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。     

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.     

原料乳中嗜冷菌及其主要热稳定性酶类的研究进展

原料乳中的嗜冷菌及其热稳定性胞外酶是引起液态乳制品多种质量问题的主要原因之一。阐述了嗜冷菌及其热稳定性蛋白酶和脂肪酶的概念及特性，对嗜冷菌在原料乳中的控制提出了自己建议，并对嗜冷菌、蛋白酶、脂肪酶的检测方法及理论进行了展望。     

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。     

鲜牛乳中乳杆菌的检测及脂肪酶、蛋白酶测定方法的研究

对鲜牛乳中乳杆菌、脂肪酶及蛋白酶的检测方法进行了初步研究 ,并以改良MRS培养基检测了 4个鲜牛乳样品中的乳杆菌数目 ,以改进的脂肪酶活力分析法测定了 4个样品的脂肪酶活力及蛋白酶活力 ,取得了较满意的结果     

耐热蛋白酶对UHT乳品质的影响及控制方法研究进展

从耐热蛋白酶对UHT乳品质的影响的角度出发．对近几年来国内外在牛乳中主要耐热蛋白酶的来源、检测方法、酶对UHT乳蛋白质水解和凝胶的影响、控制方法等方面的研究进展情况进行了综述，并提出今后深入研究应关注的方向和重点。     

水酶法提取大豆油工艺中乳状液的酶法破乳研究

为降低水酶法提取大豆油过程所产乳状液的稳定性,得到较高的游离油回收率,研究了α-淀粉酶、纤维素酶、Alcalase碱性蛋白酶、7L中性蛋白酶的破乳效果;通过破乳率、Zeta电位、粘度、粒径分布和平均粒径指标,分别考察了Alcalase碱性蛋白酶和7L中性蛋白酶对乳状液稳定性的影响。结果显示,在所选酶中Alcalase碱性蛋白酶、7L中性蛋白酶破乳效果最好,相同水解条件下,Alcalase碱性蛋白酶的破乳率高于7L中性蛋白酶。2%的7L中性蛋白酶酶解60 min时破乳率达100%,而在相同酶解时间内,1% Alcalase碱性蛋白酶即可实现100%破乳。经Alcalase碱性蛋白酶和7L中性蛋白酶水解后,乳状液的粘度变低,电位电势减弱,油滴发生聚集,导致乳状液稳定性下降。随Alcalase和7L蛋白酶浓度和酶解时间的增加,相应地,乳状液的粘度进一步降低,破乳率上升。     

响应面法优化瑞士乳杆菌产蛋白酶培养条件

为充分应用乳酸菌的产蛋白酶特性,本研究采用响应面分析法对瑞士乳杆菌产蛋白酶的培养条件进行优化。首先通过Plackett-Burman试验从7种因素中筛选发酵温度、发酵时间、起始pH为影响瑞士乳杆菌产蛋白酶的重要因素。进一步通过响应面分析得出最优产酶条件:发酵温度36℃、发酵时间16h、起始pH 6.65,瑞士乳杆菌的产蛋白酶活力为16.81U/mL和22.59U/mL,提高了34.4%,与预测结果接近,表明培养条件的优化有效,该发酵液为瑞士乳杆菌和所产蛋白酶的同步固定化研究提供了原料,也为瑞士乳杆菌产蛋白酶的应用研究奠定了基础。     

协同发酵在植物乳杆菌发酵乳中的应用研究进展

植物乳杆菌由于其益生功能受到广泛关注。植物乳杆菌蛋白水解系统不完全,存在肽转运系统和胞内肽酶,但缺乏胞壁蛋白酶,这导致该菌无法直接利用乳中蛋白质,因此在乳中生长不佳。利用蛋白酶活性强的乳酸菌和植物乳杆菌的协同发酵是一种解决方法,前者能初步水解乳中蛋白质产生多肽和游离氨基酸,为植物乳杆菌提供氮源,促进其生长。本文综述了植物乳杆菌益生功能,在乳中生长不良的原因及相关解决办法(添加营养物质、添加蛋白酶、协同发酵)的研究进展。     

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。     

Distribution of plasminogen and plasmin in fractions of bovine milk.

The relative amounts of immunoreactive plasminogen and active plasmin in different fractions of bovine milk were examined. Raw milk was centrifuged to separate skim, cream, and a somatic cell pellet. Skim milk was centrifuged to separate milk serum and casein micelles. Milk fat globule membranes were isolated from the cream fraction of bovine milk. Proteins from somatic cells were isolated following sonication of the cells. Western blot analysis showed the presence of several forms of plasminogen in bovine milk. The predominant forms of plasminogen identified following electrophoresis under nonreducing conditions were proteins with approximate molecular weights of 88,000, 152,000, and 160,000. The predominant forms of plasminogen identified after electrophoresis under reducing conditions were two proteins with approximate molecular weights of 88,000 and 50,000. The highest amount (82% of the total plasminogen), as determined by an ELISA, was associated with the casein fraction. Lower plasminogen concentrations were associated with the serum, cream fractions, and milk fat globule membranes. The SDS-PAGE of the cream and milk fat globule membranes indicated that some casein was present in both fractions. Thus, the low plasminogen concentrations in these fractions may be associated with the caseins there. No immunoreactive plasminogen was present in the somatic cells. Active plasmin was present in the same milk fractions in which plasminogen was detected: casein, serum, and cream.     

