Purification and characterization of a dipeptidase from Lactobacillus delbrueckii subsp. bulgaricus

A metal-dependent dipeptidase has been purified from a cell-free extract of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography, metal chelating affinity chromatography with immobilized Cu²⁺, and repeated FPLC anion exchange chromatography. The molecular mass of the purified enzyme was estimated to be 51 kD by SDS-polyacrylamide gel electrophoresis as well as by gel filtration, which indicates that it does not consist of subunits. The enzyme was most active at pH 7 and 50°C. Reducing agents, like dithiothreitol and β-mercaptoethanol, increased enzyme activity while metal chelating agents had an inhibitory effect. Enzyme activity, inhibited by EDTA and EGTA, could be partially restored by Co²⁺ and Mn²⁺. The enzyme was most active on dipeptides containing an aminoterminal hydrophobic amino acid such as Leu-Leu and Leu-Gly. Kinetic studies indicated that the dipeptidase had a higher affinity for the first substrate mentioned. The K_m-values for both substrates were about 0·56 and 1·23 mM, with turnover numbers of 870 and 480 s⁻¹, respectively.     

Purification and characterization of a second aminopeptidase (pepC-like) from Lactobacillus delbrueckii subsp. bulgaricus B14

A second aminopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation and two steps of anion-exchange chromatography. After SDS polyacrylamide-gel electrophoresis in the presence of β-mercaptoethanol, one protein band was detected at 54 kDa. The same molecular mass was estimated by gel filtration. SDS polyacrylamide-gel electrophoresis in the absence of β-mercaptoethanol resulted in a single band at 220 kDa, indicating that the enzyme forms complexes of four molecules under non-reducing conditions. Activity was markedly increased by reducing and metal-chelating agents. Thiol-group inhibitors, such as iodoacetic acid, inhibited the enzyme strongly. In contrast to Mg²⁺ and Ca²⁺, which had slightly activating effects, other divalent cations reduced enzyme activity at a concentration of 1 mM. The aminopeptidase showed highest activity at 50°C and pH 6·5–7 and hydrolyzed a wide range of di- and tripeptides. The most suitable substrates were Leu-Gly, Leu-Gly-Gly, Ala-Ala-Ala, and Met-Gly-Gly. For Leu-Gly and Leu-Gly-Gly, K_m-values of 1·81 mM and 2·17 mM and turnover numbers of 870 s⁻¹ were calculated, with a maximal rate of hydrolysis (V_max) of 4600 and 2780 μmol/min per mg of protein, respectively. The aminopeptidase did not cleave Lys-pNA, a substrate hydrolyzed by all type-‘N’ aminopeptidases from lactic acid bacteria with high velocities. It compared well, however, with pepC found in Lactococcus.     

Phosphate group requirement for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus

The mitogenic activity of extracellular polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 and NCFB2483 was examined in murine lymphocytes. The extracellular polysaccharide from Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 was fractionated into neutral and acidic polysaccharides by anion-exchange chromatography, while that of Lactobacillus delbrueckii ssp. bulgaricus NCFB2483 were all fractionated into neutral polysaccharide(s). The acidic polysaccharide stimulated mitogenic responses of murine splenocytes and Peyer's patches but not of thymocytes. The optimal concentration of the acidic polysaccharide at the highest stimulation was 160 μg/ml. A significant increase of mitogenic activity was initiated at 24 h, and the highest response was obtained after stimulation for 48 h. The acidic polysaccharide purified by high performance liquid chromatography also had substantial mitogenic activity, and the molecular weight was estimated to be 1.2×10⁶. The acidic polysaccharide was a phosphopolysaccharide consisting of glucose, galactose and phosphorus. Dephosphorylation by hydrofluoric acid degradation reduced the mitogenic activity in lymphocytes. The phosphopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus 1073R-1 is a potent B-cell-dependent mitogen in which the phosphate group acts as a trigger of the mitogenic induction.     

