Purification, characterization, and milk coagulating properties of ginger proteases

Ginger proteases are used as milk coagulants in making a Chinese traditional milk product (Jiangzhinai or Jiangzhuangnai), suggesting their potential as a source of rennet substitute that might be applicable in the modern dairy industry. In this study, ginger proteases were extracted from fresh ginger rhizome by using phosphate buffer and subsequently purified by ion exchange chromatography. Ginger proteases, all with a molecular weight around 31 kDa, were found to exist in 3 forms with isoelectric point values around 5.58, 5.40, and 5.22, respectively. These enzymes had very similar biochemical behavior, exhibiting optimal proteolytic activity from 40 to 60°C and maximum milk clotting activity at 70°C. They were capable of hydrolyzing isolated α_S1-, β-, and κ-casein, of which α_S1-casein was most susceptible to the enzyme; κ-casein was hydrolyzed with a higher specificity than α_S1- and β-casein. In addition, the ginger proteases exhibited a similar affinity for κ-casein and higher specificity with increasing temperature. Gel electrophoresis and mass spectra indicated that Ala90-Glu91 and His102-Leu103 of κ-casein were the preferred target bonds of ginger proteases. The milk clotting activity, affinity, and specificity toward κ-casein showed that ginger protease is a promising rennet-like protease that could be used in manufacturing cheese and oriental-style dairy foods.     

生姜蛋白酶提取、性质及应用研究进展

生姜蛋白酶是从生姜中发现的一种植物蛋白酶,属于木瓜蛋白酶类,与木瓜蛋白酶、菠萝蛋白酶及猕猴桃蛋白酶共同组成4种重要植物蛋白酶。生姜蛋白酶的提取方法主要有沉淀法中的有机溶剂法、结合有机溶剂的盐法、单宁法,以及超滤法。生姜蛋白酶的嫩肉、澄清和凝乳等作用在食品领域已被广泛研究及应用,其酶学性质、相对分子质量分布及结构也进行了检测分析和研究。近期的部分研究还表明生姜蛋白酶的一些生物活性有潜在医药价值、具有广阔的应用前景。本文对生姜蛋白酶的提取分离与纯化、酶活性及分析检测、生物活性及应用等研究现状进行了概述,并对今后应重点加强的研究方向进行展望。     

Characterization of a new isolate of Lactobacillus fermentum IFO 3956 from Egyptian Ras cheese with proteolytic activity

More than 200 isolates were obtained from 15 Egyptian traditional dairy products (Domiatti cheese, Ras cheese and Rayeb milk) collected from local markets of Alexandria, Tanta and Kafr El-Sheikh. Examination with optical microscope of these dairy samples allowed to classify 92 bacilli, 64 of which were identified as lactobacilli. The proteolytic activity of lactobacilli isolates was tested on skim milk agar. Eight isolates showing a high proteolytic activity were further tested on UHT skim milk. The strain showing the highest proteolytic activity was purified and identified as Lactobacillus fermentum IFO 3656. The specific proteolytic activity of this strain and the factors affecting it (pH, temperature and presence of inhibitors) were studied. The proteolysis targeted mainly caseins (73% of whole casein), especially β-casein (85%). Smaller portions of whey proteins were proteolyzed (20%) essentially β-lactoglobulin. The proteolysis process gave rise to medium-sized peptide populations. The optimum conditions for the proteolysis activity of the studied strains were pH 6.5 and 37 °C. Proteolytic activities were very slightly affected by the increase of the temperature to 42 °C or the pH to 8.2. The protease system of Lactobacillus fermentum IFO 3956 is most probably composed from a high amount of metalloproteases and small amount of cysteine and serine proteases.     

