Variation in activity and heterogeneity of bovine milk proteases with stage of lactation and somatic cell count

Samples of prepartum secretion, colostrum and mature milk were analysed for variation in activity of cathepsin D and cysteine protease due to lactation stage and somatic cell count (SCC). In spite of considerable variation between animals, cathepsin D activity was found to be highest in prepartum secretion, lowest in colostrum and at intermediate levels in milk throughout lactation. Levels of cysteine protease activities were highest 1 week postpartum and lowest in early/mid-lactation milk. The activity of an unidentified milk protease with specificity different from cathepsin D and cysteine proteases was also detected in milk, but had very low activity in prepartum secretions and colostrum. By immunoblotting, prepartum secretion was found to contain both cathepsin D and procathepsin D, while only procathepsin D could be detected in colostrum and milk. Cathepsin D activity was positively correlated with SCC, and immunoblotting indicated that the elevated activity derived from cathepsin D was due to increased level of immunoreactive procathepsin D in high SCC milk, and not to increased levels of mature cathepsin D. This indicates that activation of procathepsin D does not occur at elevated SCC.     

Heterogeneity of proteolytic enzyme activities in milk samples of different somatic cell count

Milk contains the alkaline proteinase plasmin and lysosomal proteinases; the significance of the latter is ill-defined. The objective of this study was to investigate composition and activities of several different proteolytic enzymes in milk samples of varying somatic cell count (SCC). Increasing milk SCC was correlated with increased plasmin, cathepsin D and cysteine protease activities, with concomitant increases in proteolysis in milk. Addition of plasmin inhibitors confirmed the heterogeneity of proteinase activities in milk, as urea-PAGE analysis of milk samples showed casein hydrolysis in milk after 7 d storage even in samples with inhibitors added; extent and heterogeneity of proteolysis was correlated with milk SCC. Rennet coagulation properties were not significantly correlated with SCC, or activities of measured enzymes. Milk of increasing SCC also exhibited decreased physical stability during incubation of milk at 37 degrees C. Pasteurized milk was more stable than raw milk, suggesting that the enzyme(s) or mechanisms leading to such instability are impaired by pasteurization. Overall, milk has a very heterogeneous proteolytic enzyme population, with a higher significance of non-plasmin enzymes, such as cathepsin D and cysteine proteinases, than perhaps previously recognised.     

Proteolysis of milk proteins lactosylated in model systems

Five different milk proteins (alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin, and lactoferrin) and a peptide substrate were applied as substrates for the investigation of how lactosylation affected proteolysis by different proteases. After a lactosylation period of 4 days in aqueous solution, at 65 degrees C and pH 6.8 in a protein: lactose ratio of 1000 the proteins were enzymatically hydrolyzed by the three milk relevant proteases plasmin, cathepsin D, and chymosin. Lactosylation of all substrates affected hydrolysis by plasmin negatively, with the largest effect on the globular proteins. This could be explained by modification of lysine residues, being the preferred cleavage site for plasmin, but also the residue generally preferred for lactosylation. Lactosylation of the caseins and of beta-lactoglobulin did not affect subsequent cleavage by cathepsin D and chymosin significantly, but for beta-lactoglobulin, both the secondary as well as the tertiary structure were affected by lactosylation. In contrast, decreased hydrolysis by cathepsin D and chymosin was observed for lactoferrin after lactosylation. Decreased hydrolysis may be caused by a more compact tertiary structure induced by lactosylation of lactoferrin, as indicated by fluorescence spectroscopy measurements.     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

Cysteine protease activity in bovine milk

Proteolytic activity of native cysteine proteases was studied in bovine milk. Five fractions (fI–fV) with cysteine protease activity were separated from acid whey prepared from raw bovine milk by ion-exchange chromatography on Q-Sepharose. The hydrolytic action of the most active fractions (fIII and fV), after further purification using gel permeation chromatography on Superdex S75, was studied against individual caseins. The two fractions contained different cysteine protease activities capable of hydrolyzing both α_s1- and β-casein. Studies of the effects of different reagents on the activity of partially purified fIII showed that the activity in this fraction was unaffected by aprotinin, slightly inhibited by p-chloromercuribenzoate and o-phenanthroline and completely inhibited by -trans-epoxysuccinyl- -leucylamido (4-guanidino) butane, consistent with identification as a cysteine protease. Phenylmethylsulphonyl fluoride and pepstatin A reduced activity of fIII by 40% and 50%, respectively. The partially purified fIII retained 20% of its cysteine protease activity after heating at 55°C, 60°C, 65°C and 72°C for 40 min, 20 min, 10 min and 30 s, respectively. Immunoblotting of fIII with antibodies to the bovine lysosomal cysteine protease, cathepsin B, clearly indicated the presence of immunoreactive cathepsin B in this fraction. This study presents strong evidence for the presence of a heterogeneous group of cysteine proteases in bovine milk, with one of these enzymes probably being cathepsin B.     

