Purification of X-prolyl dipeptidyl aminopeptidase from Lactobacillus casei subspecies.

Prolyl dipeptidylaminopeptidases from two subspecies of Lactobacillus casei were purified and biochemically characterized. L. casei ssp. casei UL21 (a debittering strain) and L. casei ssp. rhamnosus UL26 (a non-debittering strain) were the source bacteria for this study. Purification of the enzymes from both the sources was effected by a gel filtration step through Sephacryl S-300 followed by ion-exchange chromatography through DEAE Sephacel. This rendered an electrophoretically homogeneous enzyme preparation. The purified enzymes from both the sources showed similar temperature optimum (45 degrees C) and pH optimum (7.0). Their activity profiles on various substrates and the nature of inhibition by different inhibitors were also found to be similar, indicating that this enzyme is perhaps not significantly involved in the debittering process during the maturation of cheese.     

Hydrolysis of β-casein (193-209) Fragment by Whole Cells and Fractions of Lactobacillus casei and Lactococcus lactis

Whole cells and fractions of Lactococcus lactis subsp. lactis IFPL 359 and Lactobacillus casei subsp. casei IFPL731 were studied. Hydrolysis products were separated by reversed-phase, high-performance liquid chromatography (RPHPLC). Under conditions, pH 5.2 and 3% NaCl, L. casei IFPL 731 was more active in hydrolysis of the b-casein (f193-209) peptide than was L. lactis IFPL 359. This hydrolyzing activity was attributed for L. casei IFPL 731 by the cell-wall proteinase. Hydrolysis of the peptide by the intracellular extract of L. casei IFPL731 was mainly located in the fraction that contained endopeptidase and Pep N aminopeptidase activities. Results may help provide approaches and treatments to control bitterness in cheese products.     

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

干酪乳杆菌胞壁蛋白酶的分离及水解酪蛋白产物特性

通过DEAE-Sephadex A-25和Sephadex G-100对干酪乳杆菌胞壁蛋白酶粗提物进行分离纯化,分离到酶比活力为12.50U/mg的CEP-1与16.67U/mg的CEP-2两种胞壁蛋白酶。CEP-1提纯倍数达到50,回收率为33.61%;CEP-2提纯倍数为66.68,回收率55.17%。对不同时间和底物浓度下CEP-1与CEP-2的α-酪蛋白和β-酪蛋白水解特性进行研究。结果显示,酪蛋白水解产物有显著的抗ACE与抗氧化活性,产生最高ACE抑制活性的水解条件为：CEP-2水解α-酪蛋白,时间6h,酶与底物质量比1:10,此时ACE抑制活性为84.66%;在酶与底物质量比1:40,水解时间4h条件下,CEP-2水解β-酪蛋白产生最佳的O2－ ·清除能力,其IC50为0.2138mg/mL。     

不同干酪乳杆菌菌株对α-葡萄糖苷酶抑制作用的比较

从14株干酪乳杆菌中筛选出11株具有凝乳作用的菌株,采用以麦芽糖酶为靶向酶的α-葡萄糖苷酶抑制剂体外筛选模型,分析了不同干酪乳杆菌发酵脱脂乳上清对α-葡萄糖苷酶的抑制作用。结果表明,不同菌株发酵脱脂乳上清对麦芽糖酶的抑制活性具有明显的菌株特异性,且随着发酵时间延长,α-葡萄糖苷酶抑制活性呈现上升趋势。其中,干酪乳杆菌LC2W在37℃发酵10%(w/w)脱脂乳96 h后所得的上清液对麦芽糖酶抑制活性最高,达37.36%,明显高于其它被测试的干酪乳杆菌菌株,表明该菌株具有潜在的抗高血糖作用。     

Nucleotide sequencing,purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA

An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.     

Purification and characterization of extracellular caseinolytic enzyme of Micrococcus sp. MCC-315 isolated from cheddar cheese

Micrococcus sp. MCC-315, an organism isolated from Cheddar cheese, produced an extracellular calcium metalloenzyme. This protease was purified to homogeneity from culture supernatant by precipitation with ammonium sulfate (50 to 70% saturation) and gel filtration through Sephadex G-100, resulting in about 82 times increase of specific activity and 53% recovery of the enzyme. The protease exhibited a pH optimum at 10.6 for both whole casein and beta-casein. It had optimum activity for whole casein in the presence and absence of calcium++ at 60 and 50 degrees C, respectively, and at 37 to 40 degrees C for beta-casein with or without calcium++. The enzyme was stable at 45 degrees C but lost activity at higher temperatures. It was inhibited by heavy metal ions but calcium++, cobalt++, manganese++, strontium++, and iron++ had a slight stimulatory effect. The enzyme was inhibited completely and irreversibly by metal chelating agents. Calcium ions were required for maintenance of an active conformation of the enzyme. The enzyme had molecular weight of 28,900 and Michaelis constants 6.66 and 5.00 mg/ml for whole casein and beta-casein. Amino acid analysis of the hydrolyzed enzyme revealed the absence of sulfhydryl groups as was indicated also by lack of inhibition by thiol reagents.     

