Accepted Manuscript

Review

Fish viscera protein hydrolysates: production, potential applications and func-

tional and bioactive properties

Oscar Villamil, Henry Váquiro, José F. Solanilla

PII: S0308-8146(16)32083-0
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.12.057
Reference: FOCH 20349

To appear in: Food Chemistry

Received Date: 12 July 2016

Revised Date: 21 November 2016
Accepted Date: 20 December 2016

Please cite this article as: Villamil, O., Váquiro, H., Solanilla, J.F., Fish viscera protein hydrolysates: production,
potential applications and functional and bioactive properties, Food Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.foodchem.2016.12.057

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1 Fish viscera protein hydrolysates: production, potential applications and functional

2 and bioactive properties

4 Oscar Villamil, Henry Váquiro and José F. Solanilla*

5 Facultad de Ingeniería Agronómica, Universidad del Tolima. Ibagué, Colombia.

7 *Corresponding Author:

8 José F. Solanilla. Facultad de Ingeniería Agronómica, Universidad del Tolima, B. Santa

9 Helena A.A. 546, Ibagué, Colombia. Tel-Fax: +57 (8) 2771212 ext. 9712. E-mail:

10 jfsolanilla@ut.edu.co

11

12 ABSTRACT

13

14 The aquaculture and fishery chain is an important part of the economy of many countries

15 around the world; in recent years it has experienced significant growth that generates more

16 and more quantities of waste, which are mostly discarded, impacting the environment,

17 despite having a useful chemical composition in various industrial sectors. This article

18 presents a review of the agroindustrial potential of fish wastes, especially viscera, as a

19 source for obtaining native protein and hydrolysates, explaining their production process,

20 chemical composition and functional and bioactive properties that are important to the

21 agricultural, cosmetic, pharmaceutical, food and nutraceutical industry.

22

23 Keywords: Protein hydrolysates, Fish viscera, bioactivity, Functional properties, Peptides.

24
25 1. Introduction

26

27 The continuous growth of fish production, at an average annual rate of 3.2%, and the

28 increase in per capita fish consumption from 9.9 kg in 1960 to 19.2 kg in 2012 (FAO,

29 2014) have generated greater amounts of organic wastes that are discarded during primary

30 processing because they contain microorganisms and enzymes responsible for autolysis and

31 high perishability of comestible tissues (Feltes et al., 2010). These wastes generally consist

32 of viscera, carcass, head, skin and bones which have a high agroindustrial potential as a

33 source of co-products; their percentage can vary between 50 and 70% of the fresh weight,

34 according to each species (Guerard, Dufosse, De La Broise, & Binet, 2001; He, Franco, &

35 Zhang, 2013; Silva, Ribeiro, Silva, Cahú, & Bezerra, 2014). 50% of the aforementioned

36 waste is discarded and only 30% is used in activities that have low value added, such as

37 animal feed, fertilizer agents and silage production. (Arvanitoyannis, 2010; He et al., 2013;

38 Hsu, 2010; Je, Qian, Byun, & Kim, 2007). This is due to ignorance about the effective

39 management of waste and the benefits that it provides, leading to its disposal in rivers,

40 streams, seas and many more (Ezejiofor, Enebaku, & Ogueke, 2014).

41

42 Fish co-products are a significant source of protein, and other components, such as

43 polyunsaturated fatty acids, phospholipids, soluble vitamins and bioactive compounds

44 (Shirahigue et al., 2016), which make them attractive for various uses in technological

45 applications that promote product development and significant advances in the fish industry

46 (Feltes et al., 2010).

47
48 Based on the above, there have been studies and investigations directed towards finding

49 new forms of exploitation of fish waste in order to mitigate environmental problems

50 (Sheriff, Sundaram, Ramamoorthy, & Ponnusamy, 2014). Protein hydrolysates have been

51 gaining great interest in recent years (Šližytė, Daukšas, Falch, Storrø, & Rustad, 2005)

52 since the modification by enzymes or chemicals improves functional properties of native

53 proteins and their usefulness as intermediate ingredients in the cosmetics, pharmaceutical,

54 food and nutraceutical sectors (Klompong, Benjakul, Kantachote, & Shahidi, 2007;

55 Kristinsson & Rasco, 2000; Sarmadi & Ismail, 2010).

56

57 Accordingly, the aims of this study are to present a review of the agroindustrial potential of

58 fish waste, especially viscera, as a source for obtaining native protein and hydrolysates, and

59 to explain their production process, chemical composition and functional and bioactive

60 properties.

61

62 2. Fish processing co-products

63

64 Fish processing begins with size classification and removal of scales, carcass and fins by

65 washing; viscera is removed when fish is not marketed fresh or frozen, and the process then

66 continues to produce fillets, canned fish and others (Feltes et al., 2010). The co-products

67 obtained in percentage terms are composed of muscle cuts (15-20%), skin and fins (1-3%),

68 bones (9-15%), heads (9-12%), viscera (12-18%) and scales (5%) (Martínez-Alvarez,

69 Chamorro, & Brenes, 2015), all of which are susceptible to microbial degradation

70 (Arvanitoyannis, 2010; Benjakul, Yarnpakdee, Senphan, Halldorsdottir, & Kristinsson,

71 2014; Hsu, 2010). Lately, there is a particular interest in new uses of viscera, whose

72 composition is shown below in Table 1.

73

74 As seen in table 1, fish viscera have both high protein and lipid content whose variability in

75 composition depends on the species, seasonality, age, sex, nutrient intake and other factors

76 (Huss, 1995). Different fish muscles and tissues are composed by structural, myofibrillar

77 and sarcoplasmic proteins that have all the essential amino acids, among which

78 predominate lysine, phenylalanine and valine (Hayes & Flower, 2013). They also have

79 endogenous enzymes, such as pepsin, trypsin, chymotrypsin, collagenase and elastase,

80 which can be used to hydrolyze proteins (Vannabun, Ketnawa, Phongthai, Benjakul, &

81 Rawdkuen, 2014). Furthermore, collagen and gelatin, in tissues, such as bone and skin,

82 have recently gained interest in the field of research as an alternative to mammalian

83 collagen for food and pharmaceutical applications (Karim & Bhat, 2009). In addition, the

84 lipid fraction of waste contains omega-3, squalane, phospholipid, cholesterol and fat-

85 soluble vitamins (Rai, Swapna, Bhaskar, Halami, & Sachindra, 2010). Its utilization has

86 been directed towards the food and nutraceutical industries and the energy sector in the

87 production of biodiesel (Feltes et al., 2010).

88

89 3. Production of fish viscera protein hydrolysates (FVPH)

90 3.1. General
91

92 The aim of producing protein hydrolysates is the solubilization of the protein source to

93 improve its biological and nutritional value to obtain products of high added value and

94 commercial interest (Nilsang, Lertsiri, Suphantharika, & Assavanig, 2005), forming

95 different size peptides either by chemical or enzymatic methods (C. M. Silva, dos Santos da

96 Fonseca, & Prentice, 2014). Protein hydrolysis has shown continuous development over

97 time, but in a general context, this process is still in the early stages of discovering peptides

98 and individual amino acid combinations to produce desired effects for different applications

99 (Pasupuleti & Braun, 2010). Obtaining hydrolysates from co-products of the fishing and

100 aquaculture industries, such as viscera (Figure 1), involves processes of isolation or

101 pretreatments, followed by hydrolysis and protein recovery (He et al., 2013).

