Autolytic degradation of belly tissue in anchovy (Engraulis encrasicholus)

Tissue degradation in anchovy was studied during storage under different conditions. Belly bursting seems to be caused by leakage of proteolytic enzymes from the pyloric caeca and intestine to the ventral muscle. These proteases have optimum pH in the alkaline range and they are very active in the neutral range. At pH 6.5, which is the post mortem pH of the tissue, the basic proteases play an important role in the degradation of myofibrillar protein and in the solubilization of connective tissue. The rate of tissue degradation during storage was considerably reduced if the fish was chilled by refrigerated or iced sea water to about 0°C, and the pH of the medium was reduced to 5 by addition of lactic or acetic acid.     

Effect of Combining Proteolysis and Lactic Acid Bacterial Fermentation on the Characteristics of Minced Mackerel

ABSTRACT: To improve the quality of fish muscle, mackerel muscle protein was hydrolyzed by proteases from Aspergillus oryzae , and then fermented by lactic acid bacteria (LAB). The highest protease activities were obtained from A. oryzae after 72 h incubation at 25°C. Acidic protease activity was much higher than neutral and alkaline proteases. SDS-PAGE indicated the degradation of muscle proteins after 1 or 2 h hydrolysis by A. oryzae proteases at 50°C. During 48 h fermentation by Pediococcus pentosaceus L and S at 37°C, rapid growth of LAB, decline in pH, and suppression in the growth of microflora, Enterobacteriaceae, Staphylococcus , and Pseudomonas , occurred while increases in whiteness, nonprotein nitrogen, sensory quality, and free amino acids were observed. These data suggested that the acceptability of LAB -fermented mackerel hydrolysates could be substantially improved.     

ROLE OF MUSCLE PROTEINASES IN MAINTENANCE OF MUSCLE INTEGRITY AND MASS

Current evidence indicates that, of the thirteen known lysosomal peptide hydrolases, only seven, cathepsins A, B, C, D, H, L, and lysosomal carboxypeptidase B are located inside skeletal muscle cells. Only one of the reported neutral and alkaline proteases is located inside skeletal muscle cells', this neutral protease is the Ca²⁺-dependent proteinase, CAF. With the possible exception of cathepsin N, which can degrade collagen, it seems probable that any protease that contributes to postmortem tenderization needs to be located inside muscle cells. Because very little degradation of myosin or actin occurs in postmortem muscle, most of the small amount of proteolytic degradation of the myofibrillar proteins that occurs during postmortem storage must be due to CAF, which is unique in being unable to degrade myosin and actin. It is not certain that postmortem proteolysis by CAF causes increased tenderness; some recently discovered actin-fragmenting proteins could be involved.     

Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure

Postmortem tenderization is caused by enzymatic degradation of key structural proteins in myofibrils as well as in extracellular matrix, and of proteins involved in intermyofibrillar linkages and linkages between myofibrils and the sarcolemma. The function of these proteins is to maintain the structural integrity of myofibrils. Current data indicate that calpains and cathepsins may be responsible for degradation of these proteins. Other phenomena occurring in cells postmortem (pH drop, sarcoplasmic Ca2+ increase, osmotic pressure rise, oxidative processes) may act in synergy with proteases. Our understanding of the underlying mechanisms of muscle degradation should be improved for an accurate evaluation of the postmortem muscle changes and consequently of the fish quality.     

Proteolytic Activity in Muscle from Atlantic Salmon (Salmo salar)

The maximum proteolytic activity appeared in the neutral to slightly alkaline pH range. Heat-stable alkaline proteases were detected. Optimal activity was found at pH 8 and 65°C. The activity dropped off markedly below 60°C and above 70°C. Preincubation of the muscle-extract for about 10 min at 65°C stimulated the activity. Salmon muscle was especially susceptible to proteolytic degradation at elevated temperatures. No notable differences were detected in proteolytic activities of Atlantic salmon in the feeding stage (superior quality) and in the sexually mature stage.     

Effect of high pressure on the calpain-calpastatin system in fish muscle

ABSTRACT:  Calpains (calcium-activated neutral proteases) of sea bass ( Dicentrarchus labrax L.) muscle may participate in the degradation of muscle tissue during postmortem storage. These enzymes are regulated by calpastatin, their endogenous specific inhibitor. The objective of this study was to evaluate the changes encountered by the calpain system during the postmortem storage of fish muscle after high-pressure treatment. From 100 MPa, high-pressure treatment of purified calpains results in a loss of their activity as well as in the dissociation of the heterodimeric form. In muscle, the high-pressure processing decreases the initial activity of calpain. This loss in activity may be due to an inactivation by a change of structure. Initial calpastatin activity is not modified by the high-pressure treatment, but it decreases during the storage from the beginning for a treatment at 300 MPa after which calpastatin is stable during 2 d. Therefore, this study also suggests that high-pressure treatment could be a useful way to improve fish flesh quality.     

Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。     

Activity of Endogenous Muscle Proteases from 4 Australian Underutilized Fish Species as Affected by Ionic Strength,pH, and Temperature

Storage conditions may influence the hydrolytic activity of endogenous muscle enzymes postmortem, rate of autolysis of myofibrillar proteins, and biological properties of hydrolyzed end products. This study investigated the effect of ionic strength, pH, and temperature on the activity of endogenous calpain‐like, cathepsins B and B+L measured in crude extract obtained from deepwater flathead, silver warehou, ribaldo, and ribbonfish muscles. Activity of calpain‐like enzymes in 3 examined species was significantly higher at pH 6.5 than pH 6.0 or 5.5. Raising the reaction temperature increased (P < 0.05) calpain‐like activity in ribaldo. Endogenous activity of cathepsin B in ribbonfish and silver warehou declined significantly with increasing ionic strength at pH 6.5 to 6.0. The obtained results will further expand our understanding of the impact that postmortem storage conditions have on the activity of endogenous fish proteases with respect to quality and bioactivity of fish proteins and potentially diversify utilization of underutilized fish species.     

腌制鱼类中内源性酶类对制品品质影响的研究进展

鱼类腌制品在加工过程中产生一系列的品质变化(主要是风味和质构的变化),内源性酶类是引起这些变化的主要原因之一。与产品质构密切相关的酶类主要是一些降解蛋白质的内源性蛋白酶类(溶酶体组织蛋白酶、钙激活蛋白酶、基质金属蛋白酶),与产品风味密切相关的酶类主要是氨肽酶和降解脂肪的一些酶类(脂肪水解酶类、脂肪氧合酶)。本文对腌制鱼类产品中对组织蛋白降解和脂肪降解、氧化的主要内源性酶类特性和国内外研究现状进行了综述,以期为进一步革新传统腌制鱼类产品加工技术和提高产品品质提供一定的参考。      

Implication of Muscle Alkaline Proteinase in the Textural Degradation of Fish Meat Gel

Unlike the actomyosin paste, white croaker meat paste showed poor elastic gel when heated around 60°C. The hydrolysis of muscle protein by muscle proteinase was observed in the poor gel. There are at least four kinds of proteinases in white croaker muscle: cathepsin D, neutral proteinase, calpain and alkaline proteinase. Among them only alkaline proteinase could act at pH of meat paste and around 60°C. Above facts confirmed the previous work that the heat-stable alkaline proteinase is probably implicated in the textural degradation of fish meat gel around 60°C.     

酶解刺参内脏蛋白制备抗氧化肽的研究

对刺参内脏所含的内源蛋白酶的性质进行了初步研究,在此基础上分析和比较了利用内源蛋白酶和外源蛋白酶酶解内脏蛋白制备抗氧化肽的工艺。刺参内脏主要含有四种内源蛋白酶,最适pH分别为2.0、5.0、8.0和12.0。以羟自由基和超氧阴离子自由基清除率为指标,比较了内源蛋白酶和八种外源蛋白酶酶解内脏蛋白制备抗氧化肽的效果,结果表明菠萝蛋白酶为最佳用酶;用正交实验L9(34)对其水解条件进行优化,确定最佳酶解条件为:pH7.8、温度35℃、加酶量5.0g/100g、酶解时间1h,在此条件下所得酶解液对羟自由基和超氧阴离子自由基的清除率分别为79.15%和28.57%。      

RELATIONSHIP OF pH AND TEMPERATURE TO DISRUPTION OF SPECIFIC MUSCLE PROTEINS AND ACTIVITY OF LYSOSOMAL PROTEASES

From a review of the literature, and from specific data presented in this paper, it was concluded that both postmortem temperature and pH have effects on meat tenderness and on disruption of specific myofibrillar proteins. Increased postmortem temperature porduces more tender muscles and increases the disruption of troponin-T, myosin, Z-lines, connectin and gap filaments. Elevated postmortem temperature also increases the activity of enzymes which cause the disruption of myofibrillar proteins. Higher ultimate postmortem pH (above 6.0) produces more tender muscle, but also produces dark-cutting meat Except for one experiment, lower pH in the first few hours postmortem (in muscle with normal ultimate pH; i.e., 5.8 or below) improves meat tenderness. High pH increases the activity of CAF and low pH increases the activity of lsosomal cathepsins. Both high and low pH increase the degradation of troponin-T, Z-lines, gap filaments and connectin, but the degradation of these proteins (except for Z-lines) is greater at a low pH. Low pH increases the degradation of myosin; conversely, high pH retards it degradation.     

