Prevention of protein and lipid oxidation in freeze-thawed bighead carp (Hypophthalmichthys nobilis) fillets using silver carp (Hypophthalmichthys molitrix) fin hydrolysates

LWT - Food Science and Technology 123 (2020) 109050

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Prevention of protein and lipid oxidation in freeze-thawed bighead carp (Hypophthalmichthys nobilis) fillets using silver carp (Hypophthalmichthys molitrix) fin hydrolysates

T

Longteng Zhanga,b, Yuankai Shanb, Hui Hongb,d, Yongkang Luob, Xiaohui Honga,c, Weijian Yea,c,∗ a

Key Laboratory of Refrigeration and Conditioning Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, Xiamen, China Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China c Fujian Anjoyfood Share Co. Ltd, Xiamen, China d Xinghua Industrial Research Centre for Food Science and Human Health, China Agricultural University, Xinghua, 225700, Jiangsu, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Fin hydrolysates Antioxidant activity Protein oxidation Lipid oxidation Freeze-thaw cycles

Conversion of aquatic by-products into value-added food ingredients has gained great interest worldwide, and enzymatic hydrolysis is one of the most accepted methods for the recovery of by-product proteins. In our study, fins from silver carp (Hypophthalmichthys molitrix) were hydrolyzed by four enzymes (trypsin, alcalase, papain, and neutrase) and the antioxidant activity of the derived fin hydrolysates was evaluated both in vitro and in freeze-thawed bighead carp (Hypophthalmichthys nobilis) fillets. The results showed that fin hydrolysates by trypsin and alcalase exhibited strong in vitro scavenging activity against 2, 2′-azino-bis (3-ethylbenzothiazoline6-sulfonic acid) (ABTS) radicals and chelating activity to ferrous ions. Freeze-thaw-induced protein oxidation (the formation of carbonyls and disulfide bonds) and degradation (the loss of Ca2+-ATPase activity) were significantly (P < 0.05) inhibited by fin hydrolysates. In addition, fin hydrolysates reduced lipid oxidation as evidenced by decreased free fatty acid content, peroxide value, thiobarbituric acid reactive substances (TBARS), and fluorescent compounds after multiple freeze-thaw cycles. Therefore, fin hydrolysates by trypsin and alcalase could be potential natural antioxidants in preservation of fish fillets.

1. Introduction Frozen storage is a widely accepted method for long-term quality/ nutrient retention of aquatic products. Although microbial growth and enzyme activity are effectively terminated by low temperatures, the oxidation of protein and lipid still occur (Santos et al., 2019) and lead to deterioration in physicochemical properties, especially in conditions of temperature fluctuation and repeated freeze-thaw cycles (Nikoo, Benjakul, & Rahmanifarah, 2016). In order to alleviate discoloration, off-flavor, and other nutritional losses caused by lipid or protein oxidation, synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used. However, due to the concerns of toxicological effects of these synthetic antioxidants, exploration in natural plant- or animal-based antioxidants has aroused great interest (Falowo, Fayemi, & Muchenje, 2014). To achieve a sustainable development in surimi processing industry, freshwater fish species like silver carp (Hypophthalmichthys molitrix) has been recognized as great alternatives (Martin-Sanchez, Navarro, Perez∗

Alvarez, & Kuri, 2009). During surimi manufacturing, tremendous quantity of by-products with low economic value and high-quality proteins, including scales, viscera, skin, bones, fins, etc. are generated and they require further recovery and utilization (Bruno, Anta Akouan Ekorong, Karkal, Cathrine, & Kudre, 2019). Compared with the traditional direct discarding or processing into fish meal or fertilizers, better valorization technology, for example, controlled enzymatic hydrolysis, is gradually appreciated for its value-added possibility (Jayathilakan, Sultana, Radhakrishna, & Bawa, 2012). Native fish proteins can be hydrolyzed by enzymes into fish protein hydrolysates (FPH) that contained low molecular weight peptides with specific health-promoting bioactivities (Nikoo & Benjakul, 2015; Nikoo et al., 2016). A set of hydrolysates derived from fish by-products with in vitro antioxidant activity were discovered and showed promising potential as antioxidants in aquatic products (Atef & Ojagh, 2017; Chalamaiah, Kumar, Hemalatha, & Jyothirmayi, 2012; Zamora-Sillero, Gharsallaoui, & Prentice, 2018). As important parts of by-products, fins are rich in high-quality collagen (Mahboob, 2014; Nagai & Suzuki,