Characteristic Property of Low Bitterness in Protein Hydrolysates by a Novel Soybean Protease D3

ABSTRACT:  Enzymatic hydrolysis is 1 means of improving the functional properties of food protein; however, in most cases, bitter peptides are generated by such treatment, and the resulting product is therefore not acceptable as a food ingredient. We have already reported a novel cysteine protease, D3, purified from germinating soybean cotyledons. Because of its substrate specificities, most hydrophobic amino acid residues in the hydrolysate are presumed not to be located at the peptide termini. It was therefore expected that protein hydrolysate by protease D3 would taste less bitter than other enzymatic hydrolysates. The objective of this study was to demonstrate the low bitterness of protein hydrolysates by protease D3. For that purpose, soy protein and casein hydrolysates were prepared with treatment of protease D3, subtilisin, pepsin, trypsin, and thermolysin, respectively. The bitterness of these hydrolysates was evaluated by measuring points of subjective equality (PSE). The PSE value demonstrated that the protein hydrolysates by protease D3 were significantly less bitter than the other enzymatic hydrolysates, indicating that the products had a taste mild enough to be acceptable as a less-bitter peptide food ingredient. These results suggested that a prominent feature of protease D3 was its capacity to produce less-bitter peptides. Therefore, it is thought that protease D3 could be applied to produce protein hydrolysates for use as ingredients in a variety of food products.     

Cysteine protease inhibitors in various milk preparations and its importance as a food

The content of cysteine-protease inhibitors in milk preparations were reported and their contributions to bacterial growth inhibition and protection of bone resorption were discussed. Mammalian milk contains large amount of cysteine protease inhibitors, such as lactoferrin, β-casein and cystatins. Lactoferrin content in human-milk was much higher than that in cow-milk and the papain inhibition of human-milk was stronger than that of cow-milk. Since lactoferrin content in colostrum was about two to three times higher than that in mature-milk, both of the cysteine-protease inhibition and the bacterial growth inhibition by colostrum were much stronger than that by mature-milk. β-Casein content in colostrum was much lower than that in mature-milk. Lactoferrin showed strong inhibition to papain and cathepsin L, but urease to cathepsin B and H. The papain inhibition by lactoferrin was lost by 70 °C heat-treatment. Since cathepsin L inhibitors suppress bone collagen degradation, content of lactoferrin in milk is important to protect bone resorption. Lactoferrin inhibited growth of Streptococcus epidermidis, furthermore, the active site of cysteine-protease inhibition, synthesized peptide Y₆₇₉–K₆₉₅, of lactoferrin, also showed the growth inhibition.     

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.     

鱿鱼肝脏蛋白酶的鉴定及组织蛋白酶L的分离纯化

鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究。反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl?fluoride,PMSF)后,肌球蛋白重链(myosin?heavy?chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶。半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物。利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L。最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25?kD单一条带。     

The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D–spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC–tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D–spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.     

酪蛋白源降胆固醇肽的酶解制备工艺优化

以酪蛋白为原料,采用中性蛋白酶、碱性蛋白酶以及胰蛋白酶对酪蛋白进行水解,确定制备降胆固醇肽的最佳蛋白酶;通过单因素实验和响应面试验,研究水解pH、水解温度、酶与底物比、底物浓度和水解时间对酪蛋白水解度和胆固醇胶束溶解度抑制率的影响,确定最佳水解条件;而后通过超滤和凝胶过滤层析确定降胆固醇肽的初步分离工艺。结果表明:制备酪蛋白源降胆固醇肽的最佳水解工具酶是中性蛋白酶,其最佳酶解条件为反应温度51.3 ℃,酶与底物浓度比6.47%,pH6.34,底物浓度5 g/100 mL,反应时间3.5 h,胆固醇抑制率为58.25%±0.59%;Sephadex G-10分离酪蛋白降胆固醇肽条件为上样浓度80 mg/mL,上样体积2.5 mL,洗脱速度3.5 mL/min;经酶解、超滤及层析后制备的酪蛋白源降胆固醇肽峰1和峰2样品在100 μg/mL的胆固醇溶解度抑制率为24.2%±0.24%和4.3%±0.16%。经酶解制备分离后,获得具有抑制降固醇胶束溶解活性的降胆固醇肽,为降胆固醇肽的开发提供理论研究基础。     

