016].

673 674

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ACCEPTED MANUSCRIPT Highlights 

Carp skin has potential industrial application as a gelatine source



Three pre-treatment methods with potential industrial application have been studied



The best pre-treatment method was 0.2 % NaOH 2 h; 0.2 % H2SO4 2 h; 1 % C6H8O7 2 h; 21 °C