具有抗疲劳作用酪蛋白多肽水解条件的优化

用胃蛋白酶水解酪蛋白制备具有抗疲劳作用的多肽。以小鼠负重游泳时间为指标,对底物质量浓度、酶底比、酶解pH值和酶解温度等酶解参数进行优化,以获得具有最佳抗疲劳作用的酪蛋白水解物,并用毛细管电泳对各条件下水解产物进行肽谱分析。结果表明：在底物质量浓度5g/100mL、酶与底物比1:30、pH 2.5,酶解温度43℃的条件下胃蛋白酶水解酪蛋白所得水解产物,以0.048、0.24、2.4g/(kg ·d) 3个剂量灌胃小鼠28d后负重游泳,其耐受时间与同剂量的其他水解产物灌胃组相比均有显著差异(P＜0.05)。通过毛细管电泳进行肽谱分析表明,该水解条件下所得水解产物与其他各水解条件的产物相比具有明显差异,最大峰出现时间与其他各组也不同,同时在17～18min时出现其他各组未出现的峰值。     

胃蛋白酶水解酪蛋白制备ACE抑制肽的条件

研究胃蛋白酶水解酪蛋白制备ACE 抑制肽的工艺条件。采用胃蛋白酶水解酪蛋白制备ACE 抑制肽,检测其水解产物的ACE 抑制率,以ACE 抑制率为指标,通过正交试验设计法,确定最终水解条件为水解温度37℃、pH3.0、酶与底物质量比1:100、底物质量分数7%,水解产物ACE 抑制率为92.5%,其IC50 值为0.24mg/mL。酪蛋白的水解度与ACE 抑制率之间并非简单的相关性。     

不同蛋白酶水解酪蛋白及其对产物功能性质的影响

采用Alcalase 2．4L和Protamex两种蛋白酶分别水解酪蛋白酸钠（蛋白质含量88．03％）至5％、10％、15％和20％等不同的水解度（DH），并对酪蛋白酸钠及填水解产物的各种功能性质进行了分析测定。结果表明：酪蛋白酸钠经水解后，蛋白质、水分和灰分含量发生变化，游离氨基量增加且增加与DH相关；水解产物中的多肽分子量较小，平均分子量小于8103D，并且分子量随DH的增大而减小，在DH为15％和20％的水解产物中多肽分子量均低于5043D：水解产物的溶解性随DH的增大而增强，在pH4．0～5．0、DH10％～20％的范围内产物溶解度84．8％～98％，说明在等电点条件下，酪蛋白酸钠水解后溶解性得到改善：与酪蛋白酸钠相比，水解产物的乳化性和起泡性减弱；不同水解产物的氨基酸组成差异不是很大，与酪蛋白酸钠也很接近。     

乳源酪啡肽的研究进展

80年代以来，国外一些学者相继从酪蛋白的酶解产物中发现生物活性肽，证明乳除了具有营养作用外，还具有重要的生理作用。酪啡肽就是其中重要的乳源阿片肽，自从1979年Brantl等人首先从酪蛋白的水解产物中分离到β-酪啡肽-7，对于酪啡肽的制备、分离、纯化等各个方面均有广泛的研究。随着具各种生物活性的短肽的不断发现，其研究和开发日益受到各国学者的关注。     

酪蛋白抗诱变水解物生化特性的研究

采用Ams鼠伤寒沙门氏菌实验研究酪蛋白的胃蛋白酶的水解物的抗诱变性，采用超滤和等电点法分离水解物，证明水解产物比酪蛋白具有更强的抗诱变性，经快速液相色谱FPLC和质谱法分析表明具体抗诱变性短肽是由9到11个氨基酸构成的，分子量在1000到2000道尔顿范围内。     

耐热蛋白酶对UHT乳蛋白水解作用

采用SDS-PAGE、Urea-PAGE 分析耐热蛋白酶对UHT 乳蛋白的水解作用。结果表明：荧光假单胞菌(PF)蛋白酶和纤溶酶(PL)作用底物不同,产物也不同;前者优先水解κ- 酪蛋白生成副κ- 酪蛋白,而PL 主要水解β- 酪蛋白与α- 酪蛋白生成γ- 酪蛋白及胨、肽等。分别经PL和细菌蛋白酶水解3h的UHT乳水解产物,在pH4.6和12g/100mL三氯乙酸(TCA)沉淀后经反相高效液相色潽仪(RP-HPLC)分析滤液,两者呈现不同的色谱图。     

酪蛋白水解产物对不同酸奶发酵时间及品质影响

本文研究了酪蛋白水解产物对YC-380、ABT-7、FAST-89为发酵剂的酸奶发酵时间、发酵剂用量、粘度、风味、口感以及稳定性的影响.结果表明酪蛋白水解产物对这三种酸奶发酵均有明显的促进作用.在菌种用量减半时添加酪蛋白水解产物可与对照组同时或提前达到发酵终点.加入酪蛋白水解产物或在菌种用量减半时加入酪蛋白水解产物对以不同发酵剂发酵的酸奶的粘度、口感有一定的影响,能提高酸奶的稠厚感.但对酸奶的风味和乳清析出却没有明显影响.     