Corresponding author. Key Laboratory of Refrigeration and Conditioning Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, Xiamen, China. E-mail address: [email protected] (W. Ye).

https://doi.org/10.1016/j.lwt.2020.109050 Received 21 June 2019; Received in revised form 2 January 2020; Accepted 14 January 2020 Available online 18 January 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.

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2000), however, the utilization of fin collagen through enzymatic hydrolysis is rarely investigated (Ahn, Kim, & Je, 2014). Hence, in our study, in vitro antioxidant activity of fin hydrolysates of silver carp by different enzymes (trypsin, alcalase, papain, and neutrase) was examined as well as its potential inhibitory effect on lipid and protein oxidation in freeze-thawed bighead carp (Hypophthalmichthys nobilis) fillets, which might provide a better waste-handling strategy for surimi processing industry.

divided into four groups: (i) control (fillets in distilled water for 30 min), (ii) TP (fillets immersed in 0.3% tea polyphenol for 30 min), and (iii) (iv) fillets immersed in two different fin hydrolysates (2%, w/ v) that exhibited strong antioxidant activity in vitro, respectively. All fillets were packed individually in polyvinyl chloride bags and stored at −18 °C for 1 week and thawed in 4 °C until the core temperature to 0 °C (once a week, as one FT cycle) and a total of 6 FT cycles were applied. Samples were taken randomly for analysis after 0, 2, 4, and 6 FT cycles.

2. Materials and methods

2.3.2. Preparation of myofibrillar protein (MP) MP was prepared according to Lu, Liu, Zhang, Wang, and Luo (2015), with some modifications. Sarcoplasmic fractions in minced muscle (2 g) were removed by washing with cold distilled water and 0.3% NaCl. The precipitate was dispersed in 30 mL 0.6 M NaCl-20 mM Tris-maleate (pH 7.0). After centrifugation at 10, 000 g for 5 min, MP in the sediment was dissolved in 0.6 M NaCl. The protein concentration was determined by the Biuret method and then diluted to 4 mg/mL (Torten & Whitaker, 1964).

2.1. Materials Food grade alcalase from Bacillus licheniformis (280, 000 U/g) and papain from Carica papaya (240, 000 U/g) were purchased from Novozymes (Beijing, China), trypsin (200, 000 U/g) and neuturase (135, 000 U/g) were purchased from Sigma-Aldrich (Shanghai, China) and Angel Yeast Co., Ltd. (Wuhan, Hubei, China), respectively. Tea polyphenol (99.8%) was purchased from Wanbo Co., Ltd (Zhengzhou, Henan, China).

2.3.3. Determination of protein degradation and oxidation Sulfhydryl groups, disulfide bonds, and carbonyl contents were determined using the 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB), 2nitro-5-thiosulfobenzoate (NTSB), and 2, 4-dinitrophenylhydrazine (DNPH) method as described by Zhang, Li, Jia, Huang, and Luo (2018) respectively. Ca2+-ATPase activity was determined according to Benjakul, Seymour, Morrissey, and An (1997), with some modifications. The reaction was initiated after adding 0.2 mL 20 mM ATP (pH 7.0) and terminated by adding 2 mL 15% TCA. After centrifugation at 10, 000 g for 3 min, inorganic phosphate (Pi) in the supernatant was determined. Ca2+-ATPase activity was expressed as the amount of Pi generated per mg of MP per minute (μmol Pi/mg protein/min).

