Shelf-life extension of crucian carp (Carassius auratus) using natural preservatives during chilled storage

The effect of the natural preservatives, tea polyphenols and rosemary extract, on microbiological [total viable count (TVC)], chemical [pH, total volatile base nitrogen (TVB-N), K-value and thiobarbituric acid (TBA) values], texture and sensory changes of air-packaged whole crucian carp (Carassius auratus) stored at 4 ± 1 °C was investigated for 20 days. The shelf-life of crucian carp was found to be 7–8 days for untreated group (control), 13–14 days for tea polyphenols group and 15–16 days for rosemary extract treated group according to sensory assessment results, for which the corresponding microbiological assessment also showed an increased shelf-life. Meanwhile, the increases of pH, TVB-N, K-value and TBA values were significantly delayed in both treated groups of samples compared to the control group. Thus, either tea polyphenols or rosemary extract could be used as potential preservatives to extend the shelf-life of crucian carp during chilled storage.     

Purification and characterization of chymotrypsins from the hepatopancreas of crucian carp (Carassius auratus)

Two chymotrypsins (chymotrypsin A and B) have been purified to homogeneity from the hepatopancreas of crucian carp (Carassius auratus) by ammonium sulphate fractionation and chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose and SP-Sepharose. The molecular masses of chymotrypsin A and B were approximately 28 and 27 kDa, respectively, by SDS–PAGE. Purified chymotrypsins also revealed single bands by native-PAGE. Optimum temperatures of chymotrypsin A and B were 40 and 50 °C, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Both enzymes were effectively inhibited by serine proteinase inhibitors and slightly activated by metal ions such as Ca²⁺ and Mg²⁺, while inactivated by Mn²⁺, Cd²⁺, Cu²⁺, Fe²⁺ to different degrees. Apparent K_ms of chymotrypsin A and B were 1.4 and 0.5 μM, and K_cats were 2.7 S⁻¹ and 3.4 S⁻¹, respectively. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A.     

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp (Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.     

透明质酸涂膜对鲫鱼微冻贮藏保鲜效果的影响

将鲫鱼肉表面涂抹不同浓度的透明质酸(HA)后在-3℃的微冻环境中进行贮藏,并分析其挥发性盐基氮(TVB-N)、pH值、电导率、硫代巴比妥酸(TBA)、持水力、色差和质构的变化规律。结果表明,HA结合微冻贮藏能延缓鲫鱼肉品质下降的速度,经0.9%HA处理后鲫鱼肉的保鲜效果较好,TVB-N、TBA、电导率与未经HA处理鱼肉样品相比增幅较小(P0.05),持水力降幅较小(P0.05);贮藏24d时,TVB-N为9.64mg/100g,TBA为0.15mg/kg,电导率为847μS/cm,色泽和质构变化较小。     

Establishment of quality predictive models for bighead carp (Aristichthys nobilis) fillets during storage at different temperatures

Quality predictive models were developed to predict the freshness of bighead carp (Aristichthys nobilis) fillets during storage at different temperatures. Quality indices [sensory score, total volatile base nitrogen (TVB‐N), total aerobic counts (TAC) and K value] at ?3, 0, 3, 9 and 15 °C were estimated and kinetically modelled by the Arrhenius equation. The activation energy (E_A) of sensory score, TVB‐N, TAC and K value was 78.17, 75.93, 106.53 and 76.21 kJ mol^?1, and the corresponding rate constants (k₀) were 1.16 × 10¹⁵, 2.60 × 10¹⁴, 4.05 × 10¹⁹ and 1.36 × 10¹⁵. The high regression coefficients (R² > 0.87) indicated the acceptability of the zero‐order reaction for sensory score, TVB‐N, TAC and K value. Relative errors between predicted and observed freshness indicators values of sensory score, TVB‐N, TAC and K value were all below 10% except the values at 6th day of K value, 3rd day and 9th day of TVB‐N. These results indicated that the models based on sensory score, TVB‐N, TAC and K value could effectively predict the freshness indicators of bighead carp fillets at the range of ?3 to 15 °C.     

通电加热下频率、温度对草鱼鱼块和鱼皮电导率的影响

为探索通电加热作为淡水鱼类加工方式的可行性,以草鱼为研究对象,采用LCR测量仪,测定了经50 Hz,20 V交流电加热后,草鱼鱼块(并联、串联)、碎鱼肉、带皮鱼块样品在10~80℃温度范围、50~20 k Hz频率范围内的阻抗值,并计算样品电导率。实验结果表明,三种样品(并联及串联鱼块、碎鱼肉)的电导率均呈现出随频率增大而增大、随温度升高而升高的趋势,其中,受鱼块内部膜方向的影响,并联、串联样品电导率有显著差异(p<0.05);碎鱼肉样品消除了鱼肉内部膜的影响,其电导率值介于并联、串联样品之间。带皮鱼块样品由于鱼皮的存在,比起其他样品有不同的电导率值,鱼皮平行于电流放置时电导率值高于鱼皮垂直电流放置的值。      