姜汁中凝乳成分研究

以生姜和鲜牛乳为原料,将经杀菌迭一定温度的鲜牛乳冲入到适量比例的姜汁中,便制得姜汁凝乳。为了探讨姜汁凝乳的机理,研究了温度对姜汁凝乳的影响,制备了生姜蛋白酶粗提物、姜油树脂和姜精油,并分别进行了凝乳实验。实验结果表明,最佳冲浆温度为70℃,生姜蛋白酶粗提物具有凝乳作用,而后两者均无此作用。     

Biochemical Characterization of an Extracellular Heat‐Stable Protease from Serratia liquefaciens Isolated from Raw Milk

The protease Ser2 secreted by the psychrotrophic strain Serratia liquefaciens L53, a highly proteolytic strain isolated from Brazilian raw milk was purified and characterized. Using azocasein as substrate, Ser2 exhibited activity in a wide range of pH (5 to 10) and temperature (4 to 60 °C). The optimal activity was detected at pH 8.0 and at a temperature of 37 °C. This protease, still active at 4, 7, and 10 °C, was strongly inhibited by chelating agents and by dithiothreitol, a reducing agent. These results confirmed that Ser2 belongs to the peptidase family M10 and requires Ca²⁺, Zn²⁺, and disulfide bridges for stability. This protease is able to hydrolyze three kinds of casein in the preferential order of κ→ β→ α‐casein. Highly heat‐stable in skimmed, semi‐skimmed, and whole milk at 140°C with D‐values of 2.8, 3.9, and 4.5 min, respectively, Ser2 showed a residual activity between 87 and 100 percent after heat‐treatment of 65 °C for 30 min, 72 °C for 20 s, and 140 °C for 4 s that are commonly used in dairy industries. As the protease AprX that is mainly secreted by Pseudomonas genus, Ser2 could be one of the main causes of UHT milk destabilization during storage.     

几种常用食品加工试剂对生姜蛋白酶活力的影响

以部分纯化的生姜蛋白酶为研究对象,研究了几种常用 食品加工试剂对生姜蛋白酶活力的影响。结果表明,亚硫 酸氢钠和氯化钠对酶有不同程度的抑制作用,蔗糖对酶 活有促进作用。亚硫酸氢钠浓度为2.0mmol/L时,相对酶 活为50％;氯化钠浓度为7.5％时,其相对酶活为69.6％;蔗 糖浓度7％时,酶的相对活力为138％。     

LS1型科研型超滤机制备生姜蛋白酶的研究

本文研究了利用LS1型科研型超滤机制备生姜蛋白酶 ,结果证明 :操作压力为0 06Mpa时 ,酶活力和膜通量最好 ,浓缩度为80%,生姜蛋白酶得率为2 3%;膜通量随操作温度、姜汁流速的提高而增加 ,随姜汁浓度、操作时间的增加而降低 ;超滤法比单宁法、乙醇法提取的生姜蛋白酶的酶活力和得率高 ,更具有工业化生产价值。     

Proteolytic activity in ruminal fluid from cattle fed two levels of barley grain: a comparison of three methods of determination

The effects of proportion of concentrate in the ruminant diet and the effects of freezing ruminal content prior to assay on proteolytic activity in ruminal inoculum were evaluated using three analytical techniques. A novel approach for determining proteolytic activity (PA) of ruminal fluid utilising ¹⁵N‐labelled casein was compared with two published procedures. In a crossover experiment, four heifers were fed two isonitrogenous diets containing (dry matter basis) 50% barley silage, 45% rolled barley grain and 4% soybean meal (medium‐grain diet, MG) or 8% barley silage, 89% rolled barley grain and 2% soybean meal (high‐grain diet, HG). Ruminal fluid was analysed either fresh or after having been frozen at ?40 °C for 45 days. Substrates utilised in measuring PA included ¹⁵N‐labelled casein (produced by infusing (¹⁵NH)₂SO₄ into the rumen of a lactating dairy cow), ¹⁴C‐labelled casein and azocasein. Incubations were conducted in 0.2 M phosphate buffer (pH 6.8) for 20 min at 39 °C. In the ¹⁵N‐casein incubations, PA was estimated as (i) N soluble in 5% trichloroacetic acid (TCASN), (ii) N soluble in 5% TCA corrected for microbial N uptake (TCAMICR) and (iii) N depleted from the soluble protein N pool (SPR). In the ¹⁴C‐casein incubations, PA was measured as TCA‐soluble radioactivity; in the azocasein method it was measured as dye released during incubation. Across treatments the highest (P < 0.001) proteolytic activity was measured by the SPR method, followed by TCAMICR, TCASN and ¹⁴C‐casein. The lowest activity was recorded using the azocasein method. Within the ¹⁵N‐ and ¹⁴C‐casein methods, PA in previously frozen ruminal fluid was higher (P < 0.05) with the HG diet than with the MG diet, and higher (P < 0.05) in previously frozen fluid than in inoculum processed fresh. This study demonstrates that increasing the proportion of grain in the diets of cattle and freezing the ruminal inoculum both increase proteolytic activity measured in ruminal fluid. The proposed ¹⁵N‐casein method yielded higher proteolytic activity values than the ¹⁴C‐casein method. Copyright © 2002 Society of Chemical Industry. Contributions of A N Hristov, T A McAllister and Z Xu. © Minister of Public Works and Government Services, Canada 2002.     

