Participation of cathepsin L in modori phenomenon in carp (Cyprinus carpio) surimi gel

Cathepsin L (Cat L) in carp (Cyprinus carpio) dorsal muscles was purified and its molecular weight determined by SDS polyacrylamide gel electrophoresis (SDS–PAGE) was 36 kDa. Its optimal temperature and pH were 50 °C and 5.5, respectively. The results of the effects of specific substrates, activators and inhibitors on the enzymatic activity showed that Cat L belongs to the family of cysteine proteinases containing thiol. Compared to the control, the gel strength of surimi with the addition of purified Cat L decreased significantly by 24.33% while that of surimi with both purified Cat L and inhibitors increased by 13.7% and 21.6%, respectively, suggesting the participation of Cat L in the modori phenomenon occurring in carp surimi. Both the SDS–PAGE electrophoretic pattern and microstructure figure revealed that Cat L could hydrolyse the main protein in carp surimi and was one of the enzymes involved in the modori phenomenon.     

Partial purification and characterization of a yeast extracellular acid protease

During screening of 143 yeasts for proteolytic milk coagulating activity, a strain belonging to the species Cryptococcus albidus var. aerius was found which produced extracellular protease in shake culture. An enzyme preparation was obtained from the cell-free broth by ammonium sulphate precipitation. It possessed an optimum pH for milk-clotting at 5.5 to 5.7 at 35 degrees C. Maximum stability occurred between pH 3.5 and 5.5. The optimum temperature for the enzyme was 45 degrees C. Activity of the enzyme was inhibited by copper+2, iron+2, and mercury+2 ions.     

PROTEASE BIOSYNTHESIS FROM LACTOBACILLUS SPECIES: FERMENTATION PARAMETERS AND KINETICS

In the present study, 15 strains of Lactobacillus species, isolated from 25 samples of curd, were screened for their ability to produce extracellular protease. The proteolytic activities of these strains based on casein hydrolysis showed a variation of 1.26–5.80 U/mL with Lactobacillus IH₈ showing a maximum of all. Different cultural conditions for enhanced production of protease by Lactobacillus IH₈ were optimized. It was found that optimal conditions for the production of enzyme were an incubation temperature of 35C and a medium pH of 5.5. The maximum proteolytic activity of Lactobacillus IH₈ (7.28 U/mL) was achieved after 48 h of cultivation. The kinetic parameters such as product yield coefficient (Y p/x), growth yield coefficient (Y x/s), specific product yield coefficient (qp) and specific growth yield coefficient (qx) also revealed that the values of experimental results were highly significant when analyzed kinetically.     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

Partial characterization of protease from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein

Proteases are one of the most important groups of industrial enzymes, with considerable application in the food industry. The aim of this work was to study a novel protease produced by the thermophilic fungus, Thermoascus aurantiacus, through solid-state fermentation (SSF). The enzyme acted optimally at pH 5.5 and 60 °C; it was stable up to 60 °C for 1 h and in the pH range 3.0–9.5. To elucidate the enzyme’s proteolytic activity, its hydrolytic profile on bovine casein, an important protein in the food industry, was studied by enzymatic hydrolysis on skim milk, analyzed by gel electrophoresis (UREA-PAGE), which clearly showed that the protease does not have the same specificity as bovine chymosin.     

A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.)

BACKGROUND: Proteases hydrolyse storage proteins to provide precursors for perpetuating species. The aim of this study was to investigate and characterise different proteases in germinating brown rice. RESULTS: The protease activity of brown rice increased sevenfold during 7 days of germination. It was highest on day 6 when determined at pH 3.5. With casein as substrate the proteases showed two catalytic groups: acidic proteases with an optimal pH of 3.5 and alkaline proteases with an optimal pH of 8.0. The acidic protease activity was inhibited by Ba²⁺ and Pb²⁺ but stimulated by Zn²⁺, while the alkaline protease activity was inhibited by Ca²⁺ and Pb²⁺ but stimulated by Mg²⁺ and Zn²⁺. SDS‐gelatin‐PAGE assay showed two protease activity bands at pH 3.5, while two different bands with higher molecular weights were observed at pH 8.0. Inhibition assay revealed that pepstatin A and E‐64 inhibited 67.63 and 38.26% respectively of the protease activity at pH 3.5, indicating the presence of aspartic and cysteine proteases. Metalloproteases played a major role under alkaline conditions (88.37% inhibition with EDTA). CONCLUSION: Germinated brown rice proteases fall into different classes with different properties. This study is helpful for their further purification. Copyright © 2010 Society of Chemical Industry     