102

103 3.2. Pretreatments

104

105 Pretreatments comprise a group of processes whose main objective is to concentrate

106 proteins. Additionally, their objective is to prepare homogeneous mixtures of water and

107 chopped viscera with the lowest possible percentage of fat and other undesirable

108 components for subsequent hydrolysis (He et al., 2013), since these components contribute

109 to oxidation and coloration of the final product (Kristinsson & Rasco, 2000) and the

110 formation of unpleasant smells and tastes and highly toxic compounds (Dong et al., 2008).

111

112 Several methods have been used to remove fat from different tissues of fish. In the case of

113 viscera, heat treatment is a very conventional practice, because it has the dual purpose of

114 inactivating enzymes and removing fat (Guerard et al., 2001). Then the solid fraction is

115 mechanically separated by centrifugation (Bhaskar et al., 2008). On the other hand,

116 pressing has also been used for this purpose; it is followed by 2-3 hours of heat treatment to

117 yield a solid which is dried to a moisture percentage lower than 10% (Valenzuela,

118 Sanhueza, & de la Barra, 2012).

119

120 The use of solvents is another useful defatting procedure that involves the heating of a

121 mixture composed of minced raw material and alcohols, such as ethanol or isopropyl

122 alcohol, with constant stirring during a certain period of time (Dong et al., 2008; Klompong

123 et al., 2007). This technique minimizes bacterial degradation (Kristinsson & Rasco, 2000)

124 and eliminates the characteristic odour of fish and bitter flavours (Hoyle & Merritt, 1994).

125 However, protein solubility and dispersibility are usually affected, whereby lower degrees

126 of hydrolysis are obtained in comparison to non-defatted protein (Klompong et al., 2007),

127 which has a negative effect on hydrolysis efficiency and properties of protein hydrolysates

128 (Benjakul et al., 2014).

129

130 Some studies have used a new technology to isolate proteins from fish tissues. It consists of

131 solubilization of protein in acids or alkaline solutions, in addition to centrifugation and

132 filtration in order to remove insoluble compounds; once removed, the proteins are

133 precipitated by adjusting the pH to the isoelectric point and are recovered by centrifugation

134 or decantation (Nolsøe & Undeland, 2009).

135

136 3.3. Hydrolysis

137

138 Protein hydrolysis consists of the cleavage of peptide bonds to obtain free amino acids and

139 low molecular weight peptides, consuming a molecule of water for each broken bond.

140 (Rutherfurd & Gilani, 2009). The hydrolysis process (chemical or biochemical) has

141 recently been used in aquaculture and fisheries to produce more acceptable and profitable

142 products (Benjakul et al., 2014).

143

144 The conventional acid hydrolysis method is based on sample treatment in excessively

145 acidic solutions accompanied by high temperatures and, in some cases, at high pressures

146 over a given time (Kristinsson & Rasco, 2000). It is a low cost, quick and simple operation,

147 which makes it applicable at the industrial level (Gao, Hirata, Toorisaka, & Hano, 2006).

148 However, essential amino acids, such as tryptophan, methionine, cystine and cysteine, are

149 usually destroyed. Furthermore, asparagine and glutamine are converted into aspartic acid

150 and glutamic acid, respectively (Pasupuleti & Braun, 2010). Additionally, obtained

151 hydrolysates have poor functional properties due to the formation of salts after the

152 neutralization process (Kristinsson & Rasco, 2000). Therefore, several removal methods,

153 such as nanofiltration and use of ion exchange resins, have been proposed with excellent

154 results (Pasupuleti & Braun, 2010). Acid hydrolysis of fish co-products has been widely

155 studied to produce low cost fertilizers (Kristinsson & Rasco, 2000), silages (Vidotti,

156 Viegas, & Carneiro, 2003) and alternative substrate for lactic acid production using

157 improved hydrolysis in order to enhance nutritive value (Gao et al., 2006).

158

159 Alkaline hydrolysis is a very simple process, the sample is solubilized by heating and

160 mixed with alkaline solutions, then the reaction temperature is maintained until reaching

161 the desired degree of hydrolysis (Pasupuleti & Braun, 2010). Its main drawback is the

162 production of hydrolysates with low amino acids content, such as cystine, lysine, arginine,

163 serine, threonine, isoleucine and residues like lanthionine and lysinoalanine (Tavano,

164 2013). Alkaline hydrolysis has been widely used in the industrial field, but little in

165 biotechnology (Pasupuleti & Braun, 2010).

166
167 Unlike chemical methods, enzymatic hydrolysis uses mild conditions and is easy to control,

168 and is more precise in the cleavage of peptide bonds; furthermore, there are no side

169 reactions or decrease in the nutritional value (Tavano, 2013), and it exhibits greater ease in

170 protein recovery and purification of some peptides (Pasupuleti & Braun, 2010). For these

171 reasons, it has gained more and more attention to produce hydrolysates with better and

172 more defined nutritional, functional and bioactive properties (Benjakul et al., 2014).

173 Nevertheless the majority of the studies on enzymatic hydrolysis have not been scaled and

174 its biggest drawback is the high production costs compared to chemical hydrolysis (He,

175 Franco, & Zhang, 2015).

176

177 The enzymatic hydrolysis process is usually performed in a reactor with temperature, pH,

178 agitation and time controls (Benítez, Ibarz, & Pagan, 2008). Initially, the temperature and

179 pH of the homogeneous mixture obtained from pretreatment are adjusted according to the

180 ideal working conditions of the enzyme. When protease is added, the reaction between

181 enzyme and substrate causes changes in the pH of the solution due to the cleavage of

182 peptide bonds to form new amino or carboxyl groups able to release or accept protons.

183 Faced with the above, some methods rely on the addition of buffer solution in order to

184 moderate pH changes (Ovissipour et al., 2009; Ovissipour et al., 2012; Shirahigue et al.,

185 2016). However, it is considered that the presence of salts in buffer solutions can affect

186 functional properties of interest, such as the emulsifying and foaming capabilities

187 (Kristinsson & Rasco, 2000). Meanwhile, other studies choose to maintain the optimal pH

188 of enzyme activity by constant addition of neutralizing solution during the hydrolysis

189 process (Dong et al., 2008; Hsu, 2010; Kechaou et al., 2009; Klompong et al., 2007; C. M.

190 Silva et al., 2014). In both processes, when the expected degree of hydrolysis is obtained,
191 peptidase is deactivated by changes in temperature, pH or both variables simultaneously

192 (Benítez et al., 2008).

193 The choice of enzyme depends both on the final product and the price (Benjakul et al.,

194 2014). It is also important to consider the amino acid composition of the protein because

195 some proteases have preferences for the cleavage of certain peptide bonds (Pasupuleti &

196 Braun, 2010). It bears mentioning that the medium in which the enzymes work is a factor of

197 choice, for example those whose optimum pH is acidic can inhibit bacterial growth, but

198 have low recovery percentages of protein and decreased nutritional and functional value in

199 comparison to alkaline and neutral proteases (Kristinsson & Rasco, 2000). This is the

200 reason why microbial proteases with high proteolytic activity are the most commonly used

201 and suitable in the production of hydrolysates from fish tissues (Benjakul et al., 2014) as

202 can be seen in Table 2.