Purification and characterization of pyrophosphatase from bighead carp (Aristichthys nobilis)

Pyrophosphatase (PPase, EC 3.6.1.1) responsible to the hydrolysis of pyrophosphate (PPi) in muscle was purified from bighead carp (Aristichthys nobilis), and characterized in detail herein for the first time. PPase was extracted with 20 mmol/L Tris-HCl buffer (pH 7.0) containing 0.05 mol/L KCl, followed by heat treatment and ammonium sulfate precipitation. Then it was purified by deithylaminoethyl-cellulose (DE52) and Sephacryl S-300 chromatography, subsequently resulted in a 114.5-fold purification. The molecular mass of the PPase was defined to be 50 kDa with two subunits. The optimum pH of the purified PPase was around 8.0, and its optimum temperature was confirmed to be 50 °C. Magnesium ion was necessary to the activity of PPase. An excess of PPi over Mg²⁺ resulted in inhibition of PPase. When the Mg²⁺/PPi molar ratio was 1:1, the maximal enzyme activity was obtained. In addition, PPase converted PPi to orthophosphate (Pi) stoichiometrically with a K_m of 1.98 mmol/L under the condition of 5 mmol/L Mg²⁺. Ethylene diamine tetraacetate (EDTA) activated the activity of PPase at low concentration, however, it consumingly did inhibit the enzyme activity at high concentration.     

Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

An aminopeptidase was purified from the skeletal muscle of common carp (Cyprinus carpio) to homogeneity, with 1160-fold purification and a yield of 4.3%. The purification procedure consisted of ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, Phenyl Sepharose and Macro-Prep High Q columns. The molecular mass of the enzyme was estimated to be 105 kDa and 100 kDa by SDS-PAGE under reducing conditions and gel filtration chromatography, respectively, suggesting it to be a monomer. The enzymatic activity was optimal at 35 °C and pH 7.0. The metal-chelating agents EDTA, EGTA and 1,10-phenanthroline specifically inhibited the enzyme activity while inhibitors of other proteinases did not show much effect, indicating that it was a metalloproteinase. Furthermore, bestatin, a specific aminopeptidase inhibitor strongly inhibited its activity. Divalent cations Mn²⁺, Mg²⁺ and Ba²⁺ slightly enhanced the enzymatic activity, while Co²⁺, Cu²⁺, Zn²⁺, Ca²⁺ and Fe²⁺ inhibited the activity to different extents. In addition, a sulfhydryl reagent was necessary to maintain its activity. Substrate specificity study revealed that the purified enzyme preferentially hydrolyzed Leu-MCA, followed by Arg-MCA, Ala-MCA and Tyr-MCA and it was thus regarded as a leucine aminopeptidase (LAP, EC 3.4.11.1). The apparent K_m and V_max values of the enzyme were 4.6 μM and 9.6 μmol min⁻¹ mg⁻¹, respectively for substrate Leu-MCA. This is the first report concerning purification and characterization of LAP from freshwater fish.     

Antioxidant and biochemical properties of protein hydrolysates prepared from Silver carp (Hypophthalmichthys molitrix)

The antioxidant and biochemical properties of enzymatically hydrolyzed silver carp (Hypophthalmichthys molitrix) protein were studied. The molecular weight of the main peaks of the hydrolysates by both Alcalase and Flavourzyme was lower than 5000 Da. The hydrolysates treated by Alcalase for ?1.5 h (hydrolysis time) showed that the relative proportion of <1000 Da fraction was more than 60%. For the biochemical properties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range; and when the hydrolysis time was prolonged (>3 h), the colour of the hydrolysates turned yellowish. The protein hydrolysates exhibited significant hydroxyl radical-scavenging activity and inhibited linoleic acid peroxidation. For Alcalase treatment, the hydroxyl radical-scavenging activity and the inhibition of linoleic acid peroxidation of hydrolysates appeared to reach a maximum level for 1.5, 2.0 h of hydrolysis, respectively; and their antioxidant activity was close to that of α-tocopherol in a linoleic acid emulsion system, and carnosine in the 2-deoxyribose oxidation system. The hydrolysate with lower molecular weight distribution possessed stronger Fe²⁺ chelation ability at a sample concentration of 5.0 mg/mL. The results suggested that the antioxidant activity of silver carp protein hydrolysates were related to its degree of hydrolysis (DH), hydrolysis time and molecular weight.     