INHIBITION OF PACIFIC WHITING SURIMI-ASSOCIATED PROTEASE BY WHEY PROTEIN CONCENTRATE

Three whey protein concentrate (WPC) samples ranging in protein content from 34 to 95%, beef plasma protein (BPP) and bovine serum albumin (BSA) were analyzed for their effect on the autoproteolytic activity of Pacific whiting surimi. Autoproteolysis activity was determined in control and supplemented surimi containing up to 4% additive by measuring the amount of TCA-soluble peptides produced under defined reaction conditions. The degradation of specific myofibrillar proteins was followed electrophoretically. The extent of inhibition by the WPCs showed a general correlation with their respective protein contents. Autoproteolysis was essentially undetectable in surimi samples supplemented at the 2% level with either the 80% or 95% protein WPCs. Autoproteolysis was decreased by 57% relative to the nonsupplemented control in surimi containing 2% of 34% protein WPC. No proteolytic activity could be detected in surimi samples supplemented at the 1% level with BPP. Supplementation of surimi with up to 4% BSA had no demonstrable effect on autoproteolysis. The extent of proteolytic degradation of the myosin and tropomyosin fractions within surimi samples were in general agreement with the measured rates of autoproteolysis.     

Effects of Heat-Stable Alakaline Protease Activity of Atlantic Menhaden (Brevootii tyrannus) on Surimi Gels

Heat stable protease isolated from the sarcoplasmic fraction of menhaden (Brevoorti tyrannus) muscle tissue was characterized as to optimum temperature and pH against casein substrate and its degradative action on actomyosin and surimi during heating. The optimum conditions for activity were 60°C at a pH of 7.5 to 8.0. Activity dropped off remarkably at temperatures below 45°C or above 70°C and when pH was below 7.0 or above 8.0. The enzyme(s) was capable of degrading actomyosin as observed by sodium dodecyl sulfate electrophoresis. This implicated a causative role for this protease system in the texture degradation observed during thermal processing of menhaden surimi at temperatures of 50-70°C.     

白鲢鱼糜漂洗水蛋白质回收工艺研究

以白鲢鱼糜漂洗水为研究对象,采用不同沉降时间、环境温度、种类的酸、pH、絮凝剂和及其添加量对鱼糜漂洗水进行处理,研究其对蛋白质回收率、上清液透光率和絮凝蛋白质的聚丙烯酰胺凝胶电泳(SDS-PAGE)的影响。结果表明,4 ℃静置60 min有利于漂洗水的沉降;用盐酸调节pH至5.5时,蛋白质回收率和上清液透光率分别为65.8%和50.1%,且与乙酸和柠檬酸相比,盐酸沉降效果更好;经0.6 mg/mL壳聚糖进一步处理,可使漂洗水蛋白质回收率和上清液透光率分别上升至85.8%与87.8%,蛋白质回收效果加强;SDS-PAGE电泳结果表明,漂洗水中蛋白质分子量主要分布在35、42、48、63、100和190 kDa附近;经系列条件处理后(除190 kDa外),各蛋白质含量逐渐增加,且在25.0~28.0 kDa附近产生多条新的蛋白条带。综合各试验指标结果,在4 ℃环境温度、盐酸调节pH至5.5,再使用0.6 mg/mL壳聚糖处理鱼糜漂洗水60 min,可高效回收白鲢鱼糜漂洗水中的蛋白质。本试验可为工业上鱼糜漂洗水中蛋白质回收和废水净化提供一定理论依据及实际指导。     