203

204 Protein hydrolysates can also be obtained by use of proteases present in the digestive

205 system of fish, such as pepsin, trypsin, chymotrypsin, collagenase and elastase (Vannabun

206 et al., 2014). Autolysis has been widely used to obtain fish sauce and silages (Kristinsson &

207 Rasco, 2000). Bougatef et al. (2008) produced protein hydrolysates from heads and viscera

208 of sardinella Sardinella aurita by autolysis temperatures ranging from 40°C to 50°C and

209 pH 8, finding that the addition of an external enzyme can accelerate reaction and increase

210 the degree of hydrolysis. Motamedzadegan, Davarniam, Asadi, Abedian, and Ovissipour

211 (2011) optimized the autolytic sequential process with neutrase applied to yellowfin tuna,

212 concluding that enzymatic activity of 39.61 AU/kg protein, 53°C and 141 min are the

213 recommended parameters to achieve a hydrolysis degree of about 30%. Vannabun et al.

214 (2014) characterized acidic and alkaline proteases, which showed better activity in acidic
215 and alkaline media and an optimal temperature of 40°C and 60°C, respectively. However,

216 the enzymes did not show high stability in media with high salt concentration. Autolysis is

217 an economical procedure, but is difficult to standardize and control because endogenous

218 enzymes depend on several factors, including seasonality, type and amount of enzymes,

219 fish species and others (Bhaskar et al., 2008).

220

221 The process efficiency is measured by quantifying the degree of hydrolysis, which is

222 defined as the amount of peptide bonds cleaved out of the total amount of peptide bonds in

223 native protein (Benítez et al., 2008; He et al., 2013). Degree of hydrolysis is influenced by

224 different parameters, such as E/S ratio, pH, temperature and incubation time; the first three

225 have an effect on reaction rate, while the latter only impacts the degree of hydrolysis

226 (Benítez et al., 2008). It may be the most suitable measuring method as the interaction of

227 the aforementioned factors with the choice of substrate and enzyme are directly related to

228 the amount of low molecular weight peptides, protein recovery (Ovissipour et al., 2009)

229 and bioactive and functional properties of hydrolysates (Klompong et al., 2007).

230

231 3.4. Recovery

232

233 FVPH can be purified by various methods depending on the final use of the hydrolysates,

234 which are centrifugation, nano-filtration, ultrafiltration, microfiltration and ion exchange

235 chromatography (Pasupuleti & Braun, 2010). Centrifugation has been the most used of the

236 above, from which four phases are obtained: oil fraction, emulsion layer, FVPH and sludge

237 (Ramakrishnan, Ghaly, Brooks, & Budge, 2013; Šližytė et al., 2005). After separating

238 FVPH by decantation, it is dehydrated through lyophilization or spray-drying in order to

239 increase shelf life and to provide greater ease in handling, transportation and storage. The

240 final product is usually a creamy white powder with enhanced functional and bioactive

241 properties (He et al., 2013). All kinds of hydrolysates must be stored taking into account

242 parameters, such as moisture content and glass transition temperature, which generally

243 tends to decline while the degree of hydrolysis is increasing (Rao, Klaassen Kamdar, &

244 Labuza, 2016). Therefore FVPH are commonly stored below 0°C to ensure stability over

245 time (Ambigaipalan & Shahidi, 2015; Dong et al., 2008).

246

247 It is noteworthy that laboratory scale subsequent processes have been proposed to separate

248 different peptide fractions according to the molecular weight (He et al., 2013), because the

249 relationship between peptide size and certain bioactive and antioxidant properties has been

250 demonstrated in several studies (Robert et al., 2015). However, there are difficulties for

251 industrial scaling at present.

252

253 4. Chemical characteristics of fish viscera protein hydrolysates

254

255 Proximate composition of FVPH is shown in table 3. These products have high protein

256 content due to protein solubilization during hydrolysis and removal of insoluble material

257 and most of the fat layer during recovery processes (Chalamaiah, Hemalatha, &

258 Jyothirmayi, 2012). Moisture content, generally below 10%, is caused by the concentration

259 and drying processes, while ash content is linked to the neutralization process during

260 hydrolysis (Dong et al., 2008; Gbogouri, Linder, Fanni, & Parmentier, 2004; Kristinsson &

261 Rasco, 2000)

262
263 Amino acids have an important role in protein synthesis as compound carriers of hydrogen,

264 vitamins, carbon dioxide, enzymes and structural proteins (Chalamaiah et al., 2012) and

265 they also influence bioactive and functional properties. Table 4 shows amino acid

266 composition of several FVPH whose variation depends on raw material, enzyme type and

267 hydrolysis parameters (Bhaskar et al., 2008; Klompong et al., 2009). FVPH have all the

268 essential and non-essential amino acids, which is why they are considered to be high

269 nutritional sources.

270

271 5. Functional properties of FVPH and food applications

272

273 Functional properties are physicochemical characteristics that influence the performance of

274 the protein in different food systems during different stages from processing to

275 consumption (Kristinsson & Rasco, 2000; Šližytė et al., 2005). Native proteins present

276 good functionality, however their use is limited because most foods have pH close to the

277 isoelectric point, therefore protein hydrolysis has become an increasingly common

278 technique as it significantly improves the functionality of proteins to a wide range of pH

279 levels (de Castro, Bagagli, & Sato, 2015; de Castro & Sato, 2014).

280

281 The parameters that affect protein functionality are: substrates, enzymes used and degree of

282 hydrolysis. Protease specificity influences molecular weight and hydrophobic character

283 and, as a result, protein behaviour (Kristinsson & Rasco, 2000). Thus, hydrolysates with

284 different molecular weights can be obtained by appropriately selecting enzymes and

285 substrates according to desired requirements. Proteins with a lower degree of hydrolysis

286 show exceptional functional properties because they still retain amphiphilic quality, which
287 does not happen with those whose hydrolysis has been excessive despite the fact that

288 solubility, in most cases, has increased (Klompong et al., 2007). Additionally, functional

289 behaviour of proteins can also vary by extrinsic conditions, such as the nature of the matrix,

290 along with storage and processing parameters (Benjakul et al., 2014).

291

292 Protein functional properties are classified in hydrodynamic properties, such as solubility,

293 viscosity, gelling capacity and water absorption, and surface active properties, such as

294 emulsifying capacity, foaming and film formation (de Castro et al., 2015). Solubility is

295 considered by many researchers as the most important functional property because it

296 significantly affects all others (Benjakul et al., 2014). There is direct correlation between

297 solubility and degree of hydrolysis; it can be explained by the fact that longer hydrolysis

298 times produce proteins and peptides with smaller molecular weight generating greater

299 exposure of ionizable and polar groups on the protein surface, which influences the

300 improvement of their ability to form hydrogen bonds with water (de Castro & Sato, 2014;

301 He et al., 2013; Yin, Tang, Wen, Yang, & Li, 2008).

302

303 Fish protein hydrolysates present improved solubility in comparison with native proteins

304 (Dong et al., 2008; Klompong et al., 2007). Protein hydrolysates made with trypsin, pepsin

305 and chymotrypsin from muscle, skin, bones and viscera of horse mackerel and croaker

306 showed solubility greater than 65% over a wide pH range (Kumar, Nazeer, & Ganesh,

307 2012). Similar results were obtained by Liu et al. (2014), who studied solubility in surimi

308 by-products, including fish meat leftover on bones, head, skin, and viscera; they reported an

309 increase from 10% in native protein up to 65% and more in protein hydrolysates made with

310 protamex and alcalase in a wide pH range. Souissi, Bougatef, Triki-Ellouz & Nasri (2007)
311 found that alcalase hydrolysates obtained from heads and viscera of sardinella showed

312 solubility of up to 100% between pH ranging from 6 to 10 with a degree of hydrolysis

313 around 10%. These results indicate that fish protein hydrolysates can be used easily in food

314 formulations.

