生姜蛋白酶的凝乳作用及相关机理探讨

从生姜中分离得到具有凝乳活力的生姜蛋白酶,采用Urea SDS-PAGE、RP-HPLC和MALDI-TOF/MS等方法分析生姜蛋白酶对酪蛋白单体和脱脂乳中酪蛋白的水解作用。结果表明,生姜蛋白酶水解κ-酪蛋白生成的主要产物比较稳定,不会被进一步水解。温度高于60℃时生成κ-CN(f 1-90)和κ-CN(f 1-102)两个末端疏水性肽段,其次为κ-CN(f 1-121),温度较低时,还生成大量分子量高于κ-CN(f 1-121)的产物。在脱脂乳体系中,生姜蛋白酶水解脱脂乳的κ-酪蛋白,而对αS1-、αS2-和β-酪蛋白没有显著的水解作用。主要裂解κ-酪蛋白的Thr121-Ile122键,生成疏水性N末端肽段κ-CN(f 1-121)。这一结果表明生姜蛋白酶凝乳的主要机理是水解κ-酪蛋白Thr121-Ile122肽键,破坏了酪蛋白微粒的稳定性,促使酪蛋白形成凝胶。     

原料乳中体细胞数与UHT乳中酪蛋白成分的关系研究

研究了原料乳中体细胞数与15批次UHT乳样本中酪蛋白成分之间的关系。将原料乳巴氏杀菌后进行超高温处理。分别于8,30,60,90和120 d采集贮藏于室温条件下的UHT乳样本,并使用高效液相色谱法对酪蛋白成分进行分析。体细胞数范围1.97×105～8×105 mL-1。体细胞数与原料乳或UHT乳中的κ-酪蛋白质量浓度之间没有相关性(P<0.05)。原料乳中αs2-酪蛋白和β-酪蛋白与体细胞数呈负相关(P<0.05)。UHT乳中,αs1-酪蛋白(P<0.05)和β-酪蛋白(P<0.05)与体细胞数在贮藏第8天呈负相关,αs2-(P<0.01)与体细胞数在贮藏第60天呈负相关。结果表明,原料乳中体细胞数较高与β-酪蛋白和αs-酪蛋白的大量水解有关,并且可能导致UHT乳在贮藏期内出现质量问题。     

甲醇芽孢杆菌蛋白酶的水解特性及酪蛋白水解产物活性分析

首先确定甲醇芽孢杆菌LB-1蛋白酶(蛋白酶LB-1)最适水解温度和pH值,然后通过持续监测水解pH值和氧化还原电位变化、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析、质谱检测等探究蛋白酶LB-1的蛋白水解特性,并测定其酪蛋白水解产物的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS)阳离子自由基清除率、α-淀粉酶和α-葡萄糖苷酶抑制率、金属离子螯合率等生物活性。结果表明:蛋白酶LB-1的最适蛋白水解温度为52℃,最适pH 6.0～7.0;该酶对酪蛋白的水解作用显著强于乳清蛋白,水解程度为κ-酪蛋白α-酪蛋白β-酪蛋白;对κ-酪蛋白的水解位点主要位于Lys_(21)-Ile_(22)和Lys_(112)-Asn_(113);酪蛋白水解产物对DPPH自由基和ABTS阳离子自由基最高清除率分别为75.4%、48.45%,对α-淀粉酶和α-葡萄糖苷酶的最高抑制率分别为80.89%、93.12%,对钙离子和锌离子的最高螯合率分别为63.13%、35.31%;表明该酶的酪蛋白水解产物具有一定的抗氧化能力、降血糖作用以及良好的金属离子螯合能力。因此,甲醇芽孢杆菌LB-1蛋白酶在功能性干酪加工和乳源活性肽开发方面具有潜在的应用价值。     