酪蛋白糖巨肽制备及其对致龋齿菌的抑制作用

酪蛋白巨肽(CaseinGlycomacropeptide,CGMP)是一种来源于哺乳动物κ-酪蛋白,具有64个氨基酸残基(106~169)的糖肽,具有许多生理活性和营养功能,可广泛添加于保健品和医药品中。采用胃蛋白酶水解酪蛋白制备CGMP,对水解产物中CGMP进行测定,并研究CGMP抑制致龋齿菌生长的生物活性。结果显示:水解产物中确有CGMP存在,含量为30.74%;粗品CGMP对致龋齿菌-变形链球菌具有明显的抑制作用;最小抑菌浓度为24 mg/mL。     

具有免疫调节作用的酪蛋白水解产物制备条件的确定

以酪蛋白为原料,利用酶解法制备具有免疫调节作用的水解产物。用MTT 法测定不同水解条件下胰蛋白酶水解酪蛋白的水解产物对小鼠脾脏细胞免疫刺激指数,并以免疫刺激指数作为指标,确定出最佳的水解条件。结果表明,酪蛋白在不同水解条件下的水解产物,用1640 培养液配成浓度为10μg/ml,采用MTT 法测定小鼠脾脏淋巴细胞免疫刺激指数最大值为2.77,其水解条件为底物浓度4%、酶与底物比为1:90。     

Lactobacillus helveticus 9的酪蛋白水解作用研究

基于酪蛋白水解度和多肽含量数据,研究了两株瑞士乳杆菌(L.helveticus 9和L.helveticus 6)与酪蛋白共培养后的蛋白水解效果,菌株L helveticus 9蛋白水解活性显著高于菌株L.helveticus 6.在不同发酵时间下,菌株L.helveticus 9对酪蛋白的水解作用随时问延长而逐渐递增,培养48h时酪蛋白的水解度可达17.3%,多肽含量达到0.62mg/ml:SDS-PAGE分析和Sephadex G-25层析鉴定表明,酪蛋白水解产物多肽含量随培养时间延长而增加.显然,具有较高蛋白水解活力的菌株L.helveticus 9可以用于从酪蛋白中来制备潜在的生物活性肽.     

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.     

Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora

Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.     

Impact of liposome-encapsulated enzyme cocktails on cheddar cheese ripening

Cheddar cheese proteolysis and lipolysis were accelerated using liposome-encapsulated enzymatic cocktails. Flavourzyme, neutral bacterial protease, acid fungal protease and lipase (Palatase M) were individually entrapped in liposomes and added to cheese milk prior to renneting. Flavourzyme was tested alone at three concentrations (Z1, Z2 and Z3 cheeses). Enzyme cocktails consisted of lipase and bacterial protease (BP cheeses), lipase and fungal protease (FP cheeses) or lipase and Flavourzyme (ZP cheeses). The resulting cheeses were chemically, rheologically and organoleptically evaluated during 3 months of ripening at 8 °C. Levels of free fatty acids and appearance of bitter and astringent peptides were measured. Certain enzyme treatments (BP and ZP) resulted in cheeses with more mature texture and higher flavor intensity in a shorter time compared with control cheeses. No bitter defect was detected except in 90-day-old FP cheese. A full aged Cheddar flavor was developed in Z3 and ZP cheeses, while treatment BP led to strong typical Cheddar flavor by the second month and did not exhibit any off-flavor when ripening was extended for a further month.     

Chemical Profiles of Hydrolyzed Milk Samples after Treatment with Commercial Enzymes

ABSTRACT: Commercial enzymes (protease and lipase) were used to produce highly flavored cheese-like hydrolysates from fluid milk. Free fatty acids, free amino acids, degree of proteolysis, and volatile profiles were assessed to suggest the importance of proteolytic and lipolytic activity on cheese flavor development. Free fatty acid liberation was maximized with the combined Flavourzyme™ (protease) and Palatase ® (lipase) treatment incubated at 30°C, most likely due to synergism conferred by the protease. The Flavourzyme/Palatase samples incubated at 45°C generated the highest total concentration of volatile compounds. The addition of Flavourzyme generated free amino acids and low molecular weight peptides (< 1400 MW).     

