Cryoprotective Effects of Inulin on Myofibrillar Protein in Silver Carp(Hypophthalmichthys molitrix) Surimi during Frozen Storage

YI Shumin, JI Ying, YE Beibei, YU Yongming, XU Yongxia, MI Hongbo, LI Xuepeng*, LI Jianrong

(National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, National R & D Branch Center of Surimi and Surimi Products Processing, College of Food Science and Engineering,Bohai University, Jinzhou 121013, China)

Abstract: The cryoprotective effects of inulin on myofibrillar protein from silver carp surimi were evaluated for use as an alternative to sugars that are used in commercial cryoprotectants. The cryoprotective effects of inulin and its mixtures with other cryoprotectants (1.5% inulin, 1.5% inulin + 4% sorbitol, 1.5% inulin + 2.5% sucrose + 4% sorbitol, 1.5% inulin +0.3% sodium tripolyphosphate) on myofibrillar protein as well as the influence on the physical properties of surimi gels were compared with those of a commercial cryoprotectant (4% sucrose + 4% sorbitol) over 35 days of storage at -18 ℃. Inulin and its mixtures with other cryoprotectants had a cryoprotective effect on surimi during frozen storage, as noted by suppressed decrease in Ca2+-ATPase activity, total and active sulfhydryl groups and salt-extractable protein, and alleviated increase in the surface hydrophobicity of myofibrillar protein. Moreover, surimi treated with 1.5% inulin + 0.3% sodium tripolyphosphate exhibited the best physicochemical properties in contrast to surimi samples treated with any other cryoprotectant.

Keywords: inulin; silver carp; myofibrillar protein; cryoprotective property

Surimi is a protein gel derived from fish that is made up of myofibrillar proteins. The gel is produced by rinsing minced fish to eliminate soluble materials, including sarcoplasmic proteins and additional impurities. Recently,there has been an increasing demand for surimi products,although populations of marine fish, which are the principal raw materials for surimi, have concomitantly decreased.However, freshwater fish are an alternative source of surimi,cheaper to process than marine fish, and very abundant in China. In particular, silver carp (Hypophthalmichthys molitrix) is one of the most important economic freshwater aquaculture species and yielded 3.9 million tons of production in 2018[1]. Therefore, it is gradually favored by aquatic products processing enterprises.

Surimi is typically frozen prior to being processed into products. Nevertheless, functional properties may be lost during freezing because of myofibrillar protein denaturation[2].Modern surimi production began in Japan following the development of technique to halt the denaturation of Alaskan pollock (Gadus chalcogrammus) surimi via the addition of sugar and sodium polyphosphate[3]. Based on this discovery,cryoprotectants have been broadly utilized in surimi production to inhibit protein denaturation during freezing.

Sugars have long been recognized to stabilize a large group of substances throughout freeze-thawing and freeze-drying processes[4-5]. For example, the addition of monosaccharide, including sucrose, trehalose and polydextrose, have been documented to halt protein denaturation[6-7], and mixtures of sugars exhibit even greater improvement in stabilization effects. In particular,a commercial cryoprotectant agent comprising sucrose and sorbitol exhibits excellent cryoprotective effects that prevent the denaturation and aggregation of proteins and has consequently been used broadly in the surimi industry.Commercial cryoprotectants which used to slow myosin denaturation typically comprise 4% sucrose, 4% sorbitol,and 0.3% sodium tripolyphosphate. Nevertheless, the high caloric value and excessive sweetness of sugars have limited their application as cryoprotectants for surimi. Consequently,cryoprotectants lacking sweetness and high caloric values have been investigated, which includes acetic acid esterified starch and gelatin hydrolysate, in addition to protein hydrolysates from konjac glucomannan, shark skin, and Pacific hake[8-11].