315

316 Water-holding capacity consists of the ability of the protein to capture water in the food

317 matrix preventing its flow by gravitational force (Kristinsson & Rasco, 2000), it is

318 important to the food industry because it influences texture (Pires & Batista, 2013). Fish

319 protein hydrolysates from cod Gadus morhua by-products function as good water

320 scavengers in food systems by adding them to comminuted fish and then freezing the fish

321 in order to observe the influence of these components on water retention during thawing

322 (Šližytė et al., 2005). Furthermore, Balti et al. (2010) observed the increase of water-

323 holding capacity with the extent of hydrolysis, concluding that there is a direct correlation

324 between such variables probably due to the exposure of polar groups as the hydrolysis

325 process advances.

326

327 Oil holding capacity or fat absorption refers to the quantity of oil absorbed and retained by

328 the protein; it is related to emulsifying capacity and may be affected by bulk density, degree

329 of hydrolysis and enzyme – substrate specificity (Pires & Batista, 2013; Kaur & Singh,

330 2007). Oil holding capacity in fish by-product hydrolysates has been studied. FVPH

331 obtained from viscera and heads of sardinella Sardinella aurita presented improved fat

332 absorption compared to native protein and showed better results than casein at a degree of

333 hydrolysis of 9.33% (Souissi et al., 2007); similar results were collected by Balti et al.

334 (2010), hydrolysates produced from skin and viscera of cuttlefish Sepia officinalis revealed
335 excellent oil holding capacity that enables their use in formulations for the food and

336 confectionery industries.

337

338 Proteins are good emulsifiers because they have an amphipathic structure that facilitate

339 their absorption at the oil-water interface (Pires & Batista, 2013), this property in

340 hydrolysates is mainly influenced by the size and molecular weight of peptides, surface

341 hydrophobicity, enzyme, amino-acid composition (Liu et al., 2014), volume fraction of oil,

342 protein concentration, equipment used to form the emulsion (Šližytė et al., 2005) and

343 solubility (Klompong et al., 2007). Many studies have reported emulsifying properties in

344 fish co-products hydrolysates. Souissi et al. (2007) showed that fish hydrolysates from

345 heads and viscera of Sardinella aurita, at a degree of hydrolysis of 6.67%, have

346 emulsifying capacity greater than casein, but this capacity decreases with the extent of

347 hydrolysis. These results coincide with those of Klompong et al. (2007), however Balti et

348 al. (2010) reported that protein hydrolysate from skin and viscera of cuttlefish Sepia

349 officinalis at a degree of hydrolysis of 5% showed lower emulsifying capacity than those

350 with a degree of hydrolysis of about 10 and 13.5%, probably due to the liberation of

351 peptides with medium weight as hydrolysis proceeds, which increased the flexibility of

352 such peptides and consequently their surface size and ability to form emulsions. The

353 emulsifying properties of FVPH and others obtained from different co-products have

354 proven to be more effective than commercial food-grade emulsifiers, this represents a high

355 potential in the use of FVPH in food formulations (He et al., 2013).

356

357 At present, several fish protein hydrolysates have been successfully incorporated into food

358 systems, such as cereals, cookies, desserts and meat products (Chalamaiah et al., 2012;
359 Kristinsson & Rasco, 2000), however there is insufficient information about the

360 performance of hydrolysates from viscera and other co-products in food matrices and

361 commercial formulations, although they were found to be potential ingredients in food

362 formulations according to various laboratory assays (He et al., 2013).

363

364 6. FVPH as source of bioactive compounds

365

366 Current research is focused on bioactive peptides (Sarmadi & Ismail, 2010) due to their

367 biological roles which are beneficial to human health and useful to the food industry

368 (Sarmadi & Ismail, 2010; Wu et al., 2015), such peptides consist of short amino acid chains

369 that are inactive within the precursor protein. (Hayes & Flower, 2013). The performance of

370 hydrolysates from several fish protein sources as antioxidant, antihypertensive and

371 antimicrobial agents has been reported (Ryan, Ross, Bolton, Fitzgerald, & Stanton, 2011).

372 Souissi et al. (2007) evaluated hydrolysates obtained from heads and viscera of sardinella

373 Sardinella aurita in terms of scavenging effect on DPPH free radical and Inhibition of

374 linoleic acid autoxidation, the results indicate that the aforementioned peptides exhibited

375 more than 50% inhibition of linoleic acid peroxidation and an antioxidant activity of about

376 41%. Kumar, Nazeer, and Jaiganesh (2011) purified and characterized an antioxidant

377 peptide from horse mackerel Magalaspis cordyla viscera protein whose sequence was

378 determined as Ala–Cys–Phe–Leu (518.5 Da), it showed 59.1 and 89.2 percentage of

379 scavenging of hydroxyl radicals and DPPH respectively, using a concentration of 0.2

380 mg/ml. It also was found to be better than α-tocopherol in terms of inhibition of lipid

381 peroxidation. Nazeer and Kumar (2011) evidenced that fish viscera protein hydrolysates

382 obtained from Parastromateus niger have a protective effect on DNA against damage
383 caused by hydroxyl radicals. All those outcomes suggest the presence of antioxidant

384 peptides in hydrolysates obtained from fish co-products.

385

386 Hypertension is one of the main risk factors associated with cardiovascular disease; it is

387 caused by angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) which plays an important

388 role in the blood pressure control producing angiotensin II, a powerful vasoconstrictor and

389 destroyer agent of vasodilators like bradykinin (Barbana & Boye, 2010; Herpandi, Rosma,

390 & Wan Nadiah, 2011). The inhibition of this enzyme is the key factor to prevent and treat

391 hypertension. Currently, synthetized chemical compounds have been used for that purpose,

392 but their side effects have led to the search for peptides with ACE inhibitory activity from

393 several sources (Harnedy & FitzGerald, 2012).

394

395 Many studies have evidenced the ACE inhibitory activity of fish co-products peptides

396 (Harnedy & FitzGerald, 2012; Hayes & Flower, 2013; Herpandi et al., 2011; Kim, Ngo, &

397 Vo, 2012; Raghavan & Kristinsson, 2009; Wu et al., 2015). Bougatef et al. (2008)

398 produced sardinella Sardinella aurita head and viscera protein hydrolysates using alcalase,

399 chymotrypsin, crude enzyme preparation from Aspergillus clavatus ES1, alkaline proteases

400 from B. lincheniformis NH1 and crude enzyme extract viscera of sardine Sardina

401 pilchardus. The fish co-product hydrolysates were found to be good ACE inhibitors, whose

402 IC50 values ranged from 1.24 to 7.40 mg/ml. Balti et al. (2010) obtained cuttlefish Sepia

403 officinalis skin and viscera protein hydrolysates whose IC50 was about 1.00 mg/ml at 13.5%

404 degree of hydrolysis. Both studies agree that the enzyme plays a decisive role in the

405 cleavage of peptide bonds due to its specificity and that these results suggest that peptides

406 from such materials can be used as ingredients in functional products for the prevention and
407 treatment of hypertension. However, the bioactivity of fish co-products peptides has not

408 been as extensively studied as milk and plant peptides (Rustad & Hayes, 2012), more

409 research about anti-cancer, anti-atherosclerotic and anti-inflammatory activities of peptides

410 derived from different fish tissues is needed (Halim, Yusof, & Sarbon, 2016). Also, there is

411 limited information about the behaviour of these peptides on several food matrices and their

412 stability throughout industrial processing (Samaranayaka & Li-Chan, 2011), as well as

413 during gastrointestinal absorption (Sarmadi & Ismail, 2010). Additionally, the production

414 of FVPH is a huge challenge because of sensory problems, high costs and difficulties to

415 ensure reproducibility (Rustad & Hayes, 2012) which represent new fields to research.