2.2. Fin hydrolysates 2.2.1. Preparation of fin hydrolysates Silver carp (Hypophthalmichthys molitrix) (weight: 1947 ± 84 g, n = 20) were purchased from a local aquatic market in Beijing, China. After fish stunning and sampling, fins (dorsal, pelvic, caudal, and pectoral fins) were manually collected, pooled, dried, dispersed in distilled water (1:5, w/v), and heated at 121 °C for 3 h. The extracted gelatin was then cooled to room temperature and filtered through cheese cloth to remove insoluble substances. The filtrate was divided into 4 aliquots (500 mL each) and adjusted to optimum pH and temperature of enzymes (alcalase: pH 8.0 and 50 °C, trypsin: pH 8.0 and 37 °C, neutrase: pH 7.0 and 45 °C, and papain: pH 7.0 and 55 °C) and 2% enzyme was added to the protein content in filtrate (w/w). The protein content of filtrate was measured by the Kjeldahl's method (AOAC, 2002). Hydrolysates at 0.25, 0.5, 1.0, 2.0, 3.0, and 4.0 h were collected, heated at 100 °C for 15 min to inactivated enzymes, and then centrifuged at 5, 000 g for 15 min to get the supernatant. The supernatant was lyophilized by a freeze drier (FD-1PF, Detianyou Co. Ltd, Beijing, China) and stored at −18 °C prior to analysis.

2.3.4. Lipid extraction Lipid was extracted as described by Bligh and Dyer (1959), with some modifications. 70 g minced muscle was homogenized in sequence with 35 mL chloroform +70 mL methanol and 35 mL chloroform +35 mL distilled water. The residue was re-washed with 35 mL chloroform and vacuum filtered. The filtrate was then transferred to a separating funnel until completely divided. The organic phase at the bottom was collected and dried under nitrogen, obtained lipid was placed in a desiccator for 12 h prior to analysis.

2.2.2. Determination of in vitro antioxidant activity Antioxidant activity including 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, ferrous (Fe2+)chelating activity, and reducing power were examined as described by Li, Luo, Shen, and You (2012). Hydrolysates with strong antioxidant activities were chosen as additives.

2.3.5. Determination of lipid oxidation Lipid oxidation parameters including free fatty acids (FFA) content, peroxide value (PV), and thiobarbituric acid reactive substances (TBARS) were measured according to Shi, Zhang, Lu, Shen, Yu, & Luo (2017). The fluorescent compounds (FCs) were determined in the aqueous phase of lipid extraction at excitation/emission wavelengths of 393/ 463 and 327/415 nm using a fluorescence spectrophotometer (F97, Lengguang Tech., Shanghai, China). The formation of FC was expressed as the fluorescence ratio (FR) and was calculated as the ratio between fluorescence values at 393/463 and 327/415 nm (Shi et al., 2017).

2.2.3. Peptide identification The fin hydrolysates with high antioxidant activity were subjected to peptide identification by mass spectroscopy (MS) analysis using a Thermo Q-Exactive high resolution mass spectrometer (Thermo Scientific, Waltham, MA, USA) according to the method of Zhang, Zhang, Wang, Chen, and Luo (2017). The peptide mass tolerance and fragment mass tolerance were set as 20 ppm and 0.02 Da, respectively. Peptide sequences were obtained within the Uniprotein database of silver carp using a searching engine of Mascot 2.4 (Matrix Science, Boston, MA, USA).