氮源对德氏乳杆菌保加利亚亚种B61-3耐冻干能力的影响

本研究针对德氏乳杆菌保加利亚亚种B61-3,分别测定了其对16种氮源的利用情况,筛选出能够提高菌株冻干耐受的氮源种类及添加量,并进一步测定该氮源对菌株冻干粉发酵活力、菌体形态、发酵关键酶活力的影响。结果表明：氮源为30 g/L牛骨蛋白胨时可提高菌株冻干存活率及冻干粉发酵活力,冻干存活率由9.68%提高到18.90%,冻干粉平均发酵活力较对照组提高22.15%;电子显微镜显示,牛骨蛋白胨培养后菌体大小及形态有所改变,对照组发酵菌体表现出不规则、卷曲的形态且菌体较长,而牛骨蛋白胨为氮源时菌体形态呈表面光滑的短杆状,菌体长径比(l/d)和面积体积比(S/V)显著降低(P<0.05),可能与菌株更高的存活率和发酵活力有关。发酵关键酶活力测定结果表明,冷冻干燥显著降低菌株酶活力(P<0.05),牛骨蛋白胨培养菌株冻干后胞内酶活力均显著高于对照组(P<0.05),乳酸脱氢酶、Na⁺K⁺-ATP酶及β-半乳糖苷酶分别较对照组提高1.30、1.52及2.75倍,且胞外β-半乳糖苷酶活性低于对照组。结果证实,牛骨蛋白胨培养B61-3菌体能够抵...     

Purification and properties of an acid phosphatase from Lactobacillus curvatus DPC2024

An acid phosphatase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-Sephacel, Phenyl Sepharose, chelating Sepharose Fast Flow and MonoQ. The purified enzyme was a tetramer with a subunit molecular mass of 26 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. Its optimum activity was at pH 4.5 and 70°C, with more than 65% of its activity retained after pre-heating for 30 min at 70°C. The enzyme was strongly inhibited by NaF (0.1 m ) and ZnCl₂ (1.0 m ); slightly inhibited by hexametaphosphate, tripolyphosphate or pyrophosphate at 1.0 m concentrations; but unaffected by 10 m ascorbic acid. The acid phosphatase hydrolysed a number of phosphate esters but not bis(p-nitrophenyl)phosphate nor uridine-5′-monophosphate. The N-terminal amino acid sequence of the first 20 residues of this enzyme showed 65% homology with an acid phosphatase from Lactobacillus plantarum DPC2739 and some homology with other phosphatases from mammals, yeasts and Escherichia coli.     

德氏乳杆菌QS701产ACE抑制肽发酵条件的优化

通过单因素和正交实实验对德氏乳杆菌QS701产ACE抑制肽的发酵条件进行优化,以期提高ACE抑制肽的产量,并对该菌株的生长特征进行探讨。结果显示,影响ACE抑制活性的发酵条件的顺序为:发酵时间>发酵温度>接种量,在此实验中,德氏乳杆菌QS701的最佳发酵条件为:接种量3%、温度43℃、发酵时间72 h,在此条件下ACE抑制率可达到81.58%,所以将此发酵条件应用于ACE抑制肽的制备中。     

喷雾干燥对德氏乳杆菌代谢酶活与可培养性的影响

为了提高乳酸菌在喷雾干燥过程中的可培养性,以德氏乳杆菌ATx为研究对象,在分析不同热胁迫条件对其细胞可培养性的影响的基础上,以20 g/dL脱脂乳(SM)、12 g/dL脱脂乳+8 g/dL菊糖(SMS)、12 g/dL脱脂乳+8 g/dL海藻糖(SMT)、12 g/dL脱脂乳+4 g/dL菊糖+4 g/dL海藻糖(SMST)为保护剂,探讨了喷雾干燥对菌体己糖激酶、乳酸脱氢酶、细胞膜ATP酶、细胞可培养性以及产酸性能的影响。结果表明:德氏乳杆菌ATx的最佳热胁迫条件为(50℃,15 min),此时细胞活菌数仅下降0.107 lg CFU/mL(P≥0.05)。经过喷雾干燥后菌体的细胞膜ATP酶、已糖激酶与乳酸脱氢酶的活性均显著地降低(P0.05),但均高于对照组。四种菌粉在12 g/dL脱脂乳中的产酸性能良好,凝乳时间为8～10 h,说明喷雾干燥会对德氏乳杆菌ATx的细胞膜与糖代谢关键酶造成损伤,通过添加保护剂可以提高细胞对喷雾干燥中不利环境的抵抗力,从而提高可培养性。     