416

417 7. FVPH as microbial growth media

418

419 The use of fish tissues as a source of nutrients for microorganisms has been known for

420 decades; several studies with the aim of exploring the use of fish peptones as a component

421 of microbial growth substrates have been reported. Proteins obtained from tuna, cod,

422 salmon and unspecified fish were compared with casein peptone by the simulation model

423 Gompertz applied to microbial growth of six species of bacteria, yeast and mold, and

424 results revealed that fish peptones were effective (Guerard et al., 2001). Also, fish viscera

425 peptones from tuna, yellow stripe, swordfish, rainbow trout and squid were evaluated as

426 growth medium for different types of microorganisms (Pseudomonas, Vibrio and

427 Roseobacter) that are of interest to aquaculture due to their pathogenic or probiotic

428 character. The outcomes showed that the effectiveness of such culture media was even

429 better than the commercial ones (Vázquez, González, & Murado, 2004). In addition, the

430 potential of peptones from Atlantic cod viscera for growth of five different microorganisms
431 (Escherichia coli, Bacillus subtilis, Lactobacillus sakei, Saccharomyces cerevisiae and

432 Aspergillus niger) was also reported, verifying that it is a promising alternative nitrogen

433 source against currently available commercial products (Aspmo, Horn, & Eijsink, 2005).

434 Peptones obtained from cod stomachs showed higher performance than commercial

435 products for the growth of pathogens like Vibrio anguilarum and Aeromonas salmonicida

436 (Gildberg, Dahl, Mikkelsen, & Nilsen, 2010). Similarly, yellowfin tuna viscera

437 hydrolysates were effective for such purposes in Escherichia coli y Staphylococcus aureus

438 (Klompong, Benjakul, Kantachote, & Shahidi, 2012). It is worth highlighting that the

439 exploration of new resources is still open because each peptone has its own characteristics

440 and none by itself can meet all the requirements of microorganisms during cell culture

441 (Dufossé, De La Broise, & Guerard, 2001; Herpandi et al., 2011).

442

443 On the other hand, seasonal variation influences composition of biological resources like

444 fish viscera and, as a result, affects the performance of peptones as a culture media,

445 especially for fastidious microorganisms, such as Lactobacillus sakei (Horn, Aspmo, &

446 Eijsink, 2007). Likewise the influence of hydrolysis parameters, such as enzyme used, type

447 and degree of hydrolysis on its aptitude as culture media was evidenced (Klompong et al.,

448 2012). There are still questions about the standardization of the raw material as it is

449 considered a critical point because its composition can vary from one production batch to

450 another. It is also necessary to investigate about what degree of hydrolysis is suitable for

451 the growth of certain microorganisms and how to scale an economical production of

452 peptones by enzymatic hydrolysis (He et al., 2013; Klompong et al., 2012).

453

454
455 8. Conclusions

456

457 FVPH are promising high added value products due to their relevant nutritional content,

458 bioactive and functional properties that can be applied in the food and pharmaceutical

459 industries, as well as other uses as microbial growth media. However, most experiments

460 about FVPH’s bioactivity have been carried out in vitro and further investigation about

461 other bioactivities and the performance and stability of FVPH in food matrices and

462 commercial formulations are needed. The production and use of FVPH, whether chemical

463 or biological, is becoming popular as a sustainable alternative, but there are huge

464 challenges in raw material and product quality assurance, development of low-cost

465 processes, industrial scaling and isolation and recovery of peptide sequences that are

466 potentially desirable during food processing and food supplementation. Addressing these

467 concerns could generate the commercial development of FVPH products within the

468 agricultural, cosmetic, pharmaceutical, food and nutraceutical industry.

469

470 Acknowledgements

471

472 The authors acknowledge financial support from Research Fund of Universidad del Tolima

473 (Colombia) and the Administrative Department of Science, Technology and Innovation of

474 Colombia.

475

476 The authors have no conflicts of interest concerning this research or its funding.

477

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768 Tavano, O. L. (2013). Protein hydrolysis using proteases: An important tool for food

769 biotechnology. Journal of Molecular Catalysis B: Enzymatic, 90(0), 1-11.

770 Uriarte-Montoya, M. H., Santacruz-Ortega, H., Cinco-Moroyoqui, F. J., Rouzaud-Sández,

771 O., Plascencia-Jatomea, M., & Ezquerra-Brauer, J. M. (2011). Giant squid skin

772 gelatin: chemical composition and biophysical characterization. Food Research

773 International, 44(10), 3243-3249.

774 Valenzuela, A., Sanhueza, J., & de la Barra, F. (2012). El aceite de pescado: Ayer un

775 desecho industrial, hoy un producto de alto valor nutricional. Revista Chilena de

776 Nutrición, 39, 201-209.

777 Vannabun, A., Ketnawa, S., Phongthai, S., Benjakul, S., & Rawdkuen, S. (2014).

778 Characterization of acid and alkaline proteases from viscera of farmed giant catfish.

779 Food Bioscience, 6, 9-16.

780 Vázquez, J. A., González, M. P., & Murado, M. A. (2004). A new marine medium: use of

781 different fish peptones and comparative study of the growth of selected species of

782 marine bacteria. Enzyme and Microbial Technology, 35(5), 385-392.

783 Vidotti, R. M., Viegas, E. M. M., & Carneiro, D. J. (2003). Amino acid composition of

784 processed fish silage using different raw materials. Animal Feed Science and

785 Technology, 105(1), 199-204.

786 Wu, R., Wu, C., Liu, D., Yang, X., Huang, J., Zhang, J., Liao, B., He, H. & Li, H. (2015) .

787 Overview of antioxidant peptides derived from marine resources: the sources,

788 characteristic, purification, and evaluation methods. Applied Biochemistry and

789 Biotechnology, 176(7), 1815-1833.

790 Yin, S. W., Tang, C. H., Wen, Q. B., Yang, X. Q., & Li, L. (2008). Functional properties

791 and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein

792 isolate: effect of high-pressure treatment. Food Chemistry, 110(4), 938-945.

793 Zhang, Y., Liu, W., Li, G., Shi, B., Miao, Y., & Wu, X. (2007). Isolation and partial

794 characterization of pepsin-soluble collagen from the skin of grass carp

795 (Ctenopharyngodon idella). Food Chemistry, 103(3), 906-912.