Effect of various heat treatments on plasminogen activation in bovine milk during refrigerated storage

Plasmin and plasminogen‐derived plasmin activities were measured in heated milk with and without the addition of plasminogen activator, before and after storage at 4 °C for 96 h. The effect of a free sulfhydryl group donor, β‐lactoglobulin or cysteine, on plasminogen activation was investigated in a model system and milk. Heating milk to 75 °C enhanced plasminogen activation that was marked by a considerable increase in plasmin activity. Heating at 85 and 90 °C caused a significant decrease in plasmin and plasminogen‐derived plasmin activities. However, after storage, significant plasmin levels were restored because of the activation of remaining unfolded plasminogen. Both β‐lactoglobulin and cysteine significantly decreased plasmin and plasminogen‐derived plasmin activities in a model system. While endogenous β‐lactoglobulin was not sufficient to completely eliminate plasminogen activation in milk, cysteine addition prior to pasteurisation significantly decreased plasmin and plasminogen‐derived plasmin activities. Results highlighted the importance of the remaining plasminogen in heated milk systems.     

Effect of subclinical mastitis on milk plasminogen and plasmin compared with that on sodium, antitrypsin and N-acetyl-beta-D-glucosaminidase

The effect of subclinical mastitis on levels of plasminogen and plasmin in milk from cows in a high-yielding herd was investigated. Comparisons were made with levels of milk Na, antitrypsin and N-acetyl-beta-D-glucosaminidase (NAGase). In samples from mastitic quarters plasminogen activity, as measured after activation to plasmin, increased by only 21% and plasmin by 82%, while NAGase increased by 307%. Plasminogen was the only component that was normally distributed, all other components showed more or less skewed distributions. Plasmin and plasminogen were significantly related to the other components. However, plasminogen plateaued when the other components continued to increase. There was thus no further increase in plasminogen with the severity of inflammation as with the other components. Plasmin showed a similar although less pronounced tendency. Results of treatment of mastitic whey samples with acid suggested that the non-linear increase in plasmin activity was due to interaction with acid-labile proteinase inhibitors. Mastitis led to dissociation of plasminogen and plasmin from the casein micelles. The degree of activation of plasminogen was higher with casein-associated than with soluble plasminogen in both healthy and mastitic milks. Plasmin was very closely related to milk Na, which is a sensitive indicator of epithelial integrity. It is suggested that plasmin contributes to Na leakage into milk by degrading membrane proteins of the epithelial lining. Plasminogen and antitrypsin, which are both plasma proteins, were not identically affected by stage of lactation, indicating nonidentical modes of transport from plasma to milk.     

Partial purification and characterization of native plasminogen activators from bovine milk

At least four native plasminogen activators were detected in bovine milk, and two partially purified plasminogen activators were characterized. The plasminogen activators were dissociated from casein proteins by treatments with sulfuric acid and dimethylformamide. The plasminogen activators in the resulting fractions were partially purified with size exclusion, affinity, or metal chelate chromatographic techniques. Molecular weights of the two partially purified plasminogen activators were 47.2 and 30.5 kDa by gel electrophoresis. Size exclusion chromatography gave a molecular weight of 43.2 kDa for the first plasminogen activator. The isoelectric points of the two plasminogen activators were in the pH range 6.2 to 6.7. Because activity was not enhanced by the presence of fibrinogen fragments in a plasminogen activator assay mixture and decreased when human anti-urokinase Ig were added, at least some bovine milk native plasminogen activators appear to be urokinase-type plasminogen activators.     

Plasmin and plasminogen in bovine milk: a relationship with involution?

A total of 774 individual milk samples were collected from 66 Holstein cows between October 1987 and April 1988. Samples were analyzed for plasmin, plasminogen, and SCC. An increase in SCC from less than 250,000/ml to more than 1,000,000/ml resulted in an increase of plasmin, plasminogen, and serum albumin by 105, 74, and 140%, respectively. Plasminogen, plasmin, and serum albumin followed similar trends that are expected for components from blood that gain access to the alveolar lumen through ruptured epithelium caused by mastitis. Increased plasmin is the direct result of this process rather than an increase in activation of plasminogen to plasmin. The plasminogen to plasmin ratio supports this interpretation, being 4.7 at 250,000 SCC/ml and 4.0 when SCC exceeded 1 million/ml. Plasmin and plasminogen concentrations were also increased during lactation to reach peak values immediately before the dry period. However, in this case, ratio of plasminogen to plasmin was 6.55 during early lactation and decreased by half to 3.29 during the latest stage, indicating that considerable activation of plasminogen to plasmin occurred during the latter part of lactation. Mammary epithelium is not compromised at this stage, as shown by low (.8 mg/ml) serum albumin concentration in milk. Two mechanisms responsible for increased milk plasmin include influx of plasmin from blood during mastitis and increased activation of plasminogen as lactation progresses.     