酸枣仁ACE抑制肽酶解工艺优化

本试验以脱脂后的酸枣仁渣通过碱溶酸沉法提取得到的酸枣仁蛋白为研究对象,以血管紧张素转化酶(ACE)抑制率和水解度为指标,筛选复合酶种类,采用响应面分析法,以中性蛋白酶/碱性蛋白酶比例、pH、底物浓度、酶解温度、酶解时间为试验因素,优化酸枣仁ACE抑制肽最佳酶解工艺参数。结果表明:筛选出中性蛋白酶和碱性蛋白酶作为复合酶,最适酶添加量确定为6000 U/g,5个因素对ACE抑制率和水解度的影响由大到小的顺序为:酶解温度、酶解时间、pH、中性蛋白酶/碱性蛋白酶比例、底物浓度。通过拟合方程分析,得到酸枣仁ACE抑制肽酶解的最佳工艺条件为:中性蛋白酶/碱性蛋白酶比例为2.1:1、酶解温度为54 ℃,底物浓度为3.1%,pH为7.5,酶解时间为62 min。在此条件下,复合酶解酸枣仁蛋白酶解液的实际ACE抑制率和水解度分别为（79.46%±0.49%）和（31.45%±0.85%）,与理论值接近。制备得到酸枣仁ACE抑制肽与阳性对照组卡托普利对比,酸枣仁ACE抑制肽的ACE抑制率大小为（79.46%±0.49%）,与卡托普利的ACE抑制率偏差为（19.28%±0.12%）,证明酸枣仁ACE抑制肽具有显著降压效果。本研究证明了酸枣仁蛋白通过酶解有效得到ACE抑制肽并优化其酶解工艺,旨在为酸枣仁渣废物再利用提供参考方向和理论依据。     

Role of indigenous enzymes in proteolysis of casein in caprine milk

Bulk samples of caprine milk were characterized for chemical composition, enzyme activities and rheological properties; plasmin, elastase, and cathepsin D concentrations were measured as 3.22 ± 0.18, 1.14 ± 0.05, and 1.81 ± 0.06 mg L^?1, respectively. Pasteurized caprine milk was incubated with aprotinin, pepstatin, or a mix of these inhibitors at 37 °C for 7 days. Hydrolysis of α- and β-caseins was influenced by the presence of inhibitors: overall serine proteases, i.e., plasmin and possibly elastase mainly contributed to the hydrolysis of caseins whereas the limited proteolysis observed in milk incubated with aprotinin suggested a marginal role for cathepsin D. Pasteurized milk displayed a greater number of peptides than milk incubated with pepstatin, whereas no peptides were detected in samples incubated with aprotinin or a mix of aprotinin and pepstatin. Several unreported peptides were identified by mass spectrometry in caprine milk, many of which showed sequences previously described as bioactive in bovine and caprine milk.     

牦牛乳酪蛋白肽制备及乙酰化脱苦

采用碱性蛋白酶对牦牛乳酪蛋白进行水解,得到牦牛乳酪蛋白肽。以醋酸酐为酰化试剂,利用结构修饰的方法对牦牛乳酪蛋白肽进行脱苦研究,探讨醋酸酐添加量、pH对牦牛乳酪蛋白肽酰化度及苦味的影响。结果显示,碱性蛋白酶最佳水解条件为,温度55℃,pH7.0,酶添加量1.25%,反应3h。维持酰化反应pH7以上,产物乙酰化程度最高,醋酸酐的添加量与脱苦效果呈正比,当醋酸酐添加量为40%时,牦牛乳酪蛋白肽酰化产物的苦味基本消失。      