Inulin is a low-sweetness, low-calorie linear polymer of fructose molecules with a glucose moiety at its terminus.The polymer consists of oligo- and/or poly-saccharides comprising of 2-60 fructose units that are connected byβ-(2/1)-D-fructosyl-fructose bonds of different lengths[12-13].Inulin is considered a low calorie (4.18-8.36 kJ/g) compound because fructose is not released into the gastrointestinal tract, and it also features a sweetness that is about 35% that of sucrose[14-15]. Inulin is water soluble, exhibits minimal viscosity, and no off-flavors, which renders it an attractive option to enrich dietary fiber content in foods[16]. Moreover,inulin has been documented to exhibit numerous beneficial health effects, including stimulation of immune systems by functioning as bifidogenic agents, lowering pathogenic intestinal bacterial populations, relieving constipation,lowering the risk of osteoporosis by elevating mineral absorption, and lowering the risk of atherosclerosis[17-18].Further, inulin exhibits gel-formation characteristics that can also be used to make low fat cheeses, sauces, low-fat ice cream, frozen yogurt, and milk gels[12,19-22].

Following the above observations, the goals of this study were to examine 1) the cryoprotective impacts of inulin and inulin with sodium tripolyphosphate for preserving myofibrillar protein of silver carp surimi; 2) the effects of substituting inulin for sucrose as a cryoprotectant over 35 days of storage at -18 ℃, and 3) the effects of inulin and inulin mixtures on the physical properties of silver carp surimi.

1 Materials and Methods

1.1 Materials, chemicals, and reagents

Silver carp fish with an average mass between 1 000 g to 1500 g were obtained from the Jinzhou Aquatic Food Market(Jinzhou, China); Food-grade salt was obtained at nearby store (Jinzhou, China).

Food-grade inulin (with an average degree of polymerization of 26) Shaanxi Ciyuan Biotechnology Co., Ltd.; Food-grade sucrose and sorbitol Henan Zhongtai Food Co., Ltd.; Food-grade sodium tripolyphosphate Zhengzhou Xinsheng Biotechnology Co., Ltd.; Ca2+-ATPase kit the Institute of Nanjing Jiancheng Bioengineering Company;Ethylene diamine tetraacetic acid (EDTA), 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), 8-anilino-1-naphthalene-sulfonic acid (ANS), sodium dodecyl sulfate (SDS) Beijing Solarbio Science and Technology Co., Ltd.; All of the other chemical reagents were obtained from America Sigma-Aldrich Inc..

1.2 Instrument and equipment

YC200 deboner Food Machinery Factory of Kaicheng Liang-cai of Shandong Province, China; T25 digital ULTRA homogenizer IKA Instrument Equipment Co.,Ltd., Germany; Centrifuge Thermo Fisher Scientific Inc.,Germany; UV-2550 ultraviolet-visible spectrophotometer Shimadzu Company, Japan; 970 CRT fluorescence spectrophotometer Precision and Scientific Instrument Co., Ltd., Shanghai, China; CM-14 silent cutter Mainca Company, Spain; Texture analyzer Stable Micro System Company, UK.

1.3 Methods

1.3.1 Preparation of surimi

Fish were sacrificed on ice, processed, and washed. Fish meat were filleted and minced with a YC200 deboner that had a 4 mm orifice diameter. Minced meat was rinsed with cold water (＜ 10 ℃) 3 times with a mince: water ratio of 1:3 (m/V).In the third washing step, NaCl was added to a concentration of 0.15%. Water was removed from the mince after the third washing, and inulin or other cryoprotectants were added to the surimi. Surimi preservation treatments were as follows: A)a control surimi without any cryoprotectant, B) the addition of a commercial cryoprotectant (4% sucrose and 4% sorbitol),C) the addition of 1.5% inulin, D) the addition of 1.5% inulin and 4% sorbitol, E) the addition of 1.5% inulin, 2.5% sucrose,and 4% sorbitol, and F) the addition of 1.5% inulin, and 0.3%sodium tripolyphosphate. The treated surimi samples were then kept at -18 ℃ for 35 days.

