乳源嗜冷菌产胞外耐热酶检测方法的对比分析

乳及乳制品中污染的嗜冷菌可分泌耐热的胞外蛋白酶和脂肪酶,直接影响产品品质。介绍从乳体系中分离鉴定的嗜冷菌种类,指出荧光假单胞菌是产胞外蛋白酶和脂肪酶的主要嗜冷菌菌株;分别阐述乳中污染的嗜冷菌所分泌蛋白酶和脂肪酶的热稳定性,并比较乳体系中耐热酶的测定方法,期望为有效预测乳中嗜冷菌污染程度、在线检测和控制耐热酶活性、提升乳制品的质量提供理论参考。     

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。     

原料乳嗜冷菌的危害分析及控制

从相关的8个牧场的原料乳样品中分离纯化得到嗜冷菌分离物。然后重点探讨嗜冷菌对原料乳的危害及对嗜冷菌的生长控制。对嗜冷菌数动态增加与蛋白酶和脂肪酶活力变化、pH值的变化和总醛类物质浓度增加的相关性进行了分析研究。结果表明，壳聚糖和二氧化氯对嗜冷菌有较好的抑菌作用。     

原料乳中嗜冷菌快速检测新技术研究进展

嗜冷菌在原料乳低温储藏过程中能大量繁殖,其生长过程中产生的脂肪酶和蛋白酶对乳制品后期储藏的风味品质有很大影响。因此,针对原料乳中嗜冷菌的检测,国内外开展了大量的研究,其中快速、自动化的现代技术蓬勃发展。本文在整理这些主要技术的基础上,重点介绍了近些年来发展较快且灵敏度高的嗜冷菌快速检测新技术及其优缺点,并对未来该领域快速检测技术的发展做出展望。     

原料乳中嗜冷菌产脂肪酶条件的优化

研究了原料乳中嗜冷菌产生脂肪酶的几组影响条件。主要从pH值、培养温度、产酶培养基组成几个方面考虑。优化产酶条件，从而可提高脂肪酶的检测活力，为原料乳中嗜冷菌分泌的脂肪酶的研究奠定理论基础。     

原料乳中优势嗜冷菌株的确定及其微生物学特征研究

对原料乳样品进行嗜冷菌的分离筛选,结果得到4株优势嗜冷菌.本文研究了这4株优势嗜冷菌的生长特性及产脂肪酶特性,分别得到了4株优势嗜冷菌株的最佳生长条件及最佳产脂肪酶条件.     

原料乳中嗜冷菌的检测

针对原料乳中嗜冷菌的一种快速检测方法进行了初步研究。由于嗜冷菌能在乳中产生大量的耐热性脂肪酶,所以我们在脂肪酶活与嗜冷菌数之间建立了一种线性关系,进而通过4-硝基苯酚游离释放法测定脂肪酶的活力来得到嗜冷菌数。     

乳和乳制品中嗜冷菌及其耐热酶的危害与控制研究

嗜冷菌是乳低温贮藏中的主要腐败菌,嗜冷菌及其代谢酶的存在直接影响原料乳的质量,阐述了嗜冷菌及其耐热酶类对乳及乳制品的危害,并通过分析嗜冷菌的适冷机理和影响酶活的因素,探讨控制嗜冷菌及其代谢酶的方法,以保障原料乳质量。     

原料乳中嗜冷假单胞菌危害及控制研究进展

假单胞菌是对原料乳危害最大的嗜冷菌之一,极易在冷藏条件下成为优势菌群。大多数嗜冷假单胞菌有分泌蛋白酶和脂肪酶的能力,并导致原料乳及产品变质。本文对原料乳假单胞菌的危害特点,快速检测和新型控制工艺研究进展进行综述。最后探讨了嗜冷假单胞菌的研究方向。     