CHARACTERIZATION OF THE PROTEASES INVOLVED IN HYDROLYZING PADDLEFISH (POLYODON SPATHULA) MYOSIN

An extract from paddlefish surimi possessed activities of B, L, and H‐like cathepsins. The optimal pH was around 5.0 for cathepsins B and L, and was between 6.0–6.5 for the H‐like cathepsin. The enzyme activities were not impaired by heating at 40Cfor 20 min. However, the protease extract lost about 20% of its cathepsin B, 50% B+L, and 90% H‐like cathepsin activities after heating at 50C for 20 min. The activity of H‐like cathepsin was not inhibited by E‐64, suggesting that it did not belong to ike known cysteme protease group. The protease extract was capable ofhydrolyzing myosin heavy chain, producing a major fragments) around 140 kDa. Degradation of myosin by the protease extract was substantially reduced by protease inhibitors including E‐64, a protease inhibitor mixture, and bovine plasma powder.     

Physical,chemical and microbiological characteristics of fermented surimi with Actinomucor elegans

The physical, chemical and microbiological changes of surimi during surimi pehtze preparation stage, with Actinomucor elegans XH-22 (A. elegans XH-22) as fermentation starter, were investigated. Enzyme assay showed that A. elegans XH-22 could continuously produce protease and α-amylase on surimi surface. Texture profile analysis (TPA) indicated that fermentation for 48 h decreased pH and moisture content and modified the texture properties of surimi. Increases in amino nitrogen and trichloroacetic acid-soluble nitrogen (TCA-N) contents revealed obvious surimi protein degradation, which was further confirmed by size-exclusion HPLC analysis of surimi water soluble protein. Fermentation significantly increased the content of free amino acids and enriched special amino acids such as histidine, phenylalanine and glutamic acid, which could influence the nutritional and sensory properties of fermented surimi product. Considerable levels of aerobic plate count, spores and LAB were detected during the fermentation process. It is thus, from a public health point of view, advisable that the fermentation time in the first stage be controlled to be within 36 h. This research gives a new clue for improving the utilization of low-value fish.     

Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins

This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

A psychrotrophic Burkholderia cepacia strain isolated from refrigerated raw milk showing proteolytic activity and adhesion to stainless steel

The proteolytic activity of a psychrotrophic strain of Burkholderia cepacia isolated from refrigerated raw milk was characterized. Bur. cepacia produced proteolytic activity during growth at refrigeration temperature, with maximum activity at pH 6-7. The enzyme showed relative thermal stability in the range 40-50°C during 25 min, and maintained 80% its initial activity at 76°C/30 s. Milk coagulation assay showed that the crude protease from Bur. cepacia caused coagulation from day 2 for skimmed milk, whereas coagulation was observed from day 5 for whole milk. The adherence of this strain to stainless steel was evaluated, and the substrata had around 107 CFU/cm2 after 15 to 60 min incubation. Results on biofilm development suggest that this bacterium could adhere and to form biofilms even at refrigeration temperatures. These results indicate that Bur. cepacia may represent a potential hazardous to milk and dairy products.     

CHARACTERIZATION OF A SALT-TOLERANT ACID PROTEASE PRODUCED BY BACILLUS MEGATERIUM KLP-98 AND ITS POTENTIAL AS A FERMENTATION STARTER FOR THE MANUFACTURE OF FISH SAUCE

A salt-tolerant acid protease was purified from the culture broth of Bacillus megaterium KLP-98 with a molecular weight of 64 kDa. K _m and k _cat values of the protease were 2.1 mg/mL and 6.1 s⁻¹ against azocasein as a substrate, respectively. The optimal temperature for protease activity was 60C but optimal stability temperature was below 40C. The optimal pH for protease activity was 5.5, and it was stable from pH 4.5 to 6.0. Relative activity of the protease was 73, 33, and 5% at 10, 20, and 30% NaCl concentrations, respectively. Among all the substrates tested, the best substrate for B. megaterium KLP-98 was azocasein followed by Z-Phe-Arg-NMec. The protease activity was strongly inhibited by N-ethylmaleimide and E-64. This protease was therefore presumed to be a salt-tolerant acid cysteine protease. The purified protease was applied to accelerate the fermentation of anchovy sauce, and the results indicated its potential as a fermentation starter.

