嗜冷菌蛋白酶及其对乳制晶的影响

嗜冷菌蛋白酶是存在与乳中的主要的耐热性蛋白酶,通过水解乳蛋白对乳及乳制品的品质产生影响.介绍了这些蛋白酶的性质、活性测定方法、控制措施及其对UHT乳及干酪的影响.     

嗜冷菌对UHT乳的影响及预防措施

嗜冷菌代谢所产生的酶在超高温(140℃、5 s)处理下仍有部分残留,引起UHT乳变质,产生胶凝现象及风味的改变,严重影响了乳的货架期.通过研究嗜冷菌及其所产生的酶的作用机制,从原料乳预处理、加工过程到包装讨论了对嗜冷菌的预防措施.     

嗜冷茵对UHT奶品质影响的研究

嗜冷菌产生耐热蛋白酶和脂肪酶是UHT奶在长期存放过程中产生劣变的主要原因之一。原料乳中蛋白酶、脂肪酶的活性与产品长期保存时的品质呈负相关。通过控制原料奶乳中嗜冷菌数,测定蛋白酶和脂肪酶活力,对原料奶进行分级和评价是确保UHT奶品质的一种有效手段,本实验为乳品企业嗜冷菌的检测提供了理论依据。     

纤维蛋白酶及其对乳制品的影响

纤维蛋白酶及其酶原是存在于乳中的一对主要碱性蛋白酶，通过水解乳蛋白对乳及制品的品质产生形响。简要介绍了该酶的性质、活性测定方法，最后论述了对干酪及UHT乳的影响。     

UHT乳结块原因分析

总结了UHT乳产生结块问题的主要原因，并对发生结块的UHT乳进行了微生物指标与蛋白酶活性的检测。结果显示：市售UHT灭菌乳中残留蛋白酶是导致结块现象的主要原因。     

原料乳中嗜冷菌计数及产脂肪酶特性的研究

本文主要对乳中的嗜冷菌数的检测方法进行了初步研究。同时,考虑到嗜冷菌能在乳中产生大量的耐热性脂肪酶,力求在脂肪酶活与菌数之间建立联系。即对不同时间测得的酶活与菌数之间的关系进行初步探讨。     

原料乳中嗜冷菌低温蛋白酶的酶学性质研究

嗜冷菌产生的低温蛋白酶对乳品品质有重要的影响.从原料乳中通过水解酪蛋白实验筛选产低温蛋白酶的菌株,得到一株产低温蛋白酶活性较高的嗜冷菌,该菌株为片球菌属;分离得到蛋白酶的酶液,测得酶的最适生长温度和pH分别是30℃和8.0,Ca2+离子和EDTA对于酶活分别有增强的抑制作用;通过对该酶的低温和高温热稳定性的研究发现,这种蛋白酶是耐热性的低温蛋白酶.     

基于高通量测序解析UHT灭菌乳品质劣变关联微生物

针对超高温灭菌（ultra-high temperature treated,UHT）乳在货架期内出现的脂肪上浮、水乳分离、沉淀等品质劣变问题,利用高通量测序和生物信息学等技术方法,对大型乳企采集的9份在货架期内品质劣变的UHT乳和正常质量的UHT乳样品的理化指标、酶活性、微生物菌群进行了分析比较,并通过关联分析解析UHT灭菌乳品质劣变的关联微生物。结果表明：品质劣变UHT乳在理化指标、酶活性和微生物群分布方面与正常UHT乳有明显不同,假单胞杆菌、不动杆菌为品质劣变UHT乳中核心功能微生物且均属于常见的嗜冷菌,能够产生耐热酶,这可能是导致UHT乳腐败变质的主要原因。本研究为进一步解决UHT乳品质劣变问题提供了参考。     

牛乳中嗜冷菌数与胞外蛋白酶活性相关性的研究

主要研究了牛乳中嗜冷菌数与其产生的耐热性胞外蛋白酶活性的相关性.从原料乳中分离出一株优势嗜冷菌,实验室初步鉴定后用API20NE系统进行鉴定,鉴定该菌为荧光假单胞菌(Pseudomonas Fluorescens),产酶实验结果表明,该菌产耐热性胞外蛋白酶.利用该菌株研究牛乳中单一嗜冷菌菌数与蛋白酶活性的关系,结果表明菌数与蛋白酶活性呈一定正相关,回归方程为Y=0.4548ln(X) 0.4178,决定系数R2=0.8172,相关系数R=0.9039,差异极显著(P<0.01);自然状态下,原料乳中嗜冷菌数与蛋白酶活性关系实验结果表明,原料乳在不同条件下贮存,嗜冷菌数有明显的增加趋势,但原料奶中嗜冷菌数和蛋白酶活性之间没有多少相关性.     

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。     

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.     

Short communication: Variation in the composition and properties of Swedish raw milk for ultra-high-temperature processing

The composition and properties of raw milk are of great importance for the quality and shelf life of the final dairy product, especially in products with a long shelf life [e.g., ultra-high-temperature (UHT)-treated milk]. The objective of this study was to investigate the compositional variation in raw milk samples before processing at the dairy plant. Moreover, we wanted to investigate the effect of the UHT process on this variation (i.e., if the same variation could be observed in the corresponding UHT milk). The quality traits analyzed included detailed milk composition, counts of total and psychrotrophic bacteria, proteolytic activity, and color, as well as predictive measures of stability (i.e., ethanol stability and heat coagulating time). Samples of raw milk and the corresponding produced UHT milk were collected and analyzed on a monthly basis during 1 yr. Principal component analysis was used to identify months showing similarities and differences with respect to total variation. In contrast to previous studies, we observed only small variations between months and no clear effect of season for the raw milk. For the UHT milk, July and the winter months (December, January, and February) tended to separate from the other months. Quality traits showing significant variation were only to some extent identical in raw milk and UHT-processed milk. A better understanding of the natural variation in raw milk quality will provide opportunities to improve the shelf life of UHT-treated milk products.     