Evaluation of Harp Seal Gastric Protease as a Rennet Substitute for Cheddar Cheese

A crude preparation of gastric proteases from Harp Seal (Pagophilus groenlandicus) was found to coagulate milk over a wider pH range than porcine pepsin and had a higher ratio of milk clotting to proteolytic activity with hemoglobin at pH 1.8. Cheddar cheese prepared with seal gastric protease (SGP) gave significantly higher sensory scores than cheese made with calf rennet. Chemical analysis of the cheeses revealed a lower concentration of citrate-HCl soluble nitrogen and less free and peptide-bound amino acids in SGP cheese than in the cheeses made with calf rennet and Mucor miehei protease.     

SCREENING FOR EXTRACELLULAR ACID PROTEASE(S) PRODUCTION BY WINE YEASTS*

Ninety-four Saccharomyces wine yeasts were screened for their ability to produce extracellular acid protease(s) under simulated vinification conditions. Seventeen strains were selected on the basis of their higher proteolytic activity on haemoglobin and casein. These strains showed maximum activity after 20 days of fermentation on haemoglobin and 25 days on casein. Wines obtained from grape juice fermented by the above 17 strains showed significative differences in the residual protein content.     

Proteolytic and Milk Clotting Fractions in Milk Clotting Preparations

Five different milk clotting preparations were fractionated on Sephadex G-100 and then tested for milk clotting activity and for proteolysis of denatured hemoglobin. Two preparations were also tested for proteolytic activity on casein. Proteolytic activities on hemoglobin were correlated with clotting ability of bovine rennet, calf rennet, and rennin-pepsin mixture at pH 1.6 and with Mucor miehei protease at pH 5.2. Modified Mucor miehei protease activities on hemoglobin correlated equally well at pH 1.6 and 5.2. Gel filtration through Sephadex G-100 and elimination of nonclotting fractions reduced the proteolytic activities on hemoglobin at pH 5.2 of calf rennet, bovine rennet, Mucor miehei protease, modified Mucor miehei protease, and rennin-pepsin mixture by 68.6, 88.5, 3.7, 53.7, and 91.2%, respectively. At pH 1.6, proteolysis was reduced by 54.2, 41.2, 51.8, 59.5, and 60.8%. Proteolytic activities of bovine rennet and renninpepsin mixture on casein were reduced by 59.8 and 72%, respectively.     

Plasminogen activation system in goat milk and its relation with composition and coagulation properties