Contribution of somatic cell-associated activation of plasminogen to caseinolysis within the goat mammary gland

Functional regression of the mammary gland is partly reflected by proteolysis of milk protein and tissue protein. The involvement of the plasminogen activation system in degradation of milk protein and mammary tissue damage has been demonstrated under inflammatory conditions. In this study, mammary secretion from 23 dairy goats primarily grouped as lactation (milking twice daily) or involution (milking once daily or less) was used to determine the ratio of gravity-precipitated casein to total milk protein (casein ratio) as an index of caseinolysis, and activities of components of plasminogen activation system as well as their expressions on somatic cells. Based on the casein ratio, lactation goats were subcategorized as very active (71.8 ± 1.0%) or less active (29.9 ± 1.0%) in mammary function; involution goats were subcategorized as gradual (21.7 ± 1.0%) or acute (5.9 ± 0.2%) involution. This result suggests that caseinolysis occurred during regular lactation as well as during involution. On the other hand, activities of components of the plasminogen activation system in mammary secretion were increased along with the decreasing casein ratio, in contrast to the similar activities of their counterparts in circulation throughout various mammary statuses. Correlation analysis between casein ratio and activities of plasminogen activation system of goat milk indicated a significant negative relationship for plasmin (r = −0.64), plasminogen (r = −0.69), and urokinase-type plasminogen activator (uPA; r = −0.78) during involution but not during lactation. As for the cellular components of plasminogen activation system, there was an increase in immunoreactivity on somatic cells toward both monoclonal antibodies of human uPA and human uPA receptor under involution conditions suggesting their upregulation relative to lactation condition. Collectively, these results suggest that plasminogen activation system within the mammary gland differentially contribute to milk caseinolysis along the various stages of goat lactation. Meanwhile, a somatic cell-mediated local elevation of plasmin activity may be committed to extensive caseinolysis during involution.     

Activity and nature of plasminogen activators associated with the casein micelle

In fresh milk, plasminogen, the zymogen form of plasmin (PL), is the predominant form. Therefore, plasminogen activators (PA) can contribute significantly to PL activity in milk. Both tissue-type PA (tPA) and urokinase-type PA (uPA) exist in milk; however, contradictory findings have been reported for which type of PA is most closely associated with the casein micelles. Little is known about the factors that might lead to variations in the individual activities of the PA. The objective of this work was therefore to investigate possible factors that might affect the association of tPA and uPA with the casein micelle and their activities thereafter. Plasminogen activators were isolated from milk samples with different somatic cell counts following 2 different isolation protocols. Determination of uPA, tPA, and PL activities was carried out quantitatively following chromogenic assays using 2 different substrates, and qualitatively using specialized sodium dodecyl sulfate-PAGE. Different isolation methods and conditions led to differences in uPA, tPA, and PL activities. Urokinase-type PA activity was significantly higher in PA fractions isolated from milk with high somatic cell counts than from milk with low somatic cell counts. Activity results indicated that in pasteurized milk uPA could dissociate from the somatic cells and bind to casein. Moreover, a high level of PL in isolated PA fractions contributed to significantly enhanced PA activities. Overall, results confirmed the association of both uPA and tPA with the casein micelle; however, their amounts, activities, and molecular weights varied based on the nature of the milk and methods of separation, with uPA being the PA with greater potential to affect plasminogen activation in milk.     

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.     

Enzymatic assays for native plasmin, plasminogen and plasminogen activators in bovine milk.

Rapid and sensitive assays for plasmin, plasminogen and plasminogen activators (PA) were developed and applied to bovine milk. The reaction medium was clarified by addition of a dissolving agent after hydrolysis of a fluorescent substrate specific for plasmin. This final step enabled the use of larger sample amount with higher substrate concentration than other methods, and avoided previous sample preparation. The use of 4 g gelatin/l in buffers preserved plasmin activity, thus avoiding risks of overestimation of the assays results. Sensitivity, detection level, repeatability and analysis run time of plasmin and plasminogen assay were improved over previous enzymatic methods with synthetic substrates. The PA assay was assessed by measuring conversion of exogenous plasminogen into plasmin. A new kinetic approach was used to enable the direct determination of global PA activities on raw milk samples without interference from indigenous plasmin.     

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.