干酪乳杆菌发酵塑性凝块奶酪的研究

采用嗜温菌——干酪乳杆菌(Lactobacillus casei subsp.casei)作为产酸发酵剂,应用于塑性凝块奶酪,并对其发酵性能进行了研究。通过对干酪乳杆菌(L.casei subsp.casei)、保加利亚乳杆菌(Lactobacillus del—brueckii subsp.bulgaricus)和嗜热链球菌(Streptococcus thermophillus)在乳中发酵产酸性能及凝乳时凝胶的保水力进行比较,结果表明,干酪乳杆菌在脱脂乳中发酵产酸能力居中,持水力接近嗜热链球菌,且所得凝乳风味柔和,组织结构良好。采用SAS实验设计,以接种量、钙盐添加量、热缩温度为三因素进行试验,以成品得率、乳清排出率及感官综合评分为响应值,进行综合评价,结果表明,当发酵剂添加量4%,钙盐量0.04%,热缩温度55℃时,产品质量达到最优。验证试验表明,产品的乳清排出率为59.2%,成品得率为11.88%,感官评分为18.33。通过与进口Mozzarella的主要成分和融化前后产品的整体评价对比可知,产品的各项主要指标与进口产品接近,且风味更加温和。     

Purification and characterisation of cathepsin D from the digestive gland of the pelagic squid Todarodes sagittatus

A proteinase from the digestive gland of squid (Todarodes sagittatus) was purified from a neutral extract by ammonium sulphate fractionation, S-Sepharose chromatography and gel filtration on Sephadex G-75. Inhibition by pepstatin showed that the enzyme is an aspartic proteinase. The enzyme is fairly stable in the pH range 2.5-7, and optimum pH for haemoglobin digestion is 3.7. Combined results from gel filtration and sodium dodecyl sulphate electrophoresis indicate a molecular weight of 38 kDa, and that the enzyme consists of two different subunits with molecular weights approximately 10 and 28 kDa. Analytical electrofocus-ing showed an enzyme pi of 8.5. As judged from the physical properties and the amino acid composition, it is concluded that the enzyme is a cathepsin D. This enzyme probably counts for the major part of proteinase activity in the digestive gland.     

Production and evaluation of a probiotic yogurt using Lactobacillus casei ssp. casei

Skimmed milk was inoculated with the commercial starter and Lactobacillus casei ssp. casei. pH changes, viable counts, and organoleptic properties of the produced control and probiotic yogurts were analysed. The pH decrease during the fermentation period was faster in the milk inoculated with L. casei plus starter. The growth of both starters in probiotic yogurt was significantly lower than their growth in control yogurt during the fermentation period. The viable count of the probiotic bacterium remained higher than the standard limit for probiotic products. There was no significant difference between the organoleptic properties of the control and the probiotic yogurts.     

干酪乳杆菌LC2W对两种不同胃癌细胞的黏附作用

选取人胃癌细胞株MKN-45和HGC-27,采用形态学观察和显微计数的方法比较菌液浓度、悬浮液、pH值对干酪乳杆菌LC2W(Lactobacillus casei LC2W)黏附细胞的情况。结果表明,LC2W对两种细胞均具有黏附性,对MKN-45的黏附性更强;这种黏附作用具有菌悬液浓度依赖性。当加入的菌悬液浓度达到一定数量时(109 mL-1),平均每个细胞黏附的乳杆菌数量趋于饱和;悬浮介质对Lb.caseiLC2W的黏附性具有明显的影响,其中悬浮于耗尽培养上清液(SCS)的Lb.casei LC2W表现出最好的黏附性;悬浮介质的pH值对Lb.casei LC2W的黏附性也有影响,pH=4.0时黏附性最好。将不同悬浮介质的pH值调整到pH=7.0后,SCS仍能很好的促进LC2W的黏附作用,表明SCS中存在某种促进黏附的物质。由此推测,Lb.casei LC2W对胃癌细胞的黏附机理可能为黏附素与受体的特异性结合。     

Peptide hydrolases of Lactobacillus casei: isolation and general properties of various peptidase activities.

Discovery of an endopeptidase by gel chromatography and separation of 3 exopeptidases (a dipeptidase, an aminopeptidase and a specific carboxypeptidase) from Lactobacillus casei NCDO 151 by affinity chromatography is described. The 3 exopeptidases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline but were reactivated with Co2+ and Mn2+. The pH optima for aminopeptidase, dipeptidase and carboxypeptidase activities were 6.5, 7.6 and 7.2, respectively. Maximum activity was obtained at 45 degrees C for the aminopeptidase, at 30 degrees C for the dipeptidase and at 40 degrees C for the carboxypeptidase. The substrate specificities of the 3 enzymes were also studied. The properties of these 3 enzymes are compared with those of other bacteria.     

