Autolysis and biochemical properties of endogenous proteinases in Japanese sandfish (Arctoscopus japonicus)

Autolysis of Japanese sandfish ( Arctoscopus japonicus ) was studied. The highest autolysis was observed at 60 °C. Optimal pH for autolysis was found at both acidic and alkaline pH values. Autolytic activity decreased with increasing NaCl concentration. Further assay of the extract from Japanese sandfish exhibited the maximum activity at 55 °C and pH 9.5. The Japanese sandfish proteinases were markedly inhibited by pepstatin A at pH 3.5, while soybean trypsin inhibitor, PMSF and TLCK inhibited activities of proteinases at pH 9.5, suggesting Japanese sandfish proteinases were both aspartic and serine proteinases. The extracts were stable to heat treatment up to 50 °C for 15 min and still remained high relative activity after incubated at 50 °C for up to 8 h. Maximum stability of the extracts was observed between pH 6 and 11 and no changes of activity were found after incubation at pH 9.5 for up to 8 h. Activity of Japanese sandfish extract continuously decreased as NaCl concentration increased (0–30%), while activity increased as CaCl₂ concentration increased. The results suggest that major proteinases in Japanese sandfish were trypsin-like serine proteinases.     

Autolytic activity and biochemical characteristics of endogenous proteinases in Indian anchovy (Stolephorus indicus)

Maximum autolytic activity of Indian anchovy (Stolephorus indicus) was found at 60 °C. Autolytic activity decreased with increased NaCl concentration. Remaining autolytic activity at 25% NaCl (w/w) was 52%. Crude proteinase extracts exhibited the highest activity at 60 °C, using either casein or acid-denatured hemoglobin (dHb) as a substrate. Optimal pH of crude extracts was found at 8.5 for casein and 9.5 for dHb. Activity of crude extract decreased >50% when NaCl concentration was greater than 0.1 M. Crude extract was stable for up to 8 h at 4, 30, and 60 °C. Crude proteinase hydrolyzed several synthetic substrates of trypsin, including Boc-Asp(oBzl)-Pro-Arg-MCA, Boc-Val-Leu-Lys-MCA, and Boc-Gln-Ala-Arg-MCA. Soybean trypsin inhibitor (SBTI), leupeptin, phenylmethanesulfonyl fluoride (PMSF), and N-tosyl-l-lysine chloromethyl ketone (TLCK) inhibited activities of proteinase, indicating trypsin-like characteristics. Molecular weight of proteinases exhibiting caseinolytic activity at 4.0 M NaCl were estimated to be 63, 53, 46, 40, 35, and 31 kDa, using electrophoresis activity staining.     

Ka-pi-plaa fermented using beardless barb fish: physicochemical,microbiological and antioxidant properties as influenced by production processes

Production of Thai fish paste Ka-pi-plaa fermented using beardless barb, Cyclocheilichthys apogon was monitored. The physicochemical, microbiological and antioxidant properties were compared after each process, that is autolysis, salting, sun-drying and fermentation. Colour parameters L* decreased while a* and b* increased during production (P < 0.05). The Ka-pi-plaa finished product presented an intense brown colour as shown with the increase in browning intensity (A₄₂₀). Contents of formal nitrogen, ammonia nitrogen and amino nitrogen showed continuous increase (P < 0.05) indicating the formation of peptides and free amino acids, which were verified by protein patterns. Populations of total, halophilic, proteolytic, lipolytic and lactic acid bacteria (LAB) generally increased. Halophilic bacteria grew rapidly after salting. LAB counts were correlated with the pH change. It was suggested that a few biochemical reactions occurred during production, including protein hydrolysis by microbial and fish proteases, lipid oxidation as presented by the increasing thiobarbituric acid reactive substances value, and Maillard reaction based on the determined precursors and products. Antioxidant activities generally increased during production particularly fermentation, suggesting Ka-pi-plaa possessed 2,2-diphenyl-l-picrylhydrazyl and 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) scavenging and metal chelating activities, and ferric reducing power. This study provides important information on the relationship among production steps, various properties and chemical reactions for fish fermentation.     

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.     