Effect of microwave heating on the low-salt gel from silver carp (Hypophthalmichthys molitrix) surimi

Gels from silver carp (Hypophthalmichthys molitrix) surimi were obtained using microwave (MW) heating (15 W/g power intensity for 20–80 s) at different levels of salt (0 g/100 g, 1 g/100 g, or 2 g/100 g). And the gel heated by MW was compared with the gel obtained by conventional water-bath heating (85 °C for 30 min). The gel strength increased when the salt level was increased. The mechanical and functional properties of non-salted, low-salt and regular-salt products were improved by MW heating for 60 s and 80 s, significantly (p < 0.05), except for the cook loss. The content of TCA-soluble peptides indicated that the MW heating inhibited the autolysis of proteins significantly (p < 0.05) during gelling. The SDS-PAGE and total content of –SH group proved that MW enhanced the cross-linking of proteins effectively through disulphide bonds and non-disulphide covalent bonds. The microstructure of the samples revealed that a fine compact network, with particles of protein aggregates, was formed in the low-salt gels (1 g/100 g) heated by MW for 60 s. All of these properties might be responsible for the formation of a superior textural low-salt gel induced by MW.     

Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella)

Pepsin-soluble collagen was extracted from the skin of grass carp (Ctenopharyngodon idella) with a yield of 46.6%, on a dry weight basis. Electrophoretic patterns showed that the collagen contained α1 and α2 chains, similar to those of calf skin collagen. The imino acid content of the collagen from grass carp skin was much lower than those of mammalian’s collagens, as also were the transition temperature and denaturation temperature which were only 24.6 °C and 28.4 °C respectively. Peptide maps of the collagen digested by trypsin and V8 protease showed different peptide fragments from those of calf skin collagen and other fish skin collagens, suggesting differences in amino acid sequences and collagen conformation. In addition, the lyophilized collagen sponge from grass carp skin had a uniform and regular network structure, just like calf skin collagen sponge. These results suggest that grass carp skin has potential for use as a supplementary source of collagen.     

Seasonal differences in the properties of gelatins extracted from skin of silver carp (Hypophthalmichthys molitrix)

The gelatins were extracted from skins of silver carp (Hypophthalmichthys molitrix) caught in winter and summer, respectively. The physicochemical (molecular weight distribution and melting point) and rheological characteristics (viscosity property), as well as functional properties (emulsifying capacity and stability) of the gelatin from winter silver carp skin were compared with those of the summer equivalent. The results showed the properties of the summer gelatin were superior to those of the winter one, showing higher viscosity, emulsion stability, melting point and lower concentration for gelling. The summer gelatin has slightly denser strands of the gel microstructure which was observed by scanning electron microscopy (SEM). Different properties of gelatins from skin of silver carp may be attributed to the big discrepancy of the environmental temperature in the two seasons.     

Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysate from grass carp (Ctenopharyngodon idella) skin

Protein hydrolysates from grass carp skin were obtained by enzymatic hydrolysis using Alcalase^®. Hydrolysis was performed using the pH-stat method. The hydrolysis reaction was terminated by heating the mixture to 95 °C for 15 min. At 5.02%, 10.4%, and 14.9% degree of hydrolysis (DH), the hydrolysates were analyzed for functional properties. The protein hydrolysates had desirable essential amino acid profiles. Results demonstrated that the hydrolysates had better oil holding and emulsifying capacity at low DH. The water holding capacity increased with increased levels of hydrolysis. Enzymatic modification was responsible for the changes in protein functionality. These results suggest that grass carp fish skin hydrolysates could find potential use as functional food ingredients as emulsifiers and binder agents.     

The influence of superchilling and cryoprotectants on protein oxidation and structural changes in the myofibrillar proteins of common carp (Cyprinus carpio) surimi

The objective of the present study was to investigate the effects of superchilling combined with cryoprotectants (a mixture of sucrose and sorbitol, 1:1) on protein oxidation and structural changes in common carp (Cyprinus carpio) surimi. With increasing storage time, the carbonyl content of myofibrillar proteins increased from 31.4 nmol/mg of protein (0 day) to 53.4, 46.3, and 39.7 nmol/mg protein at 35 days (P < 0.05) for −1 °C superchilled, −3 °C superchilled, and −3 °C superchilled with cryoprotectants samples, respectively. The incorporation of cryoprotectants into common carp surimi was found to significantly inhibit the formation of carbonyls (P < 0.05). The protein surface hydrophobicity increased in a similar direction, and the sulfhydryl content and Ca-ATPase stability decreased (P < 0.05). Emulsifying activities, gel textural hardness, springiness, and water binding capacity were also decreased, but they exhibited significant improvements at −3 °C when combined with cryoprotectants (P < 0.05). These results suggest that superchilling treatments at −3 °C combined with cryoprotectants offer an effective approach to reducing protein oxidation in carp surimi, thereby reducing protein structural changes known to impair the texture of surimi products.     