原料乳体细胞数对UHT乳贮藏期间品质的影响

为了探究体细胞数与蛋白质、脂肪水解的关系及其对凝胶和脂肪上浮等劣变的影响。试验采集30,80万个/mL两组不同体细胞数的原料乳,制成UHT乳,20℃下做180 d的贮藏期试验,每隔30 d测定酶活、蛋白含量、游离脂肪酸、脂肪球粒径等指标。结果显示,经UHT处理,两组产品中纤维溶酶和脂肪酶基本丧失活性,贮藏期间无显著性变化(P0.05)。各酪蛋白组分发生显著水解(P0.05),κ-酪蛋白和αS2-酪蛋白降幅高达90%以上,均未发生蛋白间的相互交联,体系的黏度并无显著变化(P0.05)。脂肪球粒径在第60天迅速增加,之后无明显变化;高体细胞数组脂肪球表面的蛋白膜崩解,脂肪球聚集,从贮藏期第120天开始出现脂肪上浮;而低体细胞数组脂肪球膜蛋白也发生水解,未出现脂肪上浮。本研究表明在现有工艺条件下UHT乳出现的主要问题是脂肪上浮而非蛋白凝胶,将原料乳体细胞数控制在30万个/mL的水平,可以有效防止产品贮藏期内发生脂肪上浮,为UHT乳的品质控制提供了理论依据。     

微胶囊化蛋白酶在干酪制备中的应用及干酪成熟过程中电泳分析

将采用冷冻干燥工艺得到的明胶-阿拉伯胶-凝乳酶微胶囊用于干酪制备过程中,在排乳清之后向干酪中添加5.00 g微胶囊化蛋白酶,以促进干酪的成熟。在干酪成熟过程中通过三羟甲基氨基甘氨酸-尿素-十二烷基磺酸钠聚丙烯酰胺凝胶电泳以及毛细管电泳分析监测干酪中pH 4.6不溶性氮部分的变化情况。结果表明：干酪在成熟初期,αs1-酪蛋白较β-酪蛋白先水解,生成αs1-酪蛋白（f 102～199）和αs1-I-酪蛋白;干酪成熟21 d时,β-酪蛋白开始水解,αs1-酪蛋白继续保持水解,生成的大的水解产物同时也发生二次水解;干酪成熟90 d时干酪中β-酪蛋白比αs1-酪蛋白具有更广泛的水解程度,酪蛋白降解的同时,生成大量新的小分子肽。     

水解牛乳酪蛋白的抗原性研究

采用间接竞争抑制Elisa法测定水解酪蛋白中的残留抗原性,从而间接测定其致敏性。选择7种蛋白酶在各自适宜条件下酶解酪蛋白,观察酪蛋白抗原性随酶解过程的变化情况,并讨论了水解酪蛋白的抗原性随分子质量的变化以及深度水解酪蛋白与适度水解酪蛋白的抗原性。结果表明,不同蛋白酶对酪蛋白的抗原性影响不同,这可能是由于不同的蛋白酶具有不同特异性,其中,中性蛋白酶降低酪蛋白抗原性的效果最佳,抗原抑制率为20.91%;水解酪蛋白的分子质量越小,其残留抗原性越小,当其分子质量小于3 000Da时,抗原抑制率仅为6.61%;深度水解酪蛋白的抗原性较适度水解酪蛋白低,这主要是由于水解度及分子质量的不同。     

不同来源牛乳酪蛋白过敏原性评价及酶解消减作用研究

牛乳酪蛋白是引起婴幼儿过敏的重要来源。为分析不同来源的牛乳酪蛋白过敏原性差异,并通过蛋白酶水解实现低水解度下高效消减牛乳酪蛋白过敏的作用,研究了不同来源的酸法酪蛋白、酶法酪蛋白和胶束酪蛋白原料差异以及制备方法对牛乳酪蛋白过敏原性的影响,并分析了蛋白酶水解消减酪蛋白过敏的作用效果。结果表明,不同制备方法获取的酪蛋白过敏原性差异明显,酶法酪蛋白和胶束酪蛋白具有更高的过敏原性。对具有高过敏原性的酶法酪蛋白采用4种单酶水解时,均获得了较低的残留过敏原性,其中菠萝蛋白酶具有最优效果,残留抗原性降低至9%,对应水解度仅为8%。菠萝蛋白酶水解牛乳酪蛋白时更有利于获得低水解度下具有低致敏性的水解产品。     