2.4. Statistical analysis All measurements including hydrolysates characterization, protein and lipid oxidation, etc. were done in triplicate and data was analyzed by SPSS 19.0 software (SPSS Inc., Chicago, IL USA). One-way analysis of variance (ANOVA) and Duncan's multiple range test were performed to evaluate the significant difference at the level of P < 0.05. Significant analysis was done both in FT cycles (at the same treatment) and the treatments (at the same FT cycle), different capital letters in figures indicate significant differences (P < 0.05) in treatment at the same FT cycle and different lowercase letters indicate significant

2.3. Fillets quality during freeze-thaw (FT) cycles 2.3.1. Fillets preparation Bighead carp (Hypophthalmichthys nobilis) (weight: 1893 ± 156 g, n = 36) were purchased from a local aquatic market in Beijing, China. After fish stunning and sampling, fillets (n = 72) were randomly 2

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Fig. 1. Changes in (a) ABTS scavenging ability, (b) Fe2+-chelating activity, and (c) reducing power of fin hydrolysates prepared with alcalase, trypsin, neutrase, and papainDifferent capital letters indicate significant differences (P < 0.05) in enzyme type at the same hydrolysis time and different lowercase letters indicate significant differences (P < 0.05) in hydrolysis time by the same enzyme.

peptide sequence. According to Nikoo and Benjakul (2015), the molecular weight of antioxidant peptides were usually below 2 kDa and thus peptides lower than 2 kDa were selected. A total of 102 and 61 peptides below 2 kDa were identified in trypsin and alcalase hydrolysates, respectively (data not shown). The antioxidant properties of hydrolysates was highly dependent on the size of peptides, compared with large peptides (containing10-50 amino acids), lower molecular weight peptides containing less than 10 amino acids residues were mainly responsible for the antioxidant activities because of their easier access to quench free radicals and inhibit lipid peroxidation (Nikoo & Benjakul, 2015). As shown in Table 1 and Table 2, 25 and 18 peptides were selected as the possible cluster of peptides that determined antioxidant activity. It was noticeable that most of the identified peptides contained large amount of hydrophobic amino acids, such as alanine, leucine, isoleucine, serine, proline, etc., and those hydrophobic amino acid residues contributed to the free radical scavenging activity in hydrolysates (Chalamaiah et al., 2012). The presence of aspartic acid at the Nterminus of peptides, for example, DLEVDTTLK, explained the chelating activity of pro-oxidative transition metal ions (Wu et al., 2017). Moreover, those characterized peptides shared similar repeated structures of Gly-Pro-X, such as GDTGPSGPK, PGPIGPPGPR, GGRGPPGER (P for hydroxyproline), etc., and such repeated tripeptide structures possessed cryoprotective effects against protein denaturation (Damodaran & Wang, 2017).

differences (P < 0.05) in FT cycles at the same treatment. 3. Results and discussion 3.1. Characterization of fin hydrolysates 3.1.1. Antioxidant activity Oxidative damages in meat and meat products are believed to be initiated by free radicals (Falowo et al., 2014). Fin hydrolysates by four enzymes showed similar tendency in scavenging activity toward ABTS radicals that characterized as sharp increases (P < 0.05) within 2 h of hydrolysis and then remained stable until 4 h (Fig. 1a). Compared with fin hydrolysates by papain and neutrase, enzymatic hydrolysis by trypsin and alcalase showed significantly (P < 0.05) higher quenching activity of ABTS radicals and reaching 86.00 and 89.07% at the end of hydrolysis, respectively. As Nikoo and Benjakul (2015) illustrated, antioxidant peptides mainly contained 3–16 amino acids (mostly hydrophobic amino acids), and alcalase's ability in the cleavage of hydrophobic amino acids might result in the hydrophobic peptides with strong antioxidant activity. Transition metal ions (Cu2+, Fe2+, etc.) are regarded as pro-oxidants in accelerating oxidative modifications in meat products (Nikoo & Benjakul, 2015). As shown in Fig. 1b, Fe2+-chelating activity of fin hydrolysates from trypsin, alcalase, neutrase, and papain remarkably (P < 0.05) ascended within 1 h of hydrolysis and finally reached 91.60, 85.33, 54.01, and 43.01%, respectively, in which trypsin and alcalase hydrolysates showed excellent antioxidant activity in chelating metal ions. As Zamora-Sillero et al. (2018) reported, amino and carboxyl groups in peptides terminals and specific structure in side chain of amino acids are together responsible for the chelation of metal ions. Wu, Li, Hou, Zhang, and Zhao (2017) likewise illustrated the iron chelating activity of peptides derived from trypsin-hydrolyzed the skin of Pacific cod (Gadus macrocephalus) and proved that the amino group in lysine, imine group in histidine, and carboxylate in aspartic acid are specific binding sites for metal ions. Similar to changes in ABTS scavenging and Fe2+-chelating activity, trypsin hydrolysates showed the highest reducing power with an absorbance of 0.889 at the end of hydrolysis and the reducing power of alcalase hydrolysates was 0.735 (Fig. 1c), indicating their strong ability as electron donators to reduce ferricyanide. Significantly (P < 0.05) lower reducing power was detected in fin hydrolysates generated by neutrase and papain with absorbance of 0.473 and 0.606, respectively. Combing all in vitro antioxidant activities, fin hydrolysates by trypsin and alcalase at 3 h were chosen as additive in fillets.