796

797

798

799
800 Figure 1. Flow chart of the generic process of obtaining fish viscera protein hydrolysates (FVPH).
801

802
803 Source: (He et al., 2013; Pasupuleti & Braun, 2010) with modifications.
804 Table 1. Proximate composition of fish viscera and fish viscera protein hydrolysates
805
Proximate composition of fish viscera 806
Species Protein (%) Lipids (%) Moisture (%) Ash(%) Reference 807
Yellowfin Tuna (Thunnus albacares) Ovissipour, Kenari, Motamedzadegan,808 &
21.5±0.50 5.08±1.53 69.66±2.32 4.46±1.21
Nazari (2012) 809
Tuna (Euthynnus affinis) Salwanee, Wan Aida, Mamot, & Maskat
65.04±1.40 11.77±1.41 75.73±0.33 3.12±0.11 810
(2013)
811
Catla (Catla catla) 8.52±0.95 12.46±1.45 76.25±0.25 2.50±0.04 Bhaskar, Benila, Radha, & Lalitha (2008)
812
Cod (Gadus morhua) 14.90±2,30 2.00±0.50 60.00±0.00 4.40±0.30 Šližytė et al. (2005)
Sardine (Sardina pilchardus) 15.76±1.10 4.89±0.11 77.46±0.02 1.90±0.00 Kechaou et al. (2009) 813
Rainbow Trout (Onchorhynchus 814
15.00±0.06 13.00±0.76 71.65±0.89 2.73±0.89 Taheri, Anvar, Ahari, & Fogliano (2012)815
mykiss)
Pink Salmon (Oncorhynchus 816
15.30±0.40 2.00±0.30 81.20±0.70 1.70±0.10 Bechtel (2003)
gorbuscha) 817
Parastromateus Niger 14.40±0.50 3.90±0.30 74.00±0.50 3.40±0.40 Nazeer & Kumar (2011) 818
Tilapia (Oreochromis niloticus)* 14.62±0.79 10.75±0.97 60.44±0.27 4.90±0.61 Shirahigue et al. (2016) 819
Fish viscera protein hydrolysate 820
Persian sturgeon (Acipenser 821
65.82±7.02 0.18±0.40 4.45±0.67 7.67±1.24 Ovissipour et al. (2009)
persicus) 822
Yellowfin tuna (Thunnus albacares) 72.34±3.20 1.43±0.57 2.82±2.74 22.34±1.38 Ovissipour et al. (2012) 823
Sardinella (Sardinella aurita) (6.62% 824
75.01±1.72 8.53±1.11 1.35±0.55 14.81±0.14
hydrolysis)**
825
Sardinella (Sardinella aurita) (9.31%
72.99±1.82 10.21±1.58 2.83±0.42 13.06±0.13 826
Souissi, Bougatef, Triki-Ellouz & Nasri (2007)
hydrolysis)**
Sardinella (Sardinella aurita) (10.16% 827
73.05±1.91 10.29±1.76 4.56±0.27 12.10±0.12 828
hydrolysis)**
Rainbow trout (Onchorhynchus 829
88.32±0.07 0.80±0.60 3.45±0.02 1.14±0.88 Taheri et al. (2012) 830
mykiss)
831
832 * Total co-products (viscera, bones, head, scales), **viscera and heads
833 Table 2. Species, enzymes and parameters used for obtaining protein hydrolysates from fish
834 viscera.
835
Specie Enzyme Parameter Reference
Yellowfin tuna E/S=0.2 - 3%; T=50°C;
Alcalase 2.4L Guerard et al. (2001)
(Thunnus albacares) pH=8.0
E/S=0.1%; T=50°C;
Flavorzyme 500L
Cod (Gadus morhua pH=7.0
Šližytė et al. (2005)
L.) E/S=0.3%; T=50°C;
Neutrase 0.8L
pH=7.0
Sardinella (Sardinella E/S=727.26 U/g;
Alcalase Souissi et al. (2007)
aurita) T=50°C; pH=8.0
E/S=1.5%; T=55°C;
Catla (Catla catla) Alcalase (0.6 AU/g) Bhaskar et al., 2008
pH=8.5
Persian Sturgeon E/S=0.1 AU/g; T=55°C; Ovissipour et al.
Alcalase 2.4L
(Acipenser persicus) pH=8.5 (2009)
E/S=0.1% w/w;
Protamex
T=50°C; pH=8.0
Sardine (Sardina E/S=0.1% w/w;
Alcalase 2.4L Kechaou et al. (2009)
pilchardus) T=50°C; pH=8.0
E/S=0.1% w/w;
Flavorzyme 500MG
T=50°C; pH=8.0
Batista, Ramos,
Black scabbardfish E/S=0.5 - 4%; T=50°C;
Protamex Coutinho, Bandarra, &
(Aphanopus carbo) pH=7.5
Nunes (2010)
Alcalase (0.6 AU/g) E/S=0.5% w/v; T=40°C
Neutrase (1.5 AU/g) E/S=0.5% w/v; T=40°C
Rohu (Labeo rohita) Hathwar, Bijinu, Rai, &
Protex 7L E/S=0.5% w/v; T=40°C
Catla (Catla catla) Narayan (2011)
Protease-P-Amano
E/S=0.5% w/v; T=40°C
(60000 U/g)
Pepsin
E/S=1.0% w/v; Nazeer & Kumar
Parastromateus Niger Trypsin
T=37°C; pH=2.5 (2011)
α-Chymotrypsin
Tuna (Euthynnus E/S=1.5%; T=40°C;
Alcalase Salwanee et al. (2013)
affinis) pH=8
Tilapia (Oreochromis E/S=0.5%; T=55°C;
Neutrase Shirahigue et al. (2016)
niloticus) pH=7.0
836

837
838 Table 3. Functional properties and applications of fish wastes

Skin and Gelatin extraction

Yield (% on a
Source Pre-treatment extracting conditions Outcome Reference
weight basis)
Megrim
7.4
(Lepidorhombus boscii)
Gelatins were obtained without
Dover sole (Solea proteolytic digestion. The
8.3
vulgaris) gelling ability and thermal Gómez-Guillén et
0.05M Acetic acid. 45°C - overnight
stability of the gels were found al. (2002)
to be influenced by extrinsic
Cod (Gadus Morhua) 7.2 factors like fish species.

Hake (Merluccius
6.5
merluccius, L.)
Black tilapia Gelatins from black and red
(Oreochromis 5.39 tilapia were obtained. The
0.2% Sulfuric acid/1% citric acid. Jamilah &
mossambicus) results suggest that they can be
45ºC – 12h Harvinder (2002)
for applications different from
Red Tilapia cold water fish gelatin.
7.81
(Oreochromis nilotica)
Pangas catfish
22
(Pangasius pangasius) The fishes investigated were
Asian redtail catfish potential alternative sources of
21.28 Ratnasari, Yuwono,
(Hemibagrus nemurus) % (1:3 b/v) citric acid (pH 3) for 12 h. gelatin in spite of showing lower
Nusyam, &
Striped snake head 60°C – 6h physicochemical and rheological
20.25 Widjanarko (2013)
(Channa striata) properties compared to the
Nile tilapia commercial gelatin.
21.93
(Oreochromis niloticus)
D. gigas skin gelatin has Uriarte-Montoya et
Squid (Dosidicus gigas) 0.05M Acetic acid for 3h. 65°C - 12h 7.5
potential as a source of al. (2011)
 functional component for
different industrial applications.
Collagen
Yield (% on a
Source Pre-treatment extracting conditions Outcome Reference
weight basis)

ASC: Defatting: 10% butyl alcohol for

2 d.
Extraction: 0.5 M acetic acid for 3d.
Precipitation by the addition of NaCl
(final concentration of 2.3 M) in 0.05
M Tris–HCl (pH 7.5). Dialysis against
0.1 M acetic acid solution, distilled
water and then lyophilized.
The obtained Pepsin-Solubilized
collagen is a heterotrimer with a
Ocellate puffer fish ASC: 10.7* Nagai, Araki, &
chain composition of (α1)2α2.
(Takifugu rubripes) PSC: 44.7* Suzuki (2002)
Td: 28°C.
PSC: Residue was suspended in 0.5 M
acetic acid and digested with 10%
(w/v) pepsin for 48 h at 4°C. Dialysis
0.02 M Na2HPO4 (pH 7.2) for 3d.
Precipitation by adding NaCl (final
concentration of 2.2 M) in 0.05 M
Tris–HCl (pH 7.5). Dialysis against the
same solution and lyophilized.