Endogenous proteinases in true sardine (Sardinops melanostictus)

Characterisation of the autolytic profile of true sardine (Sardinops melanostictus) indicated the involvement of heat-activated proteinases, active at both acidic and alkaline pH values. Autolytic activity decreased with increasing NaCl concentration (0–30%). When crude proteolytic activity in true sardine was studied, two activity peaks were observed, at pH 3.5 and 9.0. Crude proteinase extracts exhibited the highest activity at 55 °C and 60 °C when assayed at pH 3.5 and 9.0, respectively. The pH 3.5 peak activity was effectively inhibited by pepstatin A, while the pH 9.0 peak activity was mostly inhibited by soybean trypsin inhibitor, PMSF and TLCK, suggesting that the various proteinases were present in true sardine. The enzymes were stable for up to 8 h at 55 °C. The activities were also stable at a pH range of 2.0–4.0 and still retained high activity toward hemoglobin after incubation at pH 3.5 for 8 h. Activities of the crude extract continuously decreased as NaCl concentration increased. ATP showed no effect on enzyme activity, while CaCl₂ and MgCl₂ activated the proteinase activity. The results implied that pepsin is a predominant enzyme responsible for autolysis in true sardine during fish sauce fermentation.     

Expression of proteases by Rhizopus species during Tempeh fermentation of soybeans

Expression of proteolytic activities by nine Rhizopus strains during soybean fermentation to produce Tempeh has been determined. All strains showed activity at acidic, neutral, and alkaline pH values. In protein solubilizates prepared from Tempeh pronounced differences in the time course and intensity of maximum protease activities were observed between the isolates when measured at pH 7, the approximate pH of Tempeh during ripening. Proteases expressed by fungi during growth on Tempeh were solubilized and the enzymes were enriched by acetone precipitation and chromatographies with Phenylsuperose, Mono S, and Mono P, respectively. They were identified as one aspartic- (∼35 kD) and one serine (∼33 kD) protease, each existing in different isoforms. Proteases isolated from Tempeh were identical to those expressed during fungal growth in a soyprotein-raffinosephytate liquid medium. The temperature stability of the Tempeh- and the cell wall-isolated proteases is very low in contrast to the proteases secreted into the medium. Proteases isolated from Tempeh, cell walls and liquid media exhibit differences in the number of alkaline isoforms.     

Characterisation of proteolytic enzymes from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii)

BACKGROUND: Fresh water prawn in Thailand is widely consumed due to its delicacy. During postmortem handling and storage, prawn meat becomes soft and mushy, probably as a result of indigenous proteases. Therefore, an understanding of prawn proteases associated with the degradation of muscle proteins from fresh water prawn could pave the way for prevention of such a phenomenon during extended storage. RESULTS: Proteolytic enzymes in the crude extract (CE) from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) were characterised. CE from muscle exhibited the highest hydrolytic activities towards haemoglobin at pH 5 and 50 °C, while that from hepatopancreas had the highest activity on casein at pH 7 and 60 °C. Based on inhibitor study, cysteine protease and serine protease were dominant in CE from muscle and hepatopancreas, respectively. CE from muscle rarely hydrolysed natural actomyosin (NAM), but could not degrade pepsin‐soluble collagen (PSC). Conversely, NAM and PSC were susceptible to hydrolysis by CE from hepatopancreas as evidenced by the marked decreases in band intensity. Activity staining using haemoglobin, casein and gelatin as substrates revealed that no proteolytic or gelatinolytic activity was observed in CE from prawn muscle, while CE from hepatopancreas exhibited pronounced hydrolytic activities towards all substrates. CE from muscle showed calpain and cathepsin L activities but CE from hepatopancreas mainly exhibited tryptic and chymotryptic activities. CONCLUSION: Serine proteases, mainly trypsin‐like or chymotrypsin‐like, from hepatopancreas were probably responsible for the softening of prawn meat during postmortem storage via the degradation of both muscle and connective tissues. Copyright © 2010 Society of Chemical Industry     