3.2. Protein degradation and oxidation 3.2.1. Sulfhydryl and disulfide bond content Sulfhydryl groups (SH) in cysteine are susceptible to be attacked in the presence of reactive oxygen species (ROS) (Lund, Heinonen, Baron, & Estevez, 2011). There were no significant differences in initial SH contents (values ranged from 81.37 to 82.57 μmol/g) among all groups (Fig. 2a). However, when FT cycles were added to 6 times, decline in SH content was more considerable in the control group and nearly 33.31% loss of SH was observed. SH decline was effectively retarded by tea polyphenol and fin hydrolysates from trypsin and alcalase with only 21.06, 21.80, and 23.90% decreases, respectively. In addition, trypsin hydrolysates showed superior antioxidant effect than alcalase, which was in accordance with its higher in vitro free radical scavenging and metal ion chelating activities. Similarly, gelatin hydrolysates from the skin of beluga sturgeon (Huso huso) also exhibited protective effect on SH oxidation in freshwater crayfish (Astacus leptodactylus) muscle that treated with multiple FT cycles (Yasemi, 2017). Corresponding to the gradual loss in SH content, continuous upward trends in disulfide bond (SS) contents were seen with prolonged FT cycles (Fig. 2b), conforming that the oxidative conversion from SH into disulfide linkages. Compared with a 2.58-fold increase in SS content in the control group after 6 FT cycles, treatments by tea polyphenol and

3.1.2. Peptide identification Fin hydrolysates by trypsin and alcalase with high in vitro antioxidant ability were then analyzed by LC-MS/MS to characterize the 3

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Table 1 Identified peptide sequence of fin hydrolysates treated by trypsin. Peptide No.

Peptide sequence

AA number

Mass

m/z

Position

PTM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

GVMGAVGA TGEAGER GMTGPIGP GETGPAGR GASGPAGPR GPVGPAGAR GGPGVVGPK AEDVNIQ FSGLDGAK GAAGLPGLK GDTGPSGPK GAEGPQGAR GFSGLDGAK GPPGPQGAR GEVGPQGAR GIVGLPGQR PGPPGPVGAR GETGEAGER LAGPPGEPGR GLQGMPGER QGPSGPSGER LPIIDIAPM PGPMGPMGPR DLEVDTTLK FQEPSVEGPR

8 7 8 8 9 9 9 7 8 9 9 9 9 9 9 9 10 9 10 9 10 9 10 9 10

660.3265 718.3246 728.3527 743.3562 768.3878 780.4242 782.4286 787.3712 793.397 798.4599 814.3821 841.4042 850.4185 851.4249 869.4355 895.5239 903.4926 904.3886 949.498 959.4495 970.4468 981.5569 995.4681 1032.534 1144.551