Grass carp The obtained collagen contained

(Ctenopharyngodon PSC: 46.6* α1 and α2 chains. Td: 28.4°C Zhang et al. (2007)
idella) Tmax: 24.6°C
 Method: fish skins were soaked in 0.1
M
Tilapia (Oreochromis
NaOH with a skin/solution ratio of
niloticus)
1:10 (w/v) for 36 h, and alkali solution Results conclude that there
was might be a correlation between
changed every 12 h. Defatted using Molecular weight the film-forming ability of
Grass carp 10% butyl alcohol with a (MW) ranging from collagen from freshwater fish
Tang et al. (2015)
(Ctenopharyngodon solid/solvent ratio of 1:10 (w/v) for 36 100 to 20 kDa skins and the primary structures
idella) h and the solvent was changed every (SDS---PAGE) and conformation of collagen
12 h. Was washed with cold tap water molecules.
and subsequentially extracted with Td: 33°C
Silver Sarp 0.5 M acetic acid with a skin/ acetic
(Hypophthalmichthys acid ratio of 1:30 (w/v) for 2 days.
molitrix)
Method I: (ASC) 10 volumes of 0.5
50
mol/L of the acetic acid for 3 days
Method II: extraction of ASC was first
extracted. Then pepsin soluble
collagen (PSC) was extracted by using 55-60
The collagen from leather jacket
0.1% (w/v) pepsin to 0.5 mol/L of Muralidharan,
skin showed good thermal
acetic acid Shakila, Sukumar,
Odonus niger properties and it can be used
Method III: PSC was extracted twice & Jeyasekaran
effectively for pharmaceutical
and then Centrifugation. Precipitation (2013)
and biomedical applications.
of collagen by using 2 mol/L NaCl for
24 h at 4 °C. and centrifuging again, 70
followed by dialysis against 0.02
mol/L phosphate buffer (pH 7.2) for 24
h at 4°C.
Antioxidant and bioactive peptides
Source Enzymes used Properties Outcome Reference
 DH 40%.
ABTS: 48 µmol
TE/g solid
FRAP: 8 µmol
Alcalase
TE/g solid
Fe+2 Chelating Hydrolysates can be used as a
activity: 19 µmol source of natural antioxidants Sai-Ut, Benjakul,
Unicorn leatherjacket
EE/g solid that are useful in the oxidation Sumpavapol, &
(Aluterus monoceros)
ABTS: 66 µmol inhibition both food and Kishimura (2014)
TE/g solid biological systems.
FRAP: 9.5 µmol
Protease from B. amyloliquefaciens
TE/g solid
H11.
Fe+2 Chelating
activity: 19 µmol
EE/g solid
DPPH: 30%
Carbon centered:
Trypsin 40-50%
Superoxide: 40-
50% An antioxidant peptide sequence
identified to be His-Gly-Pro-
Leu-Gly-Pro-Leu (797 Da) was
DPPH: 10-20%
purified by consecutive
Carbon centered:
Hoki (Johnius chromatographic separations of Mendis, Rajapakse,
α -chymotrypsin, 20%
belengerii) tryptic hydrolysate. It showed & Kim (2005)
Superoxide: 30-
positive effects on the
40%
Antioxidative Enzyme Activities
in Hep3B Cells and on the
DPPH: 20-30% inhibition of lipid peroxidation.
Carbon centered:
Pepsin
20-30%
Superoxide:40%
 Two peptides associated with
ACE inhibitory activity were
identified to be MVGSAPGVL
ACE inhibitory
Skate (Okamejei Alcalase (829Da) and LGPLGHQ (720 Ngo, Ryu, & Kim
activity: 72.8% at 2
kenojei) Da), with IC50 values of 3.09 (2014)
mg/ml
and 4.22lM, respectively. They
could be used as functional
ingredients.