1.3.2 Preparation of myofibrillar protein

Fish sample myofibrillar protein (MP) was set up as above details[23]with minor modifications. Briefly, 100 mL of buffer I (4 ℃, 0.1 mol/L KCl-20 mmol/L Tris-HCl, pH 7.2)was added to 20.0 g of surimi, and the combination was homogenized on ice for 5 min with a homogenizer. The homogenate was then centrifuged at 10 000 × g for 20 min at 4 ℃. The supernatant was then taken out and the precipitate was rinsed 3 times. Buffer II (0.6 mol/L KCl-20 mmol/L Tris-HCl, pH 7.0) was placed in with the precipitate and then homogenized. The homogenate was removed and kept for 60 min at 4 ℃ and then centrifuged at 10 000 × g for 20 min at 4 ℃ to obtain MP in the supernatant. The recovered MP samples were diluted to 4 mg/mL using buffer III (0.6 mol/L KCl) for further evaluation.

1.3.3 Determination of the Ca2+-ATPase activity

The Ca2+-ATPase activities of extracted MP were established with a Ca2+-ATPase kit. Briefly, 150 μL of MP(4 mg/mL) and chemical reagent I from the Ca2+-ATPase kit were combined and incubated for 10 min at 37 ℃.The reaction was then terminated with reagent II of the Ca2+-ATPase kit. The reaction mixture was centrifuged at 3 500 × g for 5 min. A 150 μL aliquot of the supernatant was then mixed with chemical reagent III from the Ca2+-ATPase kit and incubated for 20 min at 45 ℃, followed by cooling to room temperature. The absorbance at 660 nm was then recorded. The Ca2+-ATPase activity of the MP is expressed as μmol/(g·h).

1.3.4 Determination of the surface hydrophobicity

The protein surface hydrophobicity was established using previously detailed methods[24]. The MP was diluted to 0.125, 0.25, 0.5 and 1 mg/L with 20 mmol/L phosphate buffer (pH 7.0, containing 0.6 mol/L KCl). Diluted MP(4 mL) solutions were then combined with 20 μL of 8 mmol/L ANS in 0.1 mol/L phosphate buffer (pH 7.0). The relative fluorescence intensities of the ANS-protein conjugates were quantified with a spectrofluorometer at an excitation wavelength of 374 nm and an emission wavelength of 485 nm.The protein surface hydrophobicity was then determined from the initial slope of a linear regression for the relative fluorescence intensity versus protein concentration (g/L). The initial slope is referred to as surface hydrophobicity.

1.3.5 Determination of the total sulfhydryl content and active sulfhydryl content

The total sulfhydryl content of the protein was established with DTNB via previously described methods[24]with minor modifications. MP (0.4 mL of a 4 mg/mL solution) was mixed with 3.6 mL of buffer I (0.2 mol/L Tris-HCl, pH 6.8, containing 8 mol/L urea, 2% SDS, and 10 mmol/L EDTA). DTNB (0.4 mL of a 0.1% solution in 0.2 mol/L Tris-HCl, pH 6.8) was then added to the mixture and incubated for 25 min at 40 ℃. The absorbance was then measured at 412 nm. Active sulfhydryl content was similarly established, but without urea. The total and active sulfhydryl contents were calculated with an extinction coefficient of 13 600 L/(mol·cm).

1.3.6 Determination of the salt extractable protein content

Salt extractable protein (SEP) content was established using previously described methods[25]with slight alterations.Briefly, MP was diluted to 2 mg/mL and centrifuged at 8 500 × g for 15 min at 4 ℃. The protein concentration in the supernatant was established via the biuret method,and measurement of absorbance at 540 nm with a spectrophotometer. The SEP content is expressed as the percentage of MP relative to initial protein concentrations.