Stress resistance of biofilm and planktonic Lactobacillus plantarum subsp. plantarum JCM 1149

The purpose of this study was to investigate the change in resistance of biofilm and planktonic food spoilage lactic acid bacteria (LAB) to environmental stresses, which strongly inhibit bacterial growth and are important in food preservation or in disinfection. The stress responses of biofilm and planktonic cells of Lactobacillus plantarum subsp. plantarum JCM 1149, which was used as a model spoilage bacterium, in various organic acids (namely, acetic acid, citric acid, lactic acid, and malic acid), ethanol, and sodium hypochlorite, were investigated using survival tests. The bacterial cells in biofilms showed greater resistance to all treatments than the planktonic bacterial cells in either the stationary or logarithmic phase. The planktonic bacterial cells showed reduced resistance to acetic acid after the cell suspension was diluted; however, intriguingly, the bacterial cells in biofilms maintained their resistance to acetic acid even after they were suspended or the cell suspension was diluted. These findings suggested the risk for food spoilage due to LAB derived from biofilms and suspended or diluted in foods, and demonstrated the importance of controlling biofilms of LAB in the food industry.     

副干酪乳杆菌X12肽聚糖的理化性质及其对HT-29细胞增殖的抑制

本文对副干酪乳杆菌X12肽聚糖的理化性质进行分析,并研究其对结肠癌HT-29细胞的抑制作用。采用SDS-PAGE分析其蛋白分子量,HPLC法检测其氨基酸组成,电镜观察其形态,最后检测其对HT-29细胞增殖的抑制作用。结果显示,副干酪乳杆菌X12肽聚糖蛋白分子量低于14.4 kDa,蛋白氨基酸共有15种,其中丙氨酸、谷氨酸、甘氨酸和赖氨酸含量较高,而电镜结果显示肽聚糖保留了原菌体细胞的骨架结构,并具有抑制HT-29细胞增殖的能力,肽聚糖高剂量组(160 μg/mL)抑制HT-29细胞增殖率可达21.02%。     

Purification and characterization of a glucoamylase from Rhizopus oryzae

Glucoamylase (EC 3.2.1.3) of Rhizopus oryzae NRRL 395 was purified approximately sevenfold by sequential ammonium sulfate fractionation, Biogel P-100 gel filtration, Q-Sepharose anion exchange and S-Sepharose cation exchange. The pH and temperature optima were 4·8 and 60°C, respectively. Enzyme was stable at temperatures up to 40°C and pH values between 3 and 8. The molecular weight was 67 000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the pI was 8·7 as determined by chromatofocusing. The K_m for amylopectin and soluble starch were 0·98 and 1·34 mg/ml, respectively. The V_max for amylopectin and soluble starch were 782 and 136 μmoles of glucose produced per mg of protein per min, respectively. The enzyme activity was inhibited by Hg²⁺, Pb²⁺ and Cd²⁺, but not by EDTA.     

Release and partial characterization of cell-envelope proteinases fromLactococcus lactis subsp.lactis IFPL 359 andLactobacillus casei subsp. casei IFPL 731 isolated from raw goat,s-milk cheese