661.3317 360.1693 729.361 372.686 385.2012 391.2194 392.2209 788.379 397.7058 400.2379 408.1984 421.7101 426.2167 426.72 435.7249 448.7691 452.7534 453.2012 475.7568 480.7327 486.2303 491.7859 498.7416 517.2744 573.2839

584–591 1084–1090 749–756 896–903 1118–1126 1070–1078 497–505 1329–1335 255–262 719–727 263–271 347–355 254–262 230–238 338–346 944–952 550–559 1082–1090 990–999 710–718 970–979 1421–1429 163–172 1212–1220 93–102

/ / / / / / Hydroxylation / / Hydroxylation / / / Hydroxylation / / / / / Hydroxylation / / / / /

Note: a. P: hydroxyproline. b. Peptides from silver carp collagen (Accession number: A0A077B3P8) containing 2–10 amino acids were chosen.

fin hydrolysates (trypsin and alcalase) led to reduced SS formation with only 2.24-, 2.27-, and 2.05-fold increases than their initial values, respectively. Apart from antioxidant activity, fin hydrolysates might also have the protective effect in stabilizing the structure of myofibrillar proteins, which hindered the excessive exposure of buried SH caused by protein denaturation and thus alleviated the SH oxidation process in fillets (Yasemi, 2017).

highest carbonyl content (1.29 nmol/mg) together with a 8.58-fold increase than its initial value. The formation of carbonyls in fillets was greatly inhibited by fin hydrolysates from alcalase and trypsin and tea polyphenol with only 81.61, 81.09, and 74.61% of carbonyls were detected compared with those in the control group, indicating their excellent antioxidant activity in quenching formed ROS in fillets. Besides, hydrolysates might also lower the formation of carbonyls by binding metal ions (Lund et al., 2011).

3.2.2. Carbonyls Carbonylation of arginine, lysine, and proline are the most evident oxidative modifications occurred in meat and meat products (Lund et al., 2011). As shown from Fig. 2c, carbonyl contents in fresh fillets were relatively low in all groups (values below 0.20 nmol/mg). When fillets were treated with repeated FT cycles, remarkable carbonylation of proteins was detected in the control group as evidenced by the

3.2.3. Ca2+-ATPase activity Frozen-induced protein denaturation is responsible for the reduced integrity of myosin as characterized by the loss of Ca2+-ATPase activity in the myosin head region (Benjakul et al., 1997). As shown in Fig. 2d, tea polyphenol and fin hydrolysates did not influence the initial Ca2+ATPase activity (values around 0.45 μmol/mg/min). However, multiple

Table 2 Identified peptide sequence of fin hydrolysates treated by alcalase. Peptide No.

Peptide sequence

AA number

Mass

m/z

Position

PTM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

GFPGADGS GASGPAGPR GGPGVVGPK GFPGADGSA FSGLDGAK GDTGPSGPK GDRGFPGE GFPGLPGPS GFSGLDGAK GAPGKDGIR PGADGSAGPK GIVGLPGQR GGRGPPGER GIVGLPGQR AGRDGAPGPK PGPIGPPGPR GDRGFPGER RGFSGLDGAK

8 9 9 9 8 9 8 9 9 9 10 9 9 9 10 10 9 10

722.2871 768.3878 782.4286 793.3242 793.397 814.3821 833.3668 843.4126 850.4185 869.4719 871.4035 895.5239 897.4417 911.5189 940.4726 943.5239 1005.463 1006.52

723.2946 385.2005 392.2209 794.3314 397.7058 408.1978 417.6907 422.7137 426.2177 435.7431 436.7094 448.7695 449.7284 456.7675 471.2435 472.7698 336.1608 336.5136

479–486 1118–1126 497–505 479–487 255–262 263–271 662–669 956–964 254–262 740–748 481–490 944–952 464–472 944–952 1009–1018 1144–1153 662–670 253–262