1.3.7 Preparation of surimi gel

The surimi was thawed for 5 h at 4 ℃ and then chopped for 1 min using a CM-14 silent cutter. The surimi was combined with 2.5% salt, and then chopped again for 2 min.The moisture content of the surimi was changed to 80% with ice water and chopping was conducted again for 10 min.The temperature was precisely controlled to below 10 ℃throughout chopping. The surimi paste was then poured into a circular glass bottle (25 mm diameter, 50 mm height) after chopping. Samples were heated to 40 ℃ for 30 min, then to 90 ℃ for 20 min. Heat-set specimens were chilled right away with flowing ice water, and each of the samples were then stored at 4 ℃ prior to further analyses.

1.3.8 Determination of the gel strength

Surimi gels were sliced into 25 mm × 25 mm cylinders after the prior treatment and then equilibrated to room temperature over 30 min. The physical properties of the samples were then evaluated with a texture analyzer. The breaking force, breaking displacement and gel strength were measured using a model P/5S spherical metal probe (test speed 60 mm/min).

1.3.9 Determination of the water-holding capacity

Surimi gels were sliced into 5 mm cylindrical slices after the treatment detailed above, and every slice was weighed (m1/g) and put between two layers of Whatman paper (No.1). The specimens were set into 50 mL centrifuge tubes and centrifuged at 5 000 × g for 15 min at 4 ℃. After centrifugation, the samples were rapidly removed from the paper and reweighed (m2/g) right away. The water-holding capacity (WHC) of the samples were then calculated as follows.

1.4 Statistical analysis

Each of the experiments were conducted in three times with randomly selected samples and the data are expressed as the ± s. One-way analysis of variance and least significant difference tests were used to compare means using in the SPSS 19.0 software package. Statistical significance was established as P ＜ 0.05.

2 Results and Analyses

2.1 Ca2+-ATPase activity of MP

Fig. 1 Changes in Ca2+-ATPase activity in MP from silver carp surimi during storage at -18 ℃

Ca2+-ATPase activity is attributed to the globular heads of myosin and is considered to be a good indicator for the integrity of the myosin molecule after frozen storage[3,24].Since Ca2+-ATPase activity derives from the globular heads of myosin, sharp decreases in Ca2+-ATPase activity suggests myosin denaturation, and specifically in the head region of molecules. Thus, a decrease in Ca2+-ATPase activity is also a main indicator of protein denaturation after frozen storage and has been broadly utilized as such in fish or surimi[25].Previous analyses have indicated that surimi is especially vulnerable to denaturation and aggregation without amended cryoprotectants[26]. Moreover, similar results have been observed for other fish muscle systems when cryoprotectants are not amended[9,24,27-28].

The effects of different cryoprotectants on the Ca2+-ATPase activity of MP from silver carp surimi during 35 days of storage at -18 ℃ are revealed in Fig. 1.The Ca2+-ATPase activity clearly decreased for each of samples. The controls exhibited the largest decrease in Ca2+-ATPase activity (89.09% decrease), while the Ca2+-ATPase activities of samples B-F decreased by 53.63%,68.64 %, 50.93%, 51.83% and 40.65%, respectively. Thus,the addition of cryoprotectants increased the retention of Ca2+-ATPase activity of the MP from silver carp surimi after frozen storage. The Ca2+-ATPase activity of the controlwas significantly lower than the samples with 1.5% inulin (sample C) in addition to those with mixed cryoprotectants(samples B, D, E, and F) (P ＜ 0.05). No significant difference in Ca2+-ATPase activity was observed among treatments with 1.5% inulin in samples C, D, and E during storage (P＞ 0.05).Sample F, which contained 1.5% inulin and 0.3% sodium tripolyphosphate, exhibited the highest activity retention.

These outcomes indicate that the addition of cryoprotectants (specifically 1.5% inulin + 4% sorbitol,1.5% inulin + 2.5% sucrose + 4% sorbitol and 1.5% inulin +0.3% sodium tripolyphosphate) can mitigate decreases in Ca2+-ATPase activity during frozen storage and exhibit optimal cryoprotective effects for MP from silver carp.