UHT奶的质量与原料奶的关系

主要论述了原料奶中的微生物尤其是嗜热性芽孢，低温性细菌及蛋白质的稳定性对ＵＨＴ奶的影响。     

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.     

Proteolysis of casein micelles by heat-stable protease secreted by Serratia liquefaciens leads to the destabilisation of UHT milk during its storage

Serratia liquefaciens is a psychrotrophic species, frequently found in raw milk, which secretes Ser2, a heat-resistant protease. Involvement of this species in UHT milk destabilisation was investigated in the present study. Microfiltered milk was inoculated independently with strains S. liquefaciens L53 or L64. Then, UHT treatment was performed and stability of the corresponding UHT milk was investigated during three months of storage. The residual proteolytic activity of strain L53 led to destabilisation of UHT milk, with sedimentation and formation of aggregates. Hydrolysis of casein micelles was confirmed by the increase in the content of non-casein nitrogen and the identification of numerous peptides coming from the four caseins using mass spectrometry. For strain L64, no visual and biochemical alteration were found. This study showed that Ser2 resists UHT treatment and could be a cause of UHT milk destabilisation; however, this destabilisation by S. liquefaciens was strain-dependent.     

The diversity and proteolytic properties of psychrotrophic bacteria in raw cows' milk from North China

Most psychrotrophic bacteria have the ability to produce thermoresistant proteases that can destroy the quality of milk and dairy products. To investigate the population dynamics of psychrotrophic bacteria during refrigeration, three raw cows' milk samples (sample A comprising milk from 10 farms in Beijing, sample B comprising milk from 5 farms in Heihe, and sample C comprising milk from 7 farms in Harbin) were refrigerated at 0–5 °C and 5–10 °C. PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) analysis revealed that the bacterial community profiles varied from geographical site to site, and with refrigeration temperature. The dominant psychrotrophic bacteria among the samples after storage were affiliated with the order Pseudomonadales. Following isolation and identification, 8 psychrotrophic isolates were selected as stronger protease producers and their growth and proteolytic activities were assessed. The results indicate that the composition of psychrotrophic bacteria play an important role in the determination of the quality of milk and dairy products.     

Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk

Psychrotrophic bacteria were isolated from refrigerated raw milk from a processing plant in Southern Brazil. Psychrotrophic counts were between 4.9 and 7.8 log cfu/mL, and 5.3 to 7.2 log cfu/mL, for samples collected at the truck and the milk storage silo, respectively. Among the bacterial isolates, 90% were Gram-negative. Most strains presented low proteolytic activity, but strains of Burkholderia cepacia, Klebsiella oxytoca and Aeromonas sp. showed higher than 20 U/mL on azocasein as substrate. Crude proteases from selected strains were resistant to conventional heat treatments and caused coagulation of UHT milk after 5 days storage at room temperature.     

Detection and impact of protease and lipase activities in milk and milk powders

Freshly drawn milk contains indigenous enzymes, including proteases and lipases. During handling and processing, milk acquires contaminating bacteria that produce further proteases and lipases, adding to the enzyme loading in the milk. Even when several heat treatment steps are used to prepare milk products, these will not be sufficient to inactivate all of the enzymes. Adverse effects of heat on the product limit the extent of heat treatments that can be used. The activities of proteinases and lipases that survive the heat treatments may cause changes in functionality and flavour of milk products, including milk powders, during storage. This review focuses on the characteristics of proteases (specifically proteinases) and lipases in milk and milk powders, detection methods for such enzymes and the effects of these enzymes on milk and milk powders during storage.     

Effect of addition of CO₂ to raw milk on quality of UHT-treated milk

The objective of this study was to evaluate the effect of addition of CO(2) to raw milk on UHT milk quality during storage. Control milk (without CO(2) addition) and treated milk (with CO(2) addition up to pH 6.2) were stored in bulk tanks at 4°C for 6d. After storage, both samples were UHT processed using indirect heating (140°C for 5s). Samples were aseptically packed in low-density polyethylene pouches and stored in the dark at room temperature. Raw milk was evaluated upon receipt for physicochemical composition, proteolysis, lipolysis, standard plate count, psychrotrophic bacteria, and Pseudomonas spp. counts, and after 6d of storage for proteolysis, lipolysis, and microbial counts. After processing, UHT milk samples were evaluated for physicochemical composition, proteolysis, and lipolysis. Samples were evaluated for proteolysis and lipolysis twice a month until 120d. Peptides from pH 4.6-soluble N filtrates were performed by reversed-phase HPLC after 1 and 120d of storage. A split-plot design was used and the complete experiment was carried out in triplicate. The results were evaluated by ANOVA and Tukey's test. After 6d of storage, CO(2)-treated raw milk kept its physicochemical and microbiological quality, whereas the untreated milk showed significant quality losses. A significant increase in proteolysis occurred during 120d of storage in both treatments, but the increase occurred 1.4 times faster in untreated UHT milk than in CO(2)-treated UHT milk. In both UHT milks, the proteolysis was a consequence of the action of plasmin and microbial proteases. However, the untreated UHT milk showed higher microbial protease activity than the treated UHT milk. The addition of CO(2) to the raw milk maintained the quality during storage, resulting in UHT milk with less proteolysis and possibly longer shelf life, which is usually limited by age gelation of UHT milk.