利用Lactobacillus casei Zhang开发益生菌新鲜干酪

通过添加不同比例Lactobacillus casei Zhang发酵剂制作新鲜干酪,并对其在新鲜干酪中的活力和添加Lactobacillus后新鲜干酪的理化性质进行了研究。结果表明,Lb.casei Zhang在新鲜干酪中具有较高的活力,添加2%、1%、0.5% Lb.casei Zhang发酵剂的干酪中,4℃冷藏开始前,Lb.casei Zhang活菌数分别为2.24×10~8 cfu/g,1.38×10~8 cfu/g,5.55×10~7 cfu/g,4℃冷藏28d后,Lb.casei Zhang存活率分别为99.12%,98.31%,98.61%。在制作过程中和4℃冷藏过程中,与空白组相比,添加Lb.casei Zhang对新鲜干酪的pH值、滴定酸度、蛋白水解活性影响都不显著(P>0.05)。     

干酪乳杆菌细菌素的抗菌机制分析

目的：分析和预测干酪乳杆菌细菌素基因及其编码蛋白的分子结构和理化性质,明确细菌素的抗菌机制。方法：利用生物信息学软件进行细菌素编码蛋白的结构预测和功能分析,采用氨基酸定点突变技术对预测的氨基酸位点进行点突变,检测突变体的抗菌活性。结果：干酪乳杆菌细菌素（LacA和LacB）属于热稳定、分泌信号肽的跨膜蛋白,LacA蛋白存在典型的抗菌结构域GxxxG,构建LacA蛋白中第40位V和第57位I的突变载体,两个位点中任何一个发生突变都明显影响细菌素的抗菌活性。结论：推测V40和I57是干酪乳杆菌细菌素发挥抑菌活性的关键氨基酸位点,为今后探索干酪乳杆菌细菌素（class IIb）抗菌机制提供理论依据。     

Factors affecting the caseinolytic activity of Lactobacillus casei and Lactobacillus plantarum

Whole cell suspensions of some strains of each Lactobacillus casei and Lactobacillus plantarum were assayed for their caseinolytic activity in 0.1 M NaH₂PO₄ buffer, pH 6.5, at 30 °C, using different assay methods. Azocasein was not as sensitive as casein (Hammarsten) as a substrate. Inclusion of glucose in the assay mixture reduced the released α-amino groups as evidenced by fluorescent labelling, but generally increased the amounts of excreted amino acids. Divalent cations, including calcium ions, played only a minor role in the activation of the cell-bound proteinase, whereas NaCl inhibited it markedly. Inhibitor studies suggest that the enzyme is a serine proteinase. The different assay methods used did not give identical results. Fluorescent labelling of the free α-amino groups at pH 6.0 appears, on the contrary, to be a more reliable method.     

干酪乳杆菌胞外多糖LCP1的流变性研究

通过对干酪乳杆菌LC2W胞外多糖LCP1溶液流变性质研究发现,高浓度LCP1溶液呈剪切变稀,低浓度时,剪切速率对黏度没有影响,溶液黏度随温度升高下降明显,pH对LCP1溶液黏度影响很小,盐可以降低LCP1溶液黏度,但黏度降低与盐浓度有关,通过黏弹性研究发现LCP1水溶液不能形成胶体,乌氏黏度计测得LCP1水溶液固有黏度[η]为1.930 dL/g。     

The cell-bound proteinase system of Lactobacillus casei--purification and characterization

The cell-wall crude extract from Lactobacillus casei NCDO 151 was partially purified by DEAE-Sephacel chromatography. Three active fractions were eluted. Two major peaks (eluted with 0.05 M and 0.27 M phosphate buffer) were further investigated. Peak I represented enzymatic activity with an optimum temperature of 40 degrees C, an optimum pH of 7.0 and was strongly inhibited by the serine proteinase inhibitor phenylmethylsulfonylfluoride. Peak II represented an enzymatic activity with an optimum temperature of 45 degrees C, an optimum pH of 7.5 and was totally inhibited by p-hydroxymercuribenzoate. None of the enzymes was affected by the metal chelator ethylenediaminetetraacetic acid at a concentration up to 1 x 10(-2).     

NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation

ABSTRACT:  Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 °C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca²⁺ activated the enzyme activity but reduced enzyme stability at 65 °C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.     

Purification and characterization of a beta-galactosidase from Fusarium oxysporum var. lini

Beta-D-Galactosidase was purified from a cellular extract of Fusarium oxysporum var. lini by heat shock and successive chromatography on DEAE-cellulose DE-52 and Sephadex G-100. The purified enzyme was homogeneous on SDS gel electrophoresis. It was inhibited by divalent cations such as Zn++, Mg++, and Ca++. The Michaelis constant and maximum velocity values for o-nitrophenyl beta-D-galacto-pyranoside were 6.76 mM and 816.7 mumol X mg protein-1 X min-1. The isoelectric point was 3.83, and the optimal pH and temperature were 5.0 and 55 degrees C. The estimated molecular weight of the enzyme was 224,000 by gel filtration and 36,300 by SDS-PAGE. The enzyme was considered a hexamer. o-Nitrophenyl-beta-D-galacto-pyranoside hydrolysis was activated by lactose, suggesting an allosteric nature of the enzyme.     

Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus thermophilus,Lactobacillus acidophilus,bifidobacteria, Lactobacillus casei,Lactobacillus rhamnosus,and propionibacteria

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.