The activity of plasmin (PL), plasminogen (PG), and plasminogen activator (PA) and their correlation with goat milk components and milk clotting parameters were investigated. Seven late-lactating Saanen goats were used to provide milk samples that were analyzed for PL, PG, and PA activity (colorimetric assay) fat, protein, noncasein nitrogen, nonprotein nitrogen, casein content, and somatic cell count (SCC). Milk clotting parameters (rennet coagulating time = coagulation time; K20 = firming rate of curd; A30 = curd firmness) were measured with a formagraph. Average milk yield and composition were similar to those previously observed in other studies. Plasmin, PG, and PA activity, expressed as units/ml, were, respectively, 20.04 +/- 0.94, 3.21 +/- 0.04, and 1154 +/- 57.61. Plasminogen activity was surprisingly low compared with other species (bovine, ovine), but it was consistent with the high activity of PA. A negative significant correlation was observed between PL and milk casein content. The correlation coefficients between PL and casein/protein ratio and PA and casein/protein ratio were negative and significant. A positive significant correlation was observed between PL and rennet clotting time and PA and rennet clotting time. Also positive was the correlation between PL and K20 and PA and K20. The plasmin activity was negatively correlated with A30. High plasmin and plasminogen activator activity in goat milk appeared to be negatively related with coagulating properties in late lactation, most probably via degradation of casein due to plasmin activity.     

Effect of Heat-Stable Proteases on the Kinetic Parameters of Milk Clotting by Chymosin

The milk clotting per unit casein hydrolytic activities of proteases from 14 psychrotrophic pseudomonad isolates of raw milk ranged from 0.77 to 9.97 at 30°C. The milk clotting activities of chymodn and T16 protease were not completely additive, especially at high chymosin concentration when clotting time was relatively fast. The T16 protease was not effective in catalyzing the enzymic step of milk clbtting at O°C in the time expected on the basis of its milk clotting at 30°C. Milk incubated with the T25 motease for 8 days at 4°C and then clotted with chymosin at 30°C exhibited weak curd consistency.     

Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation

Heat-stable proteases produced by the psychrotroph Pseudomonas fluorescens M3/6 have been shown to affect the plasmin system in milk, which in turn will affect the quality of processed milk. The M3/6 proteases cause dissociation of plasmin from casein in minimally processed milk. The objective of this work was to study the effect of M3/6 protease on the plasmin system, as well as its role in plasminogen activation, under commonly applied cheese-making conditions. Isolated M3/6 protease was added to raw milk, which then was pasteurized, and subjected to pH adjustments and CaCl₂ addition. Casein and whey fractions were separated by chymosin treatment then analyzed for plasmin activity. Individual and interaction effects of M3/6 protease addition, pH treatment, and CaCl₂ addition on plasmin activity were studied. Enzyme activity assays were carried out to study individually the effect of M3/6 protease on plasmin system components. Kinetic parameters were calculated to characterize the effect of M3/6 protease on plasminogen activation. Plasmin activity increased in the curd fractions of the protease-treated milk that was subjected to conditions most resembling cheese-making conditions, indicating that M3/6 protease triggered plasminogen activation rather than dissociation of plasmin from casein micelles. Results from the studies on plasminogen activation confirmed that the observed activation of plasminogen in protease-treated samples subjected to cheese making conditions was attributed to the stimulatory effect M3/6 protease had on plasminogen activators (PA). The M3/6 protease stimulated human and bovine PA by increasing their activity 4.5- and 2.5-fold, respectively. Similarly, the catalytic efficiencies of human urokinase-type PA and bovine PA were increased in the presence of M3/6 protease by 12- and 4-fold, respectively. Our research presented a basic step toward fully understanding the effect of bacterial proteases under different processing conditions, where the gathered information can aid in better control of processing conditions based on the desired outcome.     

Purification and Characterization of Proteases from Digestive Tract of Grass Shrimp (Penaeus monodon)

Proteases in grass shrimp (Penaeus monodon) digestive tract were extracted. Four fractions, A, B, C and D, demonstrated caseinolytic activity and were purified to electrophoretic homogeneity. A, C and D were trypsin-like, while B was a chymotrypsin-like protease. Optimal temperature for proteases A, B and C were 65°C, and that for D was 55°C for hydrolysis of casein. Optimal pH of proteases A and C was 8.0, and that of D was 7.0 for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester. Optimal pH of protease B for hydrolysis of N-benzoyl-L-tyrosine ethyl ester was 8.0. Inactivation of 50 % enzyme activity in 5 min occurred at 67°C for protease B and 50°C for proteases A, C and D.     