乳及乳制品中嗜冷菌的多样性及其腐败危害研究进展

乳是一种营养丰富的物质, 同时也是微生物生长繁殖的理想培养基。生乳的质量是影响乳制品产业链的关键因素, 随着低温储存和冷链运输技术的发展, 生乳中大部分细菌的生长受到抑制, 但嗜冷菌的生长并未受到抑制, 并逐步成为生乳中的优势菌。生乳在冷藏运输或储存期间, 嗜冷菌依旧可以生长繁殖, 其分泌的蛋白酶和脂肪酶可耐高温, 经过巴氏杀菌或超高温灭菌处理后依旧保持活性, 因此, 了解嗜冷菌的多样性及其产生的酶对提高乳及乳制品质量、减少腐败和浪费具有重要作用。本文通过介绍乳及乳制品中嗜冷菌污染现状及腐败危害研究, 旨在为乳及乳制品行业的风险评估提供背景信息, 从源头控制嗜冷菌对生乳的浪费, 保证乳及乳制品的品质。     

Effects of heating temperatures and addition of reconstituted milk on the heat indicators in milk

The objective of the present study was to evaluate the effect of heating temperatures and reconstituted milk on heat treatment indicators in milk by comparing the heat damage between raw milk and raw milk plus reconstituted milk (composite milk). The contents of lactulose, furosine, beta-lactoglobulin, and lactoperoxidase were determined after the heat indicators were heated to 65 to 115 °C for 15 s both in raw milk and composite milk. In the raw milk, the lactulose and furosine contents increased with increased heating temperature, while the beta-lactoglobulin content and lactoperoxidase activity decreased. The lactulose and furosine contents were increased after the addition of reconstituted milk. The reconstituted milk also significantly (P < 0.05) reduced the concentration of beta-lactoglobulin in the milk. Both heat treatment and an addition of reconstituted milk decreased the lactoperoxidase activity significantly (P < 0.05), and the lactoperoxidase activity was undetectable at 85 °C. The ratios of lactulose to furosine in pasteurized milk were higher than that in composite pasteurized milk. It is concluded that lactulose, furosine, and beta-lactoglobulin are suitable indicators of high heat pasteurization or raw milk, while lactoperoxidase may be used in monitoring mild heat pasteurization. Practical Application: Adequate heat treatment is necessary to destroy the microbes in raw milk. However, excessive heat treatment can result in inactivation of active compounds or loss of nutrients. The present study showed that the concentrations of lactulose, furosine, beta-lactoglobulin, and the activity of lactoperoxidase are sensitive to processing temperature and can serve as indicators of milk pasteurization.     

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.     

Growth of a rifampicin-resistant strain of a proteolytic psychrotroph,Pseudomonas fluorescens,in raw and ultra heat-treated milk

Pseudomonas fluorescens UQM2490, resistant to 250 μg rifampicin/ml, was derived from P. fluorescens JC1, a proteolytic psychrotroph isolated from raw milk. Growth of UQM2490 was followed in raw and ultra heat-treated milk, by viable counting on rifampicin-containing agar medium. The growth curves obtained demonstrate slower growth in raw milk than in treated milk and the variation in growth with change in inoculum level. Generation times in ultra heat-treated milk ranged from 9.5 to 14.1 h compared with 9.6 to 33 h in raw milk.     

A gas chromatography-mass spectrometry untargeted metabolomics approach to discriminate Fiore Sardo cheese produced from raw or thermized ovine milk

Thermization is a sub-pasteurization heat treatment of cheese milk (at 57–68°C for 15–30 s) aimed to reduce the number of undesirable microbial contaminants with reduced heat damage to the indigenous milk enzymes. In this work, the effects of milk thermization on the compositional parameters, proteolysis indices, free fatty acid levels, and low molecular weight metabolite profiles of ovine cheese were studied. Cheese samples at different ripening stages and produced in 2 different periods of the year were analyzed. While the effects of milk thermization on cheese macro-compositional parameters and free fatty acid levels were not evident due to the predominant effects of milk seasonality and cheese ripening stage, the gas chromatography-mass spectrometry based metabolomics approach of ovine cheese produced from raw and thermized milk highlighted strong differences at the metabolite level. Discriminant analysis applied to gas chromatography-mass spectrometry data provided an excellent classification model where cheese samples were correctly classified as produced from raw or thermized milk. The metabolites that mostly changed due to the thermization process belonged to the classes of free amino acids and saccharides. Gas chromatography-mass spectrometry-based metabolomics has proven to be a valid tool to study the effect of mild heat treatments on the polar metabolite profile in ovine cheese.     