ROLE OF pH IN SOLUBILITY AND CONFORMATIONAL CHANGES OF PACIFIC WHITING MUSCLE PROTEINS

This study was conducted to better understand biochemical changes of fish muscle proteins as affected by novel surimi process, acid- or alkali-aided solubilization. At 10 mM NaCl, between pH 5 and 10, the solubility of Pacific whiting muscle proteins was low but increased dramatically as the pH was shifted to either acidic or alkaline pH. At 600 mM NaCl, the isoelectric point was shifted to the acidic direction by about 2 pH units, resulting in aggregation of proteins at low pH, but improving the solubility of MHC (myosin heavy chain) between pH 6 and 10. ANS surface hydrophobicity (ANS-S_O) showed much greater values than PRODAN surface hydrophobicity (PRODAN-S_O) for samples treated at pH 2 - 4 perhaps due to an enhancement of the electrostatic interactions between the ANS probe and proteins. At very high pH, according to hydrophobicity results, proteins were partially refolded when the ionic strength increased. Under acidic conditions, SDS-PAGE demonstrated the degradation of MHC at 10 mM NaCl. The formation of MHC polymers was observed under alkaline treatment with a concomitant decrease of SH content.     

Tocopherol in the lipid stability of tilapia (Oreochromis niloticus) hamburgers

Presence of tocopherol is effective for fish preservation during frozen storage, inhibiting lipid degradation by oxidation. This work evaluated the antioxidant effects of α-tocopherol in diet and postmortem addition on the final quality of hamburgers produced from tilapia fillets kept frozen for zero, 30, 60, and 90 days. Chemical composition varied within the values found for tilapia fish. The increase in α-tocopherol levels reduced the values of thiobarbituric acid reactive substances (TBARS) in the samples at all time intervals. Tocopherol supplementation in diets protected the hamburgers from lipid oxidation more effectively than postmortem addition.     

中性氧化电解水对冷鲜草鱼肉品质及质构的影响

为探讨中性氧化电解水对冷鲜草鱼肉的保鲜效果,对冷藏条件下(4℃)冷鲜草鱼肉的品质指标及质构进行检测,并对比贮藏开始和结束后草鱼的肌原纤维组织结构的变化。结果表明:中性氧化电解水能够降低冷鲜鱼肉贮藏过程中菌落总数、假单胞菌数、挥发性盐基氮及硫代巴比妥酸值等品质指标的变化,可将货架期延长4 d左右;与对照组及减菌剂Ⅰ组相比,电解水处理可以减缓冷藏过程中鱼肉肌原纤维的分解,进而减少冷鲜草鱼的硬度、弹性和回复性等质构的变化。在贮藏至第6天,中性氧化电解水处理的冷鲜鱼肉硬度、弹性及回复性分别为对照组的1.23、2.13、1.83倍,且电解水组的肌原纤维相对较长且排布较规整。利用中性氧化电解水可以有效减轻冷鲜草鱼肉贮藏过程中品质及质构的变化,可为淡水鱼冷鲜鱼肉产品的贮藏保鲜技术开发提供参考。     

酶解菜籽粕制备多肽的研究

本试验以菜籽粕为原料,从四种蛋白酶中挑选出中性蛋白酶和碱性蛋白酶对菜籽粕进行同步复合酶解。分别考察了不同的加酶量、酶比、料液比和时间对多肽得率的影响,并利用响应面分析法对混合蛋白酶水解菜籽粕的条件进行了优化。结果表明,在加酶量固定为4500 U/g,温度50℃,pH为8.0,料液比1:8及碱性蛋白酶:中性蛋白酶为3:1条件下酶解6.5 h,可使多肽得率达到54.89%。     

Effects of Alkaline and Acid Pretreatments on Alaska Pollock Skin Gelatin Extraction

Pretreatments with different alkalis and acids at different concentrations were used to determine their effects on gelatin extraction from Alaska pollock skin. The alkaline pretreatments with the OH concentrations lower than 0.5 mol/L removed noncollagenous proteins without significant loss of skin collagen. The acid pretreatments caused loss of collagen, even using a weak acid with a low H concentration at a low temperature. The presence of proteases might cause degradation of gelatin extract, but the pretreatments with NaOH or Ca(OH)₂ at 0.1 mol/L OH concentration, or acetic acid at 0.05 mol/L H concentration could significantly decrease the degradation by proteases. The combination of an alkaline pretreatment followed by an acid pretreatment not only removed the noncollagenous proteins, but also provided the proper pH condition for extraction, during which some cross‐linkages could be further destroyed but with less breakage of polypeptide chains.