Effect of Monascus Fermentation on the Characteristics of Mackerel Mince

ABSTRACT: To improve the characteristics of fish muscle, Monascus purpureus or Monascus species was inoculated in 2.1% rice powder broth containing 30% minced mackerel tissue to produce a fermented fish product. Enterbacteriaceae, Staphylococcus , and Pseudomonas were not present during the 7 d of fermentation at 25 °C. The activities of both amylase and acidic proteases were detected after 1 and 2 d of fermentation, respectively, and further increased during fermentation. The volatile base nitrogen of all Monascus fermented samples were lower than the limit of food regulation in Taiwan (< 25 mg/100 g) even after 7 d of fermentation. Hydrolysis of the muscle protein and accumulation of free amino acid were observed. The fermented fish products had good flavor and color that were highly likeable to the sensory panel.     

Autolysis of goatfish (Mulloidichthysmartinicus) mince: Characterisation and effect of washing and skin inclusion

Autolysis of goatfish mince and washed mince incubated at different temperatures (30–70 °C) was investigated. The highest autolytic activity was generally observed in mince and washed mince at 60 °C as evidenced by the highest trichloroacetic acid (TCA) soluble peptide content and the greatest disappearance of myosin heavy chains (MHCs). Autolysis of both mince and washed mince was maximised at pH 4, and lower autolytic activity was observed at pH 7. trans-epoxysuccinyl-l-leucyl-amido (4-guanidino) butane (E-64) showed the greatest inhibition of autolysis at pH 4, showing that at least one cysteine protease was active in goatfish muscle. Nevertheless, soybean trypsin inhibitor effectively inhibited the autolysis at neutral pH (pH 7), suggesting that goatfish muscle also contained at least one serine protease. Generally, autolysis of mince was more pronounced than that of washed mince, indicating that washing could lower the autolytic activity of mince. In the presence of skin, a higher autolysis was obtained with the goatfish mince. Therefore, both sarcoplasmic and myofibril-associated proteases in muscle as well as the contamination of skin likely contributed to the degradation of muscle proteins of goatfish.     

Textural and ultrastructural changes during processing and storage of lightly preserved salmon (Salmo salar) products

The effect of a controlled lactic fermentation on textural and ultrastructural properties of salmon fillets was evaluated. When compared with a non‐inoculated cured sample, fermented salmon had unique textural properties since it exhibited significantly higher force (p < 0.05) and work (p < 0.001) values in a penetration test on two sampling days but a consistently lower hardness (p < 0.05) in a Texture Profile Analysis (TPA) rheological test. Study of the myofibrillar ultrastructure showed that basic structures were drastically damaged during processing and storage of fermented and cured salmon. Nevertheless, Z‐lines were better preserved in fermented samples. It is suggested that the slight protective effect of lactic fermentation with the starter L sake may be the consequence of a lower pH, by putatively inhibiting neutral or alkaline proteolytic enzymes. © 2000 Society of Chemical Industry     

Volatile Components in Salt-Fermented Fish and Shrimp Pastes

Volatile compounds in salt-fermented anchovy, big-eyed herring, hair tail viscera, and shrimp pastes were compared by simultaneous steam distillation-solvent extraction/gas chromatography/mass spectrometry (SDE/GC/MS). A total of 155 volatile compounds were detected. Of these, 111 were positively identified consisting mainly of aldehydes, ketones, alcohols, esters, aromatics, nitrogen-, and sulfur-containing compounds. Lipid-derived components, such as aldehydes, alcohols, and esters, comprised the majority of volatile compounds from fish pastes, while heterocyclic nitrogen-containing compounds, such as pyrazines, were predominant in shrimp paste.     