Hydroxylation / Hydroxylation Hydroxylation / / / Hydroxylation / / Hydroxylation / Hydroxylation Hydroxylation Hydroxylation / Hydroxylation /

Note: a. P: hydroxyproline. b. Peptides from silver carp collagen (Accession number: A0A077B3P8) containing 2–10 amino acids were chosen. 4

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Fig. 2. Changes in (a) sulfhydryl content, (b) disulfide bond content, (c) carbonyl content, and (d) Ca2+-ATPase activity of bighead carp fillets after 0, 2, 4, and 6 freeze-thaw cycles. CK: fillets without additive; A: fillets in fin hydrolysates by alcalase; T: fillets in fin hydrolysates by trypsin; TP: fillets in tea polyphenol. Different capital letters indicate significant differences (P < 0.05) in treatment at the same FT cycle and different lowercase letters indicate significant differences (P < 0.05) in FT cycles at the same treatment.

2016), fin hydrolysates might also exhibit superior protective effect against frozen-induced quality deterioration in fillets.

FT cycles led to severe frozen-induced denaturation of myosin in terms of 72.73% loss of Ca2+-ATPase activity in the control group. Lower extent of Ca2+-ATPase activity changes were observed in the TP group with a 55.56% decrease. In addition, fin hydrolysates from trypsin and alcalase showed better protective effect against myosin denaturation as evidenced by only 48.84 and 50.00% loss of Ca2+-ATPase activity. Cryoprotective effect of fin hydrolysates was attributed to hydrophilic amino acids in peptides, which reduced the formation of ice crystals due to the water constraining effect by binding water with non-covalent forces, and thereby preventing protein denaturation at frozen temperatures (Karnjanapratum & Benjakul, 2015).

3.3.2. PV Hydroperoxide is a primary oxidative product of polyunsaturated fatty acids (Erickson, 1997). The PV of all groups peaked at the fourth FT cycle and descended drastically afterwards (Fig. 3b). The control group manifested the highest PV (16.28 meq peroxide/kg lipid) and the second highest PV (10.76 meq peroxide/kg lipid) was detected in trypsin hydrolysates-treated fillets, while the treatment of tea polyphenol led to the lowest PV (6.81 meq peroxide/kg lipid). According to Gracey, Collims, & Huey (1999, p. 407), rancidity of lipids started when PV exceeded 5 meq peroxide/kg lipid, hence, the control group reached rancidity at the second FT cycles, but additives could retard the lipid rancidity to the fourth FT cycles because of the quenching of lipid radicals. In addition to serving as free radical scavenger, fin hydrolysates also acted as physical barriers to prevent oxidants reaching lipids (Nikoo & Benjakul, 2015). The decrease in PV was attributed to the formation of secondary oxidative products (aldehydes, ketones, etc.) caused by hydroperoxide degradation (Shi et al., 2017). This kind of peroxide inhibiting effect was also illustrated by Das et al. (2017) by marine industry waste collagen hydrolysates.

3.3. Lipid oxidation 3.3.1. FFA content Fish are rich in polyunsaturated fatty acids, FFAs are degradation products of lipid hydrolysis and closely related to lipid oxidative reaction due to its higher susceptibility to oxidation than esterified fatty acids (Secci & Parisi, 2016). After applying 6 FT cycles, the accumulation of FAA was more obvious in the control group as seen from the 4.07-fold increase than the fresh value, reaching 24.11 g/100 g lipids, whereas the incline trends in FAA contents were effectively retarded by additives with only 3.08-, 3.11-, and 2.96-fold increases in tea polyphenol-, fin hydrolysates (trypsin and alcalase)-treated fillets (Fig. 3a), indicating limited lipid hydrolysis during repeated FT cycles. As FFAs formation in fish was linked with undesirable development of sensory (mainly off-odor and off-taste) and textural attributes (Méndez et al.,

3.3.3. TBARS As secondary lipid oxidation products, TBARS are expressed as equivalent malondialdehyde (MDA) content. According to Fig. 3c, initial TBARS values of all groups were all below 0.50 mg MDA/kg 5