Different methods of releasing the cell-envelope proteinase (CEP) fromLactococcus lactis IFPL 359 (Lc-CEP) andLactobacillus casei IFPL 731 (Lb-CEP) have been tested. Release of Lc-CEP was higher in Ca^2+-free buffer than in the presence of lysozyme and Ca^2+-. Lb-CEP was not soluble in Ca^2+--free buffer, making necessary the use of chelating agents such as ethylenediaminetetraacetate (EDTA) to attain release yields of 15–20%. Solubilizing the cell wall oflb.casei using lysozyme and mutanolysin improved CEP release yields, even in the presence of Ca^2+-. Two differently charged chromophoric peptides were degraded by whole cells and the soluble fractions studied at different hydrolysis rates in both the strains considered. Based on the specificity of these CEPs for the different substrates, the two proteinases can be placed in the same class as the CEP_I/III mixed-type variants that have been identified in lactococcal proteinases. In both strains ß-casein was hydrolysed more rapidly than _s-cascin.     

双歧杆菌、乳杆菌和芽孢杆菌产细菌素和多糖研究进展

双歧杆菌、乳杆菌和芽孢杆菌是益生菌的重要成员,具有很大的开发潜力。其不仅能作为优势菌调节肠道微生态平衡、防治疾病,还能产生具有广谱抗菌活性的细菌素和多糖类等化合物,其中有些化合物还具有抗肿瘤和抗氧化等特性,这些特性使得这些化合物和菌株本身有望开发成为新型膳食补充剂和保健功能性食品。但许多化合物的结构、活性、机制以及安全性尚不明确。因此,本文总结了2010~2019具有益生潜力的双歧杆菌属、乳杆菌属和芽孢杆菌属细菌中分离得到的细菌素和多糖化合物,并对其分类、结构、抗菌、抗肿瘤和抗氧化等功能进行综述,为菌株及其代谢产物的开发和应用提供参考。     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

Purification and characterization of the 3-phosphoglycerate kinase from the thermophile Lactobacillus delbrueckii subsp. lactis

The 3-phosphoglycerate kinase (PGK) of the thermophile Lactobacillus delbrueckii subsp. lactis was purified to homogeneity and found to be a monomeric enzyme with a molecular weight of 45 kDa. PGK is a Michaelis–Menten type enzyme with a K_m=0.7 mm for ATP and a K_m=2.6 mm for 3-phosphoglycerate. pH tolerance of PGK was found to be oriented towards acidic conditions with an optimum at pH 6.8 and a plateau of 90% of the total activity between pH 6.0 and 7.4. The optimum temperature for PGK activity is 45°C, which is consistent with the value expected for a thermophilic bacterium with an optimal growth temperature of 45°C.     

副干酪乳杆菌胞外多糖抗氧化活性分析

为评价干酪乳杆菌生物合成胞外多糖(exopolysaccharides,EPS)抗氧化活性,本文分离纯化高产胞外多糖的三株副干酪乳杆菌(3号、5号和7号)的胞外多糖,并对其DPPH·、·OH和O₂^-清除率分别进行测定,评价其体外抗氧化活性。通过分析EPS对H₂O₂诱导氧化损伤293T细胞的保护作用,评价其胞内抗氧化活性。结果表明,当EPS浓度为400 μg/mL时,其DPPH·、·OH和O₂^-的清除率最高,分别为8.87%(5号)、88.94%(7号)和34.55%(7号),其中对·OH清除能力高于抗坏血酸(65.87%),且三株菌株之间没有明显差异。3号副干酪乳杆菌所产EPS显著提高超氧化物歧化酶(SOD)活性、总抗氧化能力(T-AOC)活性并抑制了丙二醛(MDA)的生成(P<0.05),且与多糖浓度呈正相关。因此,3株副干酪乳杆菌胞外多糖具有良好的抗氧化活性,作为天然抗氧化剂具有良好的应用潜力。     