Monitoring Proteolysis During Cheddar Cheese Ripening Using Two-Dimensional Gel Electrophoresis

A 2-D gel electrophoretic method, consisting of isoelectric focusing and alkaline urea-PAGE was used to monitor proteolysis during ripening (180d, 5°C and 8°C) of full- and reduced-fat Cheddar cheese. The method enabled quantifying changes in levels of peptides in cheese with good spot-resolution. Results can complement those from other analyses, especially those for determining low MW peptides. Notable effects were found for cheese composition and ripening temperature on gel pattern and on relative levels of selected proteolysis products. In both cheese varieties, most peptides reached a maximum during the first 3 ripening months and gradually disappeared as ripening advanced.     

乳源ACE抑制剂(降血压肽)的研究现状

利用酶或微生物水解牛乳蛋白质可获得ACE抑制剂 (降血压肽 ) ,近来从乳酸菌发酵的酸奶中也找到了ACE抑制肽 ;经动物实验证明 ,口服一定量的ACE抑制肽或含有ACE抑制肽的酸奶可降低血压 ,而且对血压正常者没有影响。文章中综述了来源于乳蛋白质和发酵乳制品的ACE降血压肽的最新研究进展 ,对乳源ACE抑制剂的应用前景进行了展望     

Combined use of chymosin and protease from Cryphonectria parasitica for control of meltability and firmness of cheddar cheese

The combined use of chymosin and Cryphonectria parasitica protease was evaluated for manufacturing Cheddar cheese at different chymosin-to-C. parasitica protease ratios of 1:0, 0:1, 67:33, and 33:67. The degree of proteolysis over time was affected by the coagulant type. Proteolysis was thought to be the main cause of changes in functional properties of Cheddar cheeses. The meltability and hardness of cheese made with 100% C. parasitica enzyme was the highest, but it was high in bitterness. The chymosin-to-C. parasitica ratio between 0:1 to 67:33 was found suitable to independently control Cheddar cheese meltability and hardness without a significant level of bitterness.     

Acceleration of Edam cheese ripening using acid fungal protease

Edam cheese was made from a mixture of cow/goat milk (1:1, v/v). The mixture of both milks was standardized to have 3.2% fat and a casein/fat ratio of 0.7, heated at 72 °C for 15 s, immediately cooled to 40 °C and divided into four portions. Acid fungal protease (Formase 200) was added at levels of 0.5, 1.0 and 2.0 g/kg of curd to the first three portions, and the last one was manufactured without the addition of acid fungal protease to serve as a control sample. The samples of control as well as those of different treatments were analysed for chemical and organoleptic properties when fresh and after 1, 2 and 3 months of storage at 12 ± 1 °C. The obtained results pointed out that the samples of Formase-treated-cheese exhibited higher values of soluble nitrogen/total nitrogen ratio (SN/TN), total volatile fatty acids (TVFA), titratable acidity (TA), and the flavour was developed faster as compared with the samples of control. A good quality Edam cheese could be produced with a high acceptability when Formase 200 was used at level of 1.0 and 2.0 g/kg of curd and the cheese was ripened for 2 months only.     

Effect of high-pressure treatment and a bacteriocin-producing lactic culture on the proteolysis, texture, and taste of Hispánico cheese

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12°C, half of the cheeses from each vat were treated at 400 MPa for 5 min at 10°C. Ripening of high-pressure-treated and untreated cheeses continued at 12°C until d 50. High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese. Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity.     

Hydrolysis of beta-casein and peptides by intracellular neutral protease of Streptococcus diacetylactis

The endopeptidase activity of mesophilic streptococci was characterized further by investigating the specificity of an intracellular endopeptidase from Streptococcus diacetylactis for beta-casein, derived peptides, and bradykinin. The inhibitory action of phosphoramidon as well as direct determinations of metal content showed this enzyme was a metalloprotein. Hydrolysis of native beta-casein was relatively low. Peptides obtained from the fraction soluble at pH 4.6 led to the demonstration that Pro186-Ile187 and Ala189-Phe190 were hydrolyzed by the enzyme. Two peptides derived from beta-casein by the action of chymosin were hydrolyzed efficiently: we observed hydrolysis of Lys176-Ala177, Lys169-Val170, and Pro206-Ile207. The Pro7-Phe8 bond of bradykinin was hydrolyzed rapidly, showing that this enzyme was efficient for the hydrolysis of prolyl peptide bonds. The protease was slightly less sensitive to phosphoramidon than was thermolysin. Metal analyses showed the enzyme contained 580 microgram of zinc and 4,760 microgram of calcium per gram protein. This protease is thus a true metalloenzyme (E.C.3.4.24.4), and its action may complete the hydrolysis initiated by chymosin remaining active in cheese curd by hydrolyzing peptides released by chymosin.