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Fig. 3. Changes in (a) free fatty acids content, (b) peroxide value; (c) thiobarbituric acid reactive substances (TBARS), and (d) fluorescent compounds content of bighead carp fillets after 0, 2, 4, and 6 freeze-thaw (FT) cycles. CK: fillets without additive; A: fillets in fin hydrolysates by alcalase; T: fillets in fin hydrolysates by trypsin; TP: fillets in tea polyphenol. Different capital letters indicate significant differences (P < 0.05) in treatment at the same FT cycle and different lowercase letters indicate significant differences (P < 0.05) in FT cycles at the same treatment.

respectively. The incline in FCs was in agreement with excessive lipid oxidation process, and this tendency was similar to those in Atlantic mackerel (Scomber scombrus) during frozen storage (Trigo et al., 2018). Fillet without additive showed faster formation of FCs along with extensive freeze–thaw cycles (2.65-fold increases), while lower increasing trends were detected in fillets with additives and fin hydrolysates by trypsin showed the least upward tendency with only 2.24-fold changes, suggesting less lipid oxidation products were formed during freezethaw process.

muscle. However, with the prolonged FT cycles, TBARS values all significantly (P < 0.05) elevated and finally reaching 2.06, 1.45, 1.45, and 1.33 mg MDA/kg muscle in control, tea polyphenol-, trypsin hydrolysates-, and alcalase hydrolysates-treated fillets, respectively. Taking fold changes into consideration, FT for 6 times led to an obvious 4.38-fold higher TBARS value in the control group and the least increasing trend (3.74-fold) was observed when fin hydrolysates from alcalase was added, indicating its excellent antioxidant activity against lipid oxidation. As a useful indicator for the evaluation of fish quality by lipid rancidity, fish were regarded as unacceptable when TBARS content exceeded 1.51 mg MDA/kg muscle (Secci & Parisi, 2016). Hence, addition of tea polyphenols and fin hydrolysates could lower the lipid oxidation process in terms of TBARS assessment. It is noticeable that lipid oxidation products, such as MDA and 4-hydroxynonenal (4-HNE) are believed to facilitate the protein oxidation and further lead to deterioration in quality attributed of meat products (Faustman, Sun, Mancini, & Suman, 2010).

4. Conclusion Fins from silver carp were hydrolyzed using four commercial enzymes (trypsin, alcalase, neutrase, and papain). Fin hydrolysates by trypsin and alcalase showed strong in vitro ABTS radical scavenging and Fe2+-chelating activities. After being added to fillets, both fin hydrolysates effectively reduced freeze-thaw-induced protein/lipid oxidation and degradation. Therefore, fin hydrolysates could be used as promising antioxidants in preservation of aquatic products, and the research into fin hydrolysates might provide a better way of processing of by-products. However, the underlying mechanisms responsible for the antioxidant properties in fin hydrolysates remain to be explored.

3.3.4. Formation of FCs Primary and secondary lipid oxidation products could interact with nucleophilic molecules (i.e., protein, phospholipids, etc.) to form FCs (Shi et al., 2017). As shown in Fig. 3d, no significant difference was seen in initial FR values among all groups. The FC accumulation was more pronounced when multiple FT cycles were applied, and finally reached 5.15, 4.44, 4.40, and 4.38 in the control group, tea polyphenol-, trypsin hydrolysates-, and alcalase hydrolysates-treated fillets,

CRediT authorship contribution statement Longteng Zhang: Investigation, Validation, Writing - original draft, 6

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Formal analysis. Yuankai Shan: Investigation, Formal analysis. Hui Hong: Methodology, Software, Supervision. Yongkang Luo: Conceptualization, Supervision, Writing - review & editing. Xiaohui Hong: Writing - review & editing. Weijian Ye: Writing - review & editing, Supervision.

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