鼠李糖乳杆菌L-乳酸脱氢酶的生物信息学分析和基因克隆

本研究以鼠李糖乳杆菌（Lactobacillus rhamnosus）L-乳酸脱氢酶（Lr-L-LDH）为研究对象,以研究较多的Lr-L-LDH1为对照,对基因组注释的两个Lr-L-LDH1和Lr-L-LDH2基因,进行异同分析。采用在线网站和专业软件对Lr-L-LDH1和Lr-L-LDH2的一级结构、基本特性、亲疏水性、二级结构进行分析和预测,对三级结构进行同源建模以及酶和底物的分子对接分析,并对酶的编码基因进行系统发育分析,克隆表达及酶活性检测。结果显示：相较于Lr-L-LDH1,Lr-L-LDH2有着相似的分子特性,二级和三级结构,但Lr-L-LDH2编码序列短,氨基酸序列同源性低（48.08%）,有不同的进化地位;Lr-L-LDH2也含有乳酸脱氢酶催化活性位点序列和保守的NAD+结合位点序列（GXGXXG）,能够形成典型的活性三维口袋域,是NAD+依赖型四聚体结构L-乳酸脱氢酶,在细胞中需要果糖1,6-二磷酸（FBP）来激活,催化丙酮酸还原为L-乳酸;体外克隆表达和酶学分析表明,Lr-L-LDH2酶活力极显著低于Lr-L-LDH1(P<0.01)。Lr-L-LDH1和Lr...     

保加利亚乳杆菌微胶囊的制备及特性研究

为克服保加利亚乳杆菌不耐酸的缺点,建立制备保加利亚乳杆菌微胶囊的方法。本研究以海藻酸钠和壳聚糖复合包埋保加利亚乳杆菌,通过单因素实验和正交试验,确定了制备微胶囊的最佳工艺条件为:海藻酸钠浓度为3%、壳聚糖浓度为1.5%、氯化钙浓度为2%、120 min固化时间,微胶囊的包埋率为78.96%。该工艺条件下的保加利亚乳杆菌微胶囊具有良好的耐酸性和肠溶性。在人工胃液中,微胶囊在120 min,保持完好;而在人工肠液中,微胶囊60 min完全崩解。     

Purification and characterization of the pyruvate kinase of lactobacillus delbrueckii subsp. lactis

Lactic acid bacteria have evolved in a very rich and specific medium, milk, and have in the course of adaptation discarded non-essential metabolic processes. Thus, Lactobacillus delbrueckii subsp. lactis lacking both the citric cycle and the pentose-phosphate pathway relies mainly on glycolysis for ATP production and in particular, the reaction catalyzed by pyruvate kinase (PYK). In this study, we purified and characterized L. delbrueckii lactis PYK and studied its role in metabolic regulation. L. delbrueckii lactis (PYK) is a classical homotetramer and a V-type allosteric enzyme. Positive effectors are fructose 1,6-bisphosphate (K_a=2.2 mm), as well as fructose 6-phosphate and glucose 6-phosphate, which were found to be 1.3- and 1.6-fold more efficient activators than fructose 1,6-bisphosphate, respectively. L. delbrueckii lactis PYK is inhibited by inorganic phosphate, high concentrations of phosphoenolpyruvate and ATP (EC₅₀=0.75 mm). An ATP-binding motif identified on the 134-amino acid C-terminal extension of the enzyme might be involved in ATP regulation of L. delbrueckii lactis PYK. This extension also encodes a protein domain with homologies to the enzyme I of the Escherichia coli sugar-PTS system suggesting that the PYK might be involved in L. delbrueckii lactis sugar transport metabolism.     

植物乳杆菌微胶囊化研究

为保护植物乳杆菌的活性以增强乳杆菌在动物肠道内的益生功能,以天然发酵玉米青贮饲料中优良植物乳杆菌作为芯材,乳清蛋白和明胶为壁材,利用喷雾干燥法制成微胶囊,并以植物乳杆菌包埋率为响应值,研究壁材配比、壁材添加量、进风温度、进料量4个因素,进行中心组合实验(Box-Behnken),通过响应面分析对喷雾干燥法制备植物乳杆菌微胶囊条件进行优化。结果表明:最优条件为壁材配比(乳清蛋白与明胶质量比)1:2、壁材添加量22%、进风温度127℃、进料量35%,在此条件下,植物乳杆菌包埋率为62.15%。结论:本研究为应用喷雾干燥法制备植物乳杆菌微胶囊奠定了基础。