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.     

Buffalo-milk enzyme levels, their sensitivity to heat inactivation, and their possible use as markers for pasteurization

The activities and rates of inactivation of four enzymes in raw buffalo milk were measured in relation to the process of heating to determine the value of these enzymes as markers for the evaluation of milk pasteurization. The activities of the enzymes alkaline phosphatase (ALP), lactic dehydrogenase (LDH), gamma-glutamyltransferase (GGT), and aspartate aminotransferase (AST) were measured before and after heating at 50, 60, 70, and 80 degrees C for 1, 3, 5, 10, 20, and 30 min. The enzyme GGT showed the highest activity (712 +/- 601 IU/liter), followed by LDH (386 +/- 183 IU/liter), ALP (295 +/- 164 IU/liter), and AST (18 +/- 4 IU/liter). Heating the milk at 50 degrees C for 1 to 30 min resulted in no effect on the activity of any of the enzymes. At 60 degrees C, ALP showed the highest sensitivity to heat inactivation, whereas all other enzymes showed resistance. At 70 degrees C, ALP activity was abolished completely after 1 min, whereas GGT and LDH lost most activity after 10 min, and AST still maintained 50% activity even after 30 min. At 80 degrees C, the activities of LDH and GGT were lost, whereas AST still retained some of its activity. The results suggest that in addition to ALP, LDH and GGT, but not AST, are potential markers for heat denaturation in buffalo milk, with GGT having the advantage that its concentration is the highest.     

THE EFFECT OF STORAGE TEMPERATURE ON BACTERIAL GROWTH AND LIPOLYSIS IN RAW MILK

The temperature at which raw milk was stored, within the range 4°-8°C, affected the rate of growth of bacteria and the release of free fatty acids. The effects were of both statistical and practical significance and it was shown that, by maintaining milk temperatures at 4°C, a useful extension of the storage time of raw milk could be achieved. Lipolysis in stored milk was not closely related to the total concentration of psychrotrophic bacteria, but lipolytic rancidity was not observed when the psychrotroph count was below 5 × 10⁶ colony forming units/ml.     

Lipolysis in cheddar cheese made from raw, thermized, and pasteurized milks

The evolution of free fatty acids (FFA) was monitored over 168 d of ripening in Cheddar cheeses manufactured from good quality raw milk (RM), thermized milk (TM; 65°C × 15 s), and pasteurized milk (PM; 72°C × 15 s). Heat treatment of the milk reduced the level and diversity of raw milk microflora and extensively or wholly inactivated lipoprotein lipase (LPL) activity. Indigenous milk enzymes or proteases from RM microflora influenced secondary proteolysis in TM and RM cheeses. Differences in FFA in the RM, TM, and PM influenced the levels of FFA in the subsequent cheeses at 1 d, despite significant losses of FFA to the whey during manufacture. Starter esterases appear to be the main contributors of lipolysis in all cheeses, with LPL contributing during production and ripening in RM and, to a lesser extent, in TM cheeses. Indigenous milk microflora and nonstarter lactic acid bacteria appear to have a minor contribution to lipolysis particularly in PM cheeses. Lipolytic activity of starter esterases, LPL, and indigenous raw milk microflora appeared to be limited by substrate accessibility or environmental conditions over ripening.     

热处理对牛乳成分的影响以及热敏感指标的变化研究进展

热加工可以有效杀灭生牛乳中的各种致病微生物,但也会对牛乳成分产生影响。随着热处理温度的升高,乳清蛋白变性和凝集、乳糖异构化和降解、美拉德反应等理化反应会依次发生,这些反应中活性成分(例如碱性磷酸酶和乳清蛋白)的减少或反应产物的生成(例如乳果糖和糠氨酸)都可作为热加工强度的标识。本文对牛乳的热加工条件、牛乳在受热情况出现的理化变化以及相应的热敏感成分的变化进行综述。