甲醇芽孢杆菌凝乳酶的重组表达及其结构特性

为进一步了解微生物凝乳酶的结构特性,根据GenBank数据库中甲醇芽孢杆菌凝乳酶（I3EB99）的氨基酸序列和大肠杆菌密码子的偏爱性,设计合成此凝乳酶的全基因序列,构建原核表达载体,通过BL21（DE3）表达其融合蛋白,将获得的融合蛋白进行His标签特异性亲和纯化,并利用生物信息学方法研究凝乳酶的三维空间立体结构。结果表明,重组表达的甲醇芽孢杆菌凝乳酶质量浓度为0.7 mg/mL,凝乳活力为（15 870±1.17）SU/g,蛋白水解活力为（263.81±0.94）U/g,凝乳活力与蛋白水解活力比值为60.16,符合干酪生产加工的要求。结构特性研究表明,经重组表达后的甲醇芽孢杆菌凝乳酶呈现疏水特性,具有跨膜结构和信号肽,二级结构中α-螺旋少于β-折叠,在分离纯化过程中结构不稳定易降解,该凝乳酶与来自甲醇芽孢杆菌的一种未知蛋白酶是同源蛋白,高级结构与PDB蛋白数据库中的模板蛋白2ra1.1.A相似度最高。通过对甲醇芽孢杆菌凝乳酶结构特性的研究,为深入分析该凝乳酶作用机理及其功能性奠定了理论基础。     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Enhanced stability of crude protease from kiwifruit (Actinidia deliciosa) by adding hydrocolloid for organic processed food uses

Crude protease originating from kiwifruit (Actinidia deliciosa) was extracted for organic processed food uses. The protease included in the kiwifruit can be utilized for organic uses instead of current commercial enzymes from microbial origin, which are not suitable for organic processed food. Crude protease extracted by physical treatment rather than any biochemical purification methods was appropriate for the organic processed food uses. However, crude protease extract has been found to be unstable for processing and storage usage, which has to be modified to be stable by appropriate methods suitable for organic processed food uses. The proteolytic activity of the protease extracted from kiwifruit was measured using casein as a substrate. The decreased inactivation rate constant of crude protease treated with guar gum and locust bean gum within the temperature range of 30–50°C implied the enhanced stability of crude enzymes by treatment with hydrocolloid. The half-times of crude proteases treated with guar gum and locust bean gum were higher than the half-time of native crude protease at 40°C (optimum temperature of the native crude protease), with values of 55.45 min for the guar gum-treated sample, 50.23 min for the locust bean gum-treated sample and 23.26 min for the native sample, demonstrating the quantitative evidence of the enzyme stability. The relatively stable maintenance of the proteolytic activity has helped to realize hydrocolloid-treated enzyme to be used for hydrolytic function in organic processed food applications.     

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     

Production of novel dairy products using actinidin and high pressure as enzyme activity regulator

Milk clotting for the production of novel dairy products, alternative or complementary to cheese and yogurt type products can be achieved using plant sulfhydryl proteases. The objective was to apply the protease actinidin, from Actinidia chinensis, as the milk clotting agent, and High pressure (HP) technology to control excessive proteolysis. The effect of the dairy substrate and the process parameters on the coagulation rate and the texture and sensory properties of the end product, were studied. Selected values of design parameters were 25% total solids, 6.49 adjusted pH, 0.35 U activity of the clotting agent actinidin, 40 ºC process temperature and 2 h time. The selected pressure-temperature conditions, 600 MPa at 40 ºC, were applied to stop the potentially detrimental further proteolytic action of the enzyme. Results indicated that use of actinidin for milk clotting and HP to stop the enzyme activity in the final product, leads to a “fresh cheese” type dairy product.Industrial relevance: Alternative clotting methods for novel dairy products, complementary to cheese and yogurt type products, are of interest to the industry. Plant proteases can be a viable approach, provided that excessive proteolysis after structure formation is regulated. High hydrostatic pressure can be used for controlling proteolytic activity in the final products without affecting their texture and sensory characteristics.