海参肠组织蛋白酶D的提取及酶学特性研究

本研究以海参肠为原料,采用p H3.0 50 mmol/L甘氨酸-盐酸缓冲体系,按1∶6（w/v）的料液比,在4℃浸提1 h,获得海参肠组织蛋白酶D的粗酶液。以酸变性牛血红蛋白为底物,进行酶活测定,并研究了该酶的酶学性质。结果表明,海参肠组织蛋白酶D粗酶的最适p H为3.0,在p H3.0⁷.0之间稳定性较好;最适反应温度为50℃,在4⁴0℃具有较高稳定性。5 mmol/L的金属离子Mg²⁺、Ca²⁺、Ni²⁺、Zn²⁺、Cd²⁺和K⁺对该酶有激活作用,而Fe³⁺和Fe²⁺则对其有抑制作用。天冬氨酸蛋白酶抑制剂Pepstatin A对该酶活性有较强的抑制作用。上述结果说明,在本研究条件下提取获得的海参肠组织蛋白酶D粗酶是一种酸性蛋白酶,其组成以天冬氨酸蛋白酶为主。      

酶解脱脂蚕蛹蛋白制备ACE抑制肽

以实验室自制的脱脂蚕蛹蛋白为原料,利用酶工程技术,通过对中性蛋白酶、碱性蛋白酶、木瓜蛋白酶、复合蛋白酶、风味蛋白酶、胰蛋白酶等的筛选及单因素和响应面优化试验,对ACE抑制肽的制备工艺条件进行较系统的研究。结果表明：选择碱性蛋白酶作为脱脂蚕蛹蛋白制备ACE抑制肽的酶,制备ACE抑制肽的最佳工艺条件为料液比11.88:100、温度50.22℃、pH 9.46、加酶量7.03%、酶解4h。在此条件下制备的ACE抑制肽的ACE抑制率达到41.98%。     

Production and Characterization of an Enzymatic Hydrolysate of Skim Milk Lactose and Proteins

Enzymatic hydrolysis of skim milk lactose and proteins was investigated in a batch reactor; the final aim is to produce a predigested dietary product. The use of yeast β-galactosidase, a vegetable protease, a fungal protease, and a bacterial protease was investigated. Sequential and simultaneous lactose and protein hydrolysis were studied in order to diminish incubation times. In the lactose hydrolysis, 90% conversion was obtained after 4 hr using reconstituted spray-dried skim milk, and after 3 hr using fluid pasteurized skim milk. In the simultaneous hydrolysis, 82% lactose hydrolysis and a substantial peptide hydrolysis with 80% of material smaller than 5,000 molecular weight (and high in small peptides) was obtained after 5 hr. This was adequate for the preparation of a specialized dietary product to be used in enteral hyperfeeding.     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

羊乳高F值肽酶解工艺优化

目的 探究通过生物酶解技术有效得到羊乳高F值肽的关键工艺.方法 以新鲜山羊乳为原料,采用两种酶分步水解法制备高F值肽,以蛋白质水解度和F值为指标,通过单因素试验和响应面分析法分别确定两步酶解的最佳条件,酶解液经活性炭静态吸附去除游离芳香族氨基酸,对脱去芳香族氨基酸后的酶解液进行氨基酸组成分析并测定F值.结果 水解羊乳蛋...     

山羊乳酪蛋白酶解工艺及抗氧化性研究

为了制备山羊乳酪蛋白活性肽,选用中性蛋白酶、胰蛋白酶、木瓜蛋白酶和碱性蛋白酶,采用对比和正交试验方法,研究了山羊乳酪蛋白单酶和复合酶的酶解工艺,测定了山羊乳酪蛋白的总肽键摩尔数,优选出了山羊乳酪蛋白的酶解工艺参数。结果表明：山羊乳酪蛋白的总肽键摩尔数为8.5379 mmol/g。单酶中胰蛋白酶和中性蛋白酶对山羊乳酪蛋白的水解度较大,木瓜蛋白酶较小。胰蛋白酶对山羊乳酪蛋白适宜的酶解工艺：加酶量2500 U/g,pH 7.5,50 ℃下酶解2 h。中性蛋白酶和胰蛋白酶复合以及中性蛋白酶、胰蛋白酶和木瓜蛋白酶三种酶复合的水解度较大,其水解度、平均肽链长度和平均相对分子量分别为17.34%、21.16%,5.77、4.73和692、567 u。