Bones: Antioxidant and bioactive peptides

Source Pre-treatment extracting conditions Properties Outcome Reference
Pretreatment: 0.6N HCl for 24h (10%
w/v). Drying at 60°C, 76 mmHg, 24h. IC50: 0.16 mg/ml
The hydrolysate has suitable Morimura et al.
Yellowtail fish Hydrolysis: Enzyme L, 200rpm, pH: IPOX50: 0.18
properties for the food industry. (2002)
8.0, 60°C, 60 min. mg/ml
Drying at 105°C for 24h.
DPPH: 4.82%
Alcalase. Buffer 0.1 M Na2HPO4– Hydroxyl: 77.85%
NaH2PO4. pH:7.0; T:50°C Superoxide:
22.53% An antioxidant peptide was
purified and identified to be
DPPH: 17.38 %
VKAGFAWTANQQLS
α -Chymotrypsin. Buffer 0.1 M Hydroxyl: 83.25 %
(1519Da). Results suggest that Je, Qian, Byun, &
Tuna backbone Na2HPO4–NaH2PO4. pH:8.0; T:37°C Superoxide:
these peptides fractions could be Kim (2007)
32.25%.
used as diet nutrients, food
DPPH: 36.72%
additives and pharmacological
Papain. Buffer 0.1 M Na2HPO4– Hydroxyl: 78.23%
agents.
NaH2PO4 pH:6.0 T:37°C Superoxide:
21.89%
Pepsin. Buffer: 0.1 M Glycine–HCl. DPPH: 35.82%
 pH:2.0; T:37°C. Hydroxyl: 80.91%
Superoxide:
31.92%
DPPH: 5.13%
Neutrase. Buffer 0.1 M Na2HPO4– Hydroxyl: 65.23%
NaH2PO4 pH:8.0 T:50°C Superoxide:
27.63%
DPPH: 6.89%
Trypsin Buffer 0.1 M Na2HPO4– Hydroxyl: 81.08%
NaH2PO4. pH:8.0 37°C Superoxide:
29.89%.
A low molecular peptide was
recovered from pepsinolytic
hydrolysates and it was
identified to be Val-Leu-Ser-
Calcium solubility:
Alaska Pollack Gly-Gly-Thr-Thr-Met-Ala-Met-
Pepsin. pH:2.0; T:37°C; E/S:1/100; 32 mg/l at a
(Theragra Tyr-Thr-Leu-Val (1442 Da). Its Jung et al. (2006)
Substrate concentration: 1% concentration of the
chalcogramma) affinity to calcium suggests that
peptide of 250 mg/l
the peptides might be used in
nutraceutical products to oriental
people with lactose indigestion
and intolerance.
Viscera: Proteolytic Enzymes
Optimum
Source Pre-treatment extracting conditions conditions and Outcome Reference
parameters
Proteases were successfully used
- Acid protease: 10 mM citrate/HCl
to hydrolyze bovine muscle and
pH 3.0 Centrifugation at 10000g Vannabun,
Acid proteases: pH farmed giant catfish skin.
for 10 min at 4°C. Ketnawa,
Giant catfish 3.0; 40°C Conclusions suggest that they
- Alkaline protease: 10 mM Tris- Phongthai,
(Pangasianodon gigas) Alkaline proteases: could be used as
HCl pH 8.0, 10mM CaCl2 (1:5w/v) Benjakul, &
pH 9.0; 60°C biotechnological alternative for
Centrifugation at 10000g for 10 Rawdkuen (2014)
gelatin hydrolysate production
min at 4°C.
and others.
 A protein hydrolysate derived
Hybrid catfish (Clarias 10 mM Tris-HCl pH 8.0, 1 mM CaCl2 from toothed ponyfish with Klomklao,
microcephalus × (1:50 w/v). Centrifugation at 10000g pH 9.0; 50°C desirable composition of Kishimura, &
Clarias gariepinus) for 10 min at 4°C. aminoacids and peptides was Benjakul (2013)
obtained.
10 mM Tris-HCl pH 8.0, 1 mM CaCl2
(1:50w/v). Centrifugation at 10000g Klomklao,
Hybrid catfish (Clarias
for 10 min at 4°C. A 24KDa purified trypsin was Benjakul,
microcephalus × pH 8.0; 60°C
Purification by using ammonium obtained. Kishimura, &
Clarias gariepinus)
sulfate fractionation and a series of Chaijan (2011).
chromatographies.
0.1 M Tris-HCl pH 8.0 (200 mg A thermostable alkaline protease
tissue/ml buffer). Centrifugation at with great activity and stability
10000g for 20 min at 4°C. over a wide alkaline pH range
Pirarucu (Arapaima Purification by a four step procedure: and high salt concentrations was Freitas-Júnior et al.
pH 9.0; 65°C
gigas) heat treatment, ammonium sulphate obtained from pyloric caeca of (2012)
fractionation, molecular size exclusion pirarucu. Its characterization
chromatography and affinity proved that this protease is a
chromatography. trypsin.
Castillo-Yánez,
Viscera extract from Monterey
100 g sample was homogenized with Pacheco-Aguilar,
Monterey sardine sardine found to be a promising
200 ml ice-cold distilled water and García-Carreño, &
(Sardinops sagax pH 2.5; 45°C biotechnological alternative to
centrifuged at 26000g for 20 min at 2- de los Ángeles
caerulea) the food industry due to its high
4°C. Navarrete-Del
activity detected.
(2005)
10 mM Tris-HCl buffer, pH 7.0,
(1:2w/v). Centrifugation at 8000g for
The crude acid protease was
15 min at 4°C. Bkhairia, Mhamdi,
Golden grey mullet (L. proven to be effective in gelatin
Supernatant was collected and adjusted pH 3.0; 40°C Jridi, & Nasri
aurata) extraction from golden grey
at pH 2.0 with 1 M HCl, then (2016)
mullet skin.
centrifuged for 30 min at 4 °C at
5000g.
Nile tilapia Viscera homogenized 40 mg of Enzymes from Nile tilapia Bezerra et al.
pH 8.0; 50°C
(Oreochromis niloticus) tissue/mL (w/v) in 0.9% (w/v) NaCl viscera could be isolated at low (2005)
 Centrifugation at 10000g for 10 min at cost due to the large amount of
10.8°C. this co-product in industrial
Purification by a three step procedure: processing. It can be used in the
heat treatment, ammonium sulphate production of fish protein
fractionation and gel filtration. hydrolysates, fish sauce and as a
laundry detergent additive.
Scale: Collagen
Source Pretreatment extracting conditions Yield (%) Outcome Reference

Sardine (Sardinops
50.9
melanostictus) Decalcification 0.05 m Tris–HCl (pH
7.5) containing 0.5 m EDTA-4Na for 2
Fish scales have potential as an
d. Disaggregation 0.1 m Tris–HCl (pH
alternative source of collagen Nagai, Izumi, &
8.0) containing 0.5 m NaCl, 0.05 m
for use in the cosmetic and Ishii (2004)
Red sea bream (Pagrus EDTA-2 Na and 0.2 m 2-
37.5 medical fields.
major) mercaptoethanol (2-ME) for 3 d and
limited pepsin digestion.

Japanese sea bass

41.0
(Lateolabrax japonicus)
839 *Dry weight basis, Td: denaturation temperature, Tt: transition temperature, ASC: Acid-Solubilized Collagen, PSC: Pepsin-Solubilized Collagen, DH:
840 Degree of hydrolysis
841

842
843 Table 4. Fish viscera protein hydrolysates amino acid composition

Fish specie

albacares) (Ovissipour
(Bhaskar et al., 2008)

morhua) (Horn et al.,

morhua) (Horn et al.,

et al., 2012) Alc 2.4L
(Acipenser persicus)

Atlantic cod (Gadus

Atlantic cod (Gadus

Atlantic cod (Gadus

Catla (Catla catla)
(Ovissipour et al.,

Aspmo, & Eijsink,

Persian Sturgeon

morhua) (Horn,
Tuna (Thunnus
2009) Alc 2.4L

2005) Endo
2005) Pap

2005) Alc
Alc 2.4L
Amino acids (g / 100 g)
Histidine 2.08 2.06 8.45 1.20 1.00 1.10
Isoleucine 3.80 3.60 6.93 3.30 2.90 3.10
Leucine 7.13 7.17 7.70 5.30 5.10 5.10
Lysine 6.80 7.07 1.87 3.30 3.70 3.70
Essential

Methionine 10.30 2.02 1.48 2.20 2.00 2.10

Phenylalanine 3.14 3.53 3.85 2.70 2.60 2.60
Threonine 3.50 4.02 5.90 4.10 3.40 3.70
Tryptophan - - - N.R. 0.50 0.50
Valine 5.79 4.79 8.93 4.00 3.60 3.80
Arginine 7.28 10.82 8.81 3.40 3.90 3.30
Tyrosine 2.34 2.57 1.31 2.40 2.40 2.50
Aspartate/Asp
8.30 8.50 11.83 6.30 6.10 6.00
aragine
Glutamate/Gl
13.70 15.01 15.31 9.70 9.40 9.70
Non-essential

utamine
Glycine 5.40 10.99 5.87 7.10 7.40 6.60
Alanine 6.30 7.04 2.23 5.10 4.90 4.80
Proline/hydro
3.46 6.24 N.R. 4.30/1.40 4.20/1.60 4.00/1.20
xyproline
Cystine N.R. 0.23 N.R. N.R. N.R. N.R.
Taurine N.R. N.R. N.R. 2.70 2.50 3.30
Serine 4.20 4.34 6.81 4.10 4.00 4.10
844 N.R.: No report, Alc: Alcalase, Pap: Papain, Endo: Endogenous enzymes
845

846

847
848

849

850

851 Graphical abstract

852

853
854 Highlights

855 Fish viscera protein hydrolysates (FVPH) are promising high added value products.

856 Nutritional, bioactive and functional properties of FVPH are useful to the industry.

857 Further research about the performance and stability of FVPH are needed.

858 FVPH production is considered sustainable, however there are huge challenges ahead.

859