胃蛋白酶中性蛋白酶水解酪蛋白的研究

本实验主要研究双蛋白酶(胃蛋白酶-中性蛋白酶)催化酪蛋白水解的反应过程。实验中首先设计正交试验确定了中性蛋白酶和胃蛋白酶各自单独水解酪蛋白的最佳反应条件,分别为酶浓度0.6%、温度55℃、pH 8.0、底物浓度2%和酶浓度1.5%、温度55℃、pH 1.6、底物浓度10%。在此基础上以双蛋白酶作为催化剂水解酪蛋白的反应过程进行研究,并利用层析分离技术和液相色谱初步验证了水解产物中含有β-啡肽-7等多肽类物质。     

日本曲霉酸性蛋白酶的分泌表达、性质及其在猪肉嫩化中的应用

为研究日本曲霉酸性蛋白酶的分泌表达、性质及其在猪肉嫩化中的应用。将日本曲霉来源的酸性蛋白酶基因(AjproA1)在毕赤酵母中高效表达,经5L发酵罐高密度发酵后测得该酸性蛋白酶酶活力为669.6U·mL^-1,蛋白质量浓度为6.64mg·mL^-1。氨基酸多重序列比对结果表明:该酸性蛋白酶属于天冬氨酸蛋白酶A1家族,与海枣曲霉来源的酸性蛋白酶同源性最高(73%),是一个新型的酸性蛋白酶。采用强阴离子交换层析柱对该酸性蛋白酶进行纯化得到电泳级纯酶,纯化后分析测得该重组酸性蛋白酶(AjproA1)的最适催化条件为pH值3.0,反应温度为45℃,在pH值为3.0~6.0及温度为40℃以下具有良好的稳定性。该酸性蛋白酶对不同蛋白质水解的分析结果表明,该酶底物水解特异性广泛,对酪蛋白组分中的κ-酪蛋白表现出最高水解活力。利用该酸性蛋白酶对猪肉进行处理,发现该酶能够有效降低猪肉剪切力,起到嫩化效果。研究结果旨在为新型酸性蛋白酶的开发及其在肉类嫩化中的应用提供理论参考。     

酱油曲蛋白酶的分离及催化性质研究

酱油曲提取液,经硫酸铵盐析后,用Sephadex G-75柱层析分离,测定收集液中的蛋白含量并用两种方法测定蛋白酶活力。结果显示,福林法酶活力峰出现在蛋白吸收曲线第二高峰,酶解酪蛋白显示的酶活力峰值出现在蛋白含量第三高峰的左肩。表明酱油曲含有不同分子量大小的蛋白酶,分别作用于蛋白质的不同位点,分子量较大(在20 kDa到14.3 kDa之间)的蛋白酶福林法酶活力最高,蛋白酶水解酪蛋白产生的酪氨酸较多;分子量比较小(小于14.3 kDa)的蛋白酶福林法检测酶活力不高,但分解酪蛋白产生的氨基酸态氮(ANN)含量较高,表明该蛋白酶酶解酪蛋白产生酪氨酸之外的其它氨基酸或肽类较多。     

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.     

乳中蛋白酶与UHT乳贮存中的胶凝现象

乳中的蛋白酶有2个主要的来源途径，乳中天然存在的蛋白酶和由某些微生物产生的蛋白酶，其中纤维蛋白溶酶和嗜冷菌产生的耐热性蛋白酶是存在于UHT乳中的主要蛋白酶，这些蛋白酶非常耐热，经UHT灭菌处理仍可存活。耐热性蛋白酶在UHT乳贮存中水解乳蛋白质从而导致了UHT乳的胶凝．简要介绍了纤维蛋白溶酶、嗜冷菌耐热性蛋白酶的性质、活性测定方法，论述了这些酶引起的UHT乳的胶凝性质、形成原因及影响因素，并提出了一些防止措施。     