Effects of konjac glucomannan on physicochemical properties of myofibrillar protein and surimi gels from grass carp (Ctenopharyngodon idella)

The cryoprotective effect of konjac glucomannan (KGM) on myofibrillar protein from grass carp (Ctenopharyngodon idella) during frozen storage at −18 °C and the influence of five levels of KGM (0%, 0.5%, 1%, 1.5%, and 2%) on texture properties, water-holding capacity, and whiteness of grass carp surimi gels were investigated. KGM as a novel cryoprotectant could significantly mitigate the decrease in salt extractable protein (SEP), Ca²⁺-ATPase activity, and total sulphydryl and active sulphydryl contents of myofibrillar protein during frozen storage. KGM at the level of 1% showed the same good cryoprotective effect as a conventional cryoprotectant (10% sucrose–sorbitol, 1:1, w/w). As the levels of KGM increased, breaking force and deformation of grass carp surimi gels increased significantly. Water-holding properties of the surimi gels are improved with the increasing addition of KGM, but the whiteness decreased and the colour became darker. The optimum addition level of KGM was suggested to be 1%.     

Pink discoloration and quality changes of squid (Loligo formosana) during iced storage

Pink discoloration and quality changes of squid (6-10 squids/kg) with and without deskinning during iced storage at different squid/ice ratios (1:1 and 1:2, w/w) for 16 days were investigated. The increases in a* and b*-values of squid mantle were observed with increasing storage time (p < 0.05), indicating the formation of pink color on the mantle. The increase was more pronounced in squid without deskinning with a squid/ice ratio of 1:1 (p < 0.05). No changes in a*-value were observed in deskinned squid throughout the storage, regardless of squid/ice ratio (p > 0.05). However, the slight increase in b*-value was found in the squid with deskinning during the storage. Psychrophilic bacteria counts of squid increased continuously as the storage time increased. Coincidental increases in total volatile base (TVB), trimethylamine (TMA) and ammonia contents were observed during the storage. The rates of increase were greater in the samples with a squid/ice ratio of 1:1 than those found in the samples kept in ice with a squid/ice ratio of 1:2. Pink discoloration, psychrophilic bacteria count, TVB and TMA contents were much lowered when the squid without deskinning was treated with 0.1 g/100 mL sodium azide (NaN₃) prior to storage, suggesting that microbial growth was associated with those changes. Therefore, deskinning together with icing using a sufficient amount of ice as well as the use of safe antimicrobial agent could be a means to lower the pink discoloration and retard the losses in quality of squid stored in ice.     

Effects of superchilling and cryoprotectants on the quality of common carp (Cyprinus carpio) surimi: Microbial growth,oxidation, and physiochemical properties

The objective of the present study was to investigate the effect of superchilling with cryoprotectants (a mixture of sucrose and sorbitol) on microbial growth, lipid oxidation, and proteolytic degradation in common carp (Cyprinus carpio) surimi. With increasing storage time, the microbial count of surimi increased from 4.4 log₁₀ CFU/g (0 days) to 7.2, 6.2, 5.9, and 5.5 log₁₀ CFU/g (35 days; P < 0.05) in the samples superchilled at −1 °C, superchilled at −3 °C, superchilled at −3 °C with cryoprotectants, and frozen at −18 °C, respectively. The total volatile basic nitrogen and thiobarbituric acid-reactive substances exhibited an increasing trend (P < 0.05) similar to that obtained with the microbial growth, whereas the whiteness and lightness stability decreased (P < 0.05) with increasing storage time. The SDS-PAGE analysis revealed that the degree of protein degradation increased with increasing storage time. Compared with the sample frozen at −18 °C, superchilling at −1 °C and −3 °C resulted in a marked reduction in the microstructure deterioration of the myofibrillar protein gels, and the addition of cryoprotectants further reduced this deterioration. These results suggest that superchilling with cryoprotectants offers an effective approach for reducing microbial growth and lipid oxidation and limiting proteolytic degradation in common carp surimi.     

Effect of concentration,pH and ionic strength on the kinetic self-assembly of acid-soluble collagen from walleye pollock (Theragra chalcogramma) skin

The effects of concentration, pH value and ionic strength on the kinetic self-assembly of acid-soluble collagen from walleye pollock (Theragra chalcogramma) skin were investigated. A two-phase kinetic process was provided which included the formation of nucleus center and nucleus growth, the first phase being the controlled step for collagen self-assembly. Collagen showed marked assembly behavior when concentration reaching and above 0.6 mg/mL, and higher concentration could accelerate collagen self-assembly. Rate constants of the first and second assembly phase both increased with pH to a maximum around pH 7.2 and then decreased, indicating that pH 7.2 was the optimum pH value for collagen self-assembly. The kinetics of collagen self-assembly could be modulated by NaCl concentration. The concentration of NaCl from 30 to 60 mM was more suitable to self-assemble for pollock skin collagen.