多酶法在鱼露生产工艺中的应用

应用正交试验优选了碱性蛋白酶和中性蛋白酶对青鳞鱼下脚料 (鱼头、鱼骨、内脏、鱼皮等 )的水解条件 ,在此基础上 ,同时用碱性蛋白酶和中性蛋白酶 ,再用风味酶对青鳞鱼下脚料蛋白质进行水解 ,确定了以多酶法生产鱼露的新工艺。结果表明 ,青鳞鱼下脚料中蛋白质含量达 14 8% ,经多酶水解和适当调配可制得风味浓郁的鱼露。多酶法水解蛋白质的条件为 :同时用 1 5 %碱性蛋白酶和 1 5 %中性蛋白酶在 pH 7 0、5 0℃条件下水解 12 0min后再加入 2 %风味酶继续水解 60min ;新工艺生产鱼露中必需氨基酸含量达 40 3 % ,总氮达 19mg/L ,氨基氮达 11mg/g ,占总氮的 61 0 % ,呈味氨基酸含量达 49 5 %。因此新工艺鱼露营养丰富 ,味道鲜美。     

Autolysis of shrimp head by gradual temperature and nutritional quality of the resulting hydrolysate

Autolysis method was investigated for protein recovery from shrimp head (SH) of white shrimp (Penaeus vannamei). Autolysis of SH by several temperature conditions (40 °C, 50 °C, 60 °C and gradual temperature) at its initial pH showed protein recovery of 43.6%, 73.6%, 50.3% and 87.4%, respectively. Autolysis of SH by gradual temperature (temperature was risen 5 °C every half an hour, from 40 °C to 70 °C) served best for the preparation of autolysis hydrolysate (AH) with the maximum degree of hydrolysis (DH) 48.6% and the maximum protein recovery 87.4%. Subsequently, the chemical components of SH and its AH obtained by gradual temperature were investigated and comparably evaluated. Protein contents of SH and AH by gradual temperature method amounted to 60.6% and 88.8% (dry basis), respectively. Both SH and AH had high contents of tasting or functional amino acids. The contents of essential amino acids (EAA) of SH and AH were 340.26 and 405.30 mg/g protein, respectively. The amino acids scores of SH and AH were 73 and 96, respectively. The EAA of AH covered 96-100% of the FAO/WHO ideal protein. The high performance size exclusion chromatography of AH showed that with the increasing of DH values, there occurred a larger amount of smaller polypeptides with molecular weight <5000 Da. The nutritional value of SH could be greatly enhanced by gradual temperature autolysis.     

不同发酵豆制品中生物胺调查分析

目的 调查腐乳、豆酱和豆豉3类豆制品中生物胺含量, 初步了解发酵豆制品中生物胺存在情况。方法 参照GB 5009.208-2016《食品安全国家标准 食品中生物胺的测定》, 采用高效液相色谱法测定6种腐乳、3种豆酱、6种豆豉中的生物胺含量。结果 腐乳中的生物胺含量明显超出另两类, 其总生物胺平均含量达472.35 mg/kg; 豆酱略高于豆豉, 分别为196.77 mg/kg和171.46 mg/kg。腐乳、豆豉和豆酱样品中含量最高的生物胺均是酪胺, 腐乳样品中居其次的是腐胺, 豆豉和豆酱样品中是章鱼胺, 3种品种样品亚精胺和精胺含量均较少。结论 从整体而言, 3类发酵豆制品生物胺含量在安全范围内。     

发酵鱼中菌群组成及发酵特性研究进展

发酵是鱼类保藏和加工的重要手段。发酵赋予鱼肉独特的风味和质地,微生物在发酵过程中扮演着重要的角色。本文介绍了发酵鱼中微生物组成和在抑制杂菌、物质降解、风味形成和生物胺积累中的作用,以帮助了解优势微生物及其发酵特性,制备工业发酵剂,提高发酵鱼产量和品质。此外,对发酵鱼深入研究进行了展望。     

糖化增香曲在发酵酱油和酱制品中的应用探讨

糖化增香曲是用于酿造酱油及酱制品的复合曲种,针对传统制曲菌种单一、酶活力不全等问题,利用现代生物工程技术,筛选、遗传诱变、纯化出复合菌种,具有较强的糖化力、酯化力和增色能力,可明显改善产品口感、色泽、香味,并显著提高酱油、酱制品的全氮和氨基酸态氮含量。该文论述了糖化增香曲的研发过程及原理,并在低盐固态发酵法的酱油生产和酱制品生产中使用糖化增香曲及复合制曲技术进行了应用试验,提高了产品质量及出品率。     

The effect of proteinase inhibitors in food protein hydrolysis by digestive proteinases of white shrimp (Penaeus vannamei) larvae