2.2 Surface hydrophobicity of MP

Fig. 2 Changes in surface hydrophobicity of MP from silver carp surimi during storage at -18 ℃

Increased surface hydrophobicity indicates that the hydrophobic portions of proteins that were previously hidden inside the molecule have become exposed because of protein denaturation or degradation[27,29-30]. The changes in surface hydrophobicity of MP from silver carp surimi are shown in Fig. 2. Increased surface hydrophobicity were observed in all of the MP samples from silver carp surimi during 35 days of storage at -18 ℃, despite the addition of various cryoprotectectants (Fig. 2). The surface hydrophobicity of the MP from the control treatment increased dramatically after frozen storage by 844.78%, while the surface hydrophobicity of samples B-F increased by 542.74%, 605.67%, 494.06%,508.06% and 457.15%, respectively. The marked elevation of surface hydrophobicity in the control samples is likely attributable to protein denaturation from freezing. Likewise,the variation in surface hydrophobicity elevation reflects the efficacy of various cryoprotectants towards protein stabilization. Sample C with 1.5% inulin exhibited a higher surface hydrophobicity than the samples with other cryoprotectants, but it was still lower than that for the control sample. No significant differences were observed for surface hydrophobicity values among samples B, D, E, and F. Sample F, which was amended with 1.5% inulin + 0.3% sodium tripolyphosphate, exhibited the lowest increases in surface hydrophobicity. Interestingly, sample D (1.5% inulin + 4%sorbitol) and sample E (1.5% inulin + 2.5% sucrose + 4%sorbitol) exhibited an even lower surface hydrophobicity than sample B, which was amended with commercial cryoprotectant (4% sucrose + 4% sorbitol).

Overall, these outcomes suggest that the cryoprotectants including 1.5% inulin + 4% sorbitol, 1.5% inulin + 2.5%sucrose + 4% sorbitol, and 1.5% inulin + 0.3% sodium tripolyphosphate, could mitigate increase in surface hydrophobicity and thereby have optimal cryoprotective effects on MP from silver carp.

2.3 Total sulfhydryl and active sulfhydryl contents of MP

Protein sulfhydryl groups play critical roles in protein structure and function. In silver carp, myosin contains 42 sulfhydryl groups that are oxidized to disulfide groups after denaturation, thereby resulting in decreases in total and active sulfhydryl contents, particularly after storage by freezing[31-33].Further, quantitation of sulfhydryl groups can indicate the degree of protein aggregation[34]. Consequently, the total and active sulfhydryl content of MP from silver carp surimi were evaluated after the use of various cryoprotectants.

Fig. 3 Changes in total sulfhydryl content of MP from silver carp surimi during storage at -18 ℃

Fig. 4 Changes in active sulfhydryl content of MP from silver carp surimi during storage at -18 ℃

The overall and active sulfhydryl contents of the MP decreased among all of the treatments, with similar trends among those treatments incorporating cryoprotectants (Fig. 3 and Fig. 4). The overall sulfhydryl content of the control was lowered by 68.55%, while those of samples B-F were lowered by 43.24%, 46.70%, 42.82%, 44.17% and 26.90%,respectively. The active sulfhydryl content of samples in the A-F treatments were lowered relative to the initial contents by 68.88%, 32.81%, 46.44%, 22.57%, 32.00% and 17.72%,respectively. The total and active sulfhydryl contents of the control samples were significantly lower compared to the specimens with cryoprotectants (samples B-F). (P＜ 0.05)The decreases in the total sulfhydryl content were greater than those for active sulfhydryl contents, which is likely due to some sulfhydryl groups that were originally hidden within proteins becoming exposed and oxidized.