IMPROVED SHELF LIFE ESTIMATION OF UHT MILK BY PREDICTION OF PROTEOLYSIS

ABSTRACT

During growth in raw milk, many psychrotrophic bacteria produce proteases that can retain activity following ultra‐high temperature (UHT) treatment. In this study, casein and skim milk powder assays for detecting very low levels of protease in UHT milk were optimized, and the suitability of azocasein and fluorescein isothiocyanate‐casein (FITC‐casein) as substrates was investigated. The strongest correlations of protease activity with proteolysis in stored UHT milk were observed when FITC‐casein was used as substrate in the assays. Assays using casein and FITC‐casein as substrates yielded the highest activities. To determine sensitivity, crude protease was added at low concentrations to UHT milk, and the milk was assayed for progress of proteolysis over 12 months and for protease activity using the casein and FITC‐casein assays. With long assay incubation times, the FITC‐casein assay was more sensitive than the casein assay and may be suitable for detecting very low levels of protease activity and predicting progress of proteolysis in stored UHT whole milk.

PRACTICAL APPLICATIONS

This study contributes to the development and evaluation of practical assays for the detection of protease activity in the industry to identify potential premature spoilage of contaminated UHT milk before it is distributed for sale. The developed assays are also useful for assessing the quality of milk powder as active protease can persist in milk powder to cause spoilage in reconstituted milk. Although the assays require up to 14 days to complete, this is not an excessive time, compared with the time required for microbiological clearance and total shelf life of the product. High protease activity can be identified with less incubation time. The cost of protease detection assays developed during this work is quite low and, although 20 min analysis time is required per sample, the tests can be very cost efficient when run in batches, as would be expected in a commercial testing facility.

     

Changes in proteins,physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.     

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6 h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12 h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α_S1-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α_S1-casein. Two-dimensional gel electrophoretograms of milk 6 h after infusion with the toxin confirmed hydrolysis of β- and α_S1-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C₁₈ columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α_s1- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.     

Investigation of the ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides from cheese

The ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides found in Cheddar cheese was investigated. The purified protease was incubated with α_s1-casein f1–9 and β-casein f193–209 in a model system (pH 6.8, 30°C) to determine hydrolysis. Residual substrate and hydrolysis products were determined by capillary electrophoresis. Both peptides were hydrolyzed by the protease during the 90-min assay. α_s1-Casein f1–9 was hydrolyzed into two products and β-casein f193–209 was degraded completely in 90 min to three hydrolytic products. This protease hydrolyzed bitter peptides that are known to accumulate in Cheddar and Gouda cheese during aging, suggesting a possibility to debitter Cheddar cheese.     

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.     

The effect of legume protease inhibitors on native milk and bacterial proteases

Protease inhibitors from legume seed extracts (soybean, cowpea and marama beans) and purified soybean protease inhibitor were evaluated with regards to their abilities to inhibit proteases produced by important milk contaminating bacteria, i.e. Bacillus spp. and Pseudomonas spp., and native milk protease, plasmin. Although heat treatment is the most common mean of inactivating enzymes, some heat-stable enzymes can survive the ultra-high temperature (UHT) processing of milk and cause sensory and consistency defects during storage at room temperature. The legume protease inhibitors reduced the activity of plasmin and proteases produced by Bacillus spp. by up to 94% and 97%, respectively, while it showed low inhibitory activity towards Pseudomonas fluorescens proteases (19%) in a buffer system. The protease inhibitors reduced the activity of plasmin (41%) and Bacillus proteases (50%) in UHT milk, however to a lesser extent as compared to inhibition in the buffer system; while it had little or no effect on proteases form Pseudomonas spp. Legume protease inhibitors show great potential in preventing or reducing proteolytic activity of Bacillus proteases and plasmin and may be exploited in various applications where these proteases cause sensory or consistency defects in the product.     

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra‐high‐temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat‐resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf‐life.     

苦味UHT乳原因分析

通过对发生苦味的UHT乳进行分析，将引起UHT乳贮存中出现苦味的原因分为两大类：一是受残留的微生物污染导致UHT乳产生苦味和其它异味，这类微生物包括耐热性强的解淀粉芽孢杆菌和青霉菌等；二是由于牛乳中存在的水解酶分解蛋白质和脂肪．生成肽、氨基酸和脂肪酸类，导致苦味的出现。针对这些影响因素，可以通过提高原料乳的卫生质量、严格控制生产加工过程和环境卫生，以避免UHTSL出现苦味的质量问题。