PRACTICAL APPLICATIONS

The B. megaterium KLP-98 protease accelerated the fermentation of anchovy sauce and greatly improved its quality only during 2 days of fermentation, thus will decrease its production cost. Hence, fish or soy sauce industries can benefit from this protease as a fermentation accelerant.     

Partial properties of an aspartic protease in bitter gourd (Momordica charantia L.) fruit and its activation by heating

Bitter gourd (BG fruit) is usually heated in hot water to reduce bitterness and improve flavour before being served. Protein extract from BG was analyzed for protease activity by gelatin-gel electrophoresis. The study showed that the proteolytic activity in BG flesh was enhanced by heat-treatment at temperatures ranging from 50 °C to 75 °C. An aspartic protease (AP) was characterized by gel electrophoresis. The optimal AP activity was at pH 7; the pI of the AP was demonstrated to be 4.8; the protein molecular weight of the BG–AP was estimated to be 60 KD by SDS–PAGE. The AP was implicated in the proteolysis of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase.     

Inhibition of proteases during fermentation improves xylanase production by alkali tolerant Aspergillus fumigatus ARl

An alkali-tolerant Aspergillus fumigatus ARl produced a high level of xylanase (228 U/ml) at 42 h of submerged fermentation at pH 9.0. During later culture periods, xylanase activity declined considerably and reached 45 U/ml at 90 h. This trend is because of proteolytic degradation of xylanases since A. fumigatus co-produced multiple proteases with high activity (120-159 U/ml) during submerged fermentation. Xylanase and protease were found to be active over a wide range of pH from 5.5 to 9.0. In contrast, protease lost its activity completely in the presence of CuSO4 (25 mM) while xylanase remained active. The selective inhibition of proteases by the addition of CuSO4 to the culture medium prevented proteolytic degradation of xylanase and improved the production of xylanase by 3-fold.     

Physicochemical characteristics,protein hydrolysis,and textual properties of surimi during fermentation with Actinomucor elegans

Solid-fermentation is a promising way of producing novel surimi-based food. In the present study, changes in physicochemical characteristics, protein hydrolysis, and textual properties of surimi during the fermentation, including pehtze preparation, salting, and ripening stages, were studied. The protease produced by Actinomucor elegans led to continuous hydrolysis of surimi protein, reflected from the high value of protease activity which reached 69.34 U/g after pehtze preparation stage, the decrease of protein bands intensity in sodium dodecyl sulfate polyacrylamide gel electrophoresis image especially for bands of 200, 43, and 36 KDa, and the increasing contents of free amino acids and trichloroacetic acid-soluble nitrogen. Hardness, springiness, and cohesiveness of the surimi all decreased during pehtze preparation stage, increased after salting, and decreased again after ripening, while adhesiveness kept increasing until the ripening stage started. Scanning electron microscopy images showed a dense and soft microstructure of surimi after ripening. The final surimi product showed high overall acceptability and no Enterobacteriaceae was detected.     

CHARACTERIZATION OF STOMACH AND PYLORIC CAECA PROTEINASES OF TAMBAQUI (COLOSSOMA MACROPOMUM)

The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of crude extracts from the stomach, liver, pyloric caeca, and intestines of Colossoma macropomum were investigated. The highest acid and alkaline proteolytic activities were found in stomach and pyloric caeca, respectively. The optimum pH for the acid and alkaline proteases were 1.8 and 7.0–9.0, respectively, while the optimum temperatures were 35C and 65C. This alkaline protease thermal stability remained unaltered after 90 min incubation at 55C. A pepsin‐like protease was responsible for most of the acidic proteolytic activity (Pepstatin A inhibited approximately 90%), whereas PMSF inhibited about 40% of the alkaline protease. The alkaline proteolytic activity has attractive thermal properties for industrial applications.