2.4 Salt extractable protein contents of MP

Fig. 5 Changes in salt extractable protein content of MP from silver carp surimi during storage at -18 ℃

Decreases in SEP content are a main indicator of protein denaturation throughout frozen storage of meat that occurs due to the formation of hydrogen bonds, hydrophobic bonds, disulfide bonds, and ionic interactions[28-29,35-36]. The SEP content of all silver carp surimi samples was reduced throughout frozen storage, with the control decreasing by 54.81%, and the contents of samples B-F decreasing by 22.94%, 31.18%, 23.60%, 23.08% and 22.61%, respectively(Fig. 5). The SEP content was significantly lower in the control samples after frozen storage compared to samples B-F with cryoprotectants amended (P＜ 0.05). Further, the rate of decrease in the specimens with cryoprotectants was significantly slower than that in the control, and particularly so throughout the initial storage. The sharp decrease in SEP content in the absence of a cryoprotectant indicated that frozen storage prompted protein denaturation. Moreover,SEP contents of samples containing 1.5% inulin remained at low levels compared to samples with mixed cryoprotectants(samples B, D, E, and F).

These results suggest that the addition of cryoprotectant mixtures have advantageous protective effects on protein denaturation in silver carp surimi, as compared to the use of inulin alone. However, SEP contents were no significantly different among samples with different cryoprotectant mixtures. These observations suggest that inulin is a novel cryoprotectant with a distinctive cryoprotective impact on silver carp surimi throughout frozen storage. Moreover, the cryoprotective impact of mixed cryoprotectants (i.e., inulin with sorbitol and inulin with sodium tripolyphosphate)were greater than those from a commercial cryoprotectant(a sucrose-sorbitol mixture) that is broadly utilized in the surimi industry.

2.5 Gel strength and WHC of MP

Table 1 Effects of different cryoprotectants on textural properties and water-holding capacity of silver carp surimi gels

Gelation is a ubiquitous phenomenon in surimi processing, and the molecules typically responsible for gelation are proteins or polysaccharides[37]. The gel strength is a pivotal index for surimi products that reflects the formation of gel structures, and is related to customer acceptability.The WHC of a surimi gel reflects the sum of surimi protein and water, and is commonly utilized as an objective quality indicator for surimi products. Thus, the breaking force, the breaking displacement, the gel strength, and the WHC of silver carp surimi gels were evaluated after frozen storage with the use of various cryoprotectants. The breaking force, the breaking displacement, the gel strength, and the WHC of the control samples were 216.54 g, 4.23 mm,915.96 g·mm, and 70.56%, respectively (Table 1). In contrast,the synergistic effects were obvious for inulin and sucrose,sorbitol, sodium tripolyphosphate. The gel strength and the WHC of the surimi gel from sample F containing 1.5% inulin and 0.3% sodium tripolyphosphate were the highest among the treatments at 3 410.64 g·mm and 77.52%, respectively.The gel strength and the WHC of sample E that was preserved with 1.5% inulin, 2.5% sucrose, and 4% sorbitol was better than those for the sample B containing 4% sucrose and 4%sorbitol. These differences may possibly reflect that sodium tripolyphosphate has an optimal cryoprotective effect[35].Overall, these results indicate that inulin can increase the gel-forming ability of surimi gels as noted by improvement in both gel strength and WHC.

3 Conclusions

Overall, the results reported here indicate that inulin can prevent MP denaturation during frozen storage. In particular,a 1.5% concentration of inulin had a cryoprotective effect on surimi, and it was more effective than commercial cryoprotectants. Moreover, surimi containing 1) 1.5% inulin +4% sorbitol or 2) 1.5% inulin + 2.5% sucrose + 4% sorbitol or 3) 1.5% inulin + 0.3% sodium tripolyphosphate retained the best physicochemical properties in contrast to surimi samples preserved with other treatments. Importantly,inulin can be used as a cryoprotectant alternative to sugars since it is non-caloric, exhibits low sweetness, requires low dosages, has good cryoprotective effects, and improves surimi gel properties.

Future investigations should evaluate the optimal concentrations of these compounds to use in cryoprotection in addition to the sensory properties of the resulting surimi products. Further, the mechanisms underlying the synergistic cryoprotective effects of inulin with other ingredients (e.g.,sucrose, sorbitol, and sodium tripolyphosphate) must be considered in upcoming work.