This study describes the digestive capacities of 10‐day‐old white shrimp postlarvae (PL10) and how some inhibitors of proteinases affect the digestion of protein in aquafeeds by using in vitro hydrolysis techniques. Biochemical data showed eight active proteinases in the PL10 hepatopancreas extract. Enzymes belong to the metallo‐ and serine‐proteinase classes. The effect of inhibitors present in protein ingredients and aquafeeds on PL10 proteinases showed that ovalbumin alone and in commercial microcapsules yielded a significant inhibition in proteolytic activity of PL10 hepatopancreas enzymes. The capacity of PL10 proteinases to hydrolyse the protein fraction within different sources and microcapsules was demonstrated by two in vitro approaches, the pH‐stat (degree of hydrolysis, DH) and electrophoresis (coefficient of protein degradation, CPD). It was shown that PL10 proteinases hydrolyse, in different extent protein sources and microcapsules. Casein, cuttlefish meal and feeds containing these ingredients are quickly hydrolysed. By contrast, ovalbumin and microcapsules containing ovalbumin are not hydrolysed. Copyright © 2006 Society of Chemical Industry     

Partial purification and characterization of trypsin-like proteinases in Indian anchovy (Stolephorus spp.)

Four fractions (P111, P21, P31, and P4) of proteinases were obtained from various purification steps including heat treatment (60 °C, 10 min), 30–60% ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. Optimal temperature and pH of all fractions were 50–60 °C and 8.5, respectively. All partially purified proteinases preferably hydrolyzed substrates containing Arg at the P₁ position. All proteinases were inhibited by soybean trypsin inhibitor, leupeptin, and N-tosyl-l-lysine chloromethyl ketone. Partially purified proteinases were stable at 35 °C up to 12 h. However, their activity decreased about 40% when incubated at the optimal temperature (50–55 °C) for 2 h. Only P111 was stable at its optimal temperature (60 °C) up to 12 h. Molecular weight (MW) of P111, P21, and P31 was estimated to be 27, 33, 37, 43, 48, 55, 60, and 65 kDa, while MW of P4 was 39 kDa based on activity staining. All partially purified proteinases hydrolyzed washed anchovy mince at 4.0 M NaCl, pH 8.5, at 35 °C and at their optimal temperatures (50–60 °C).     

顶空-固相微萃取两种传统面酱挥发性成分的气相色谱-质谱联用分析

采用顶空固相微萃取和气-质联用(GC-MS)技术,对2种传统面酱(M1和M2)中的挥发性成分进行提取、分析。从M1中鉴定出53种挥发性化合物,占色谱流出峰总面积的77.408%,包括醛类6种,酯类5种,酸类14种,烃类2种,醇类2种,杂环类化合物12种,酮类2种,其他化合物10种。从M2中鉴定出48种挥发性化合物,占色谱流出峰总面积的64.455%,包括醛类6种,酯类8种,酸类6种,烃类4种,醇类5种,杂环类化合物9种,酮类4种,其他化合物6种。     

蓝圆鲹肌肉中丝氨酸蛋白酶的分离纯化及性质研究

鱼类死后肌肉容易发生软化现象。研究表明,这与肌肉中的丝氨酸蛋白酶有着密切的关系。本研究通过硫酸铵盐析、DEAE-Sephacel、Q-Sepharose及Capto Q等柱层析相结合的方法,从蓝圆鲹肌肉中纯化得到一种具有分解明胶能力的丝氨酸蛋白酶,SDS-PAGE结果显示其分子量约为60ku,该酶最适温度及最适pH分别为40℃和9.0。丝氨酸蛋白酶抑制剂Pefabloc SC、Benzamidine、MBTI、PMSF和LBTI均能明显的抑制该酶的活性,而其他蛋白酶抑制剂对其活性没有明显的影响。底物特异性表明其能有效的降解丝氨酸蛋白酶荧光底物Boc-Leu-Lys-Arg-MCA,但进一步研究发现,该酶对I型胶原蛋白及明胶有明显的分解能力,同时对肌球蛋白重链也有一定的分解作用,说明该酶可能参与鱼肉保鲜中肌肉软化的过程。