Selective enumeration and identification of mixed cultures of Streptococcus thermophilus,Lactobacillus delbrueckii subsp. bulgaricus,L. acidophilus,L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk

This study describes selective plating methodologies for enumeration of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk based on selective antibiotic-free media. Enumeration of S. thermophilus was performed using M17-lactose. MRS-fructose was suitable for enumeration of L. bulgaricus and MRS-maltose for differentiation between L. acidophilus and L. paracasei. The selective enumeration of B. lactis was obtained using MRS-raffinose containing 0.05% LiCl. The bacterial counts obtained using selective methods were equivalent to those under optimum culture conditions at a probability level of 95%. Performance of the methods was verified in fermented milk products where identification of the enumerated species was confirmed by species-specific polymerase chain reaction. This study shows that combination of species-specific polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis has great detection and identification potential for verification of accurate species labelling in fermented milk without prior isolation of the bacteria.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Lactobacillus delbrueckii subsp. bulgaricus和Streptococcus thermophilus接种比例对Bifidobacterium lactis益生菌发酵乳品质的影响

将Lactobacillus delbrueckii subsp.bulgaricus ND02(LB-ND02)和Streptococcus thermophilus ND03(ST-ND03)按1∶1、1∶10、1∶100、1∶1000接种于脱脂乳中,同时接入益生菌Bifidobacterium lactis V9(B.lactis V9,接种量为2.0×107g-1),于42℃进行发酵。通过对发酵及贮藏过程中发酵乳指标的测定,评价LB-ND02和ST-ND03的接种比例对发酵乳品质的影响。结果表明,随着LB-ND02接种比例减小,凝乳时间显著延长,B.lactis V9活菌数显著提高。4℃贮藏28 d后,随LB-ND02接种比例减小,B.lactis V9存活率差异显著,后酸化也显著减弱。研究发现,LB-ND02和ST-ND03的接种比例,显著影响发酵乳的发酵时间、B.lactis V9活菌数、后酸化及黏度。     

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.     

Effects of glutathione on acid stress resistance and symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Streptococcus thermophilus SDMCC18 contains a novel bi-functional glutathione synthetase gene gshF, and it can produce glutathione (GSH). In the present study, we demonstrated that the produced GSH by S. thermophilus SDMCC18 could protect cells against acid challenge and be secreted into the medium. Moreover, using a gshF-defective mutant strain as the control, we found that the GSH conferred a protective role against acid stress to Lactobacillus delbrueckii subsp. bulgaricus ATCC11842. In addition, L. bulgaricus ATCC11842 could import the exogenous GSH into the cytoplasm, leading to an improved growth of strain ATCC11842. Taken together, our study reported a novel proto-cooperation relationship between S. thermophilus and L. bulgaricus in yoghurt fermentation.     

嗜热链球菌和德氏乳杆菌保加利亚亚种共生关系的研究进展

嗜热链球菌和德氏乳杆菌保加利亚亚种是组成酸奶发酵剂的主要菌种,在长期共处于牛乳环境的过程中形成多种协同共生关系.这些协同共生关系赋予发酵剂菌株产酸快、黏度高、活性物质丰富等优良性能,同时对酸奶品质产生重要影响.该文旨在从营养供给、抵抗环境胁迫及酸奶品质改良等方面讨论两菌的协同共生关系,为提高发酵剂菌株活力及酸奶品质提供...     

Purification and characterization of X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus lactis and from Streptococcus thermophilus

X-Prolyl-dipeptidyl-aminopeptidase recently was found in several lactic acid bacteria. This article describes the purification of the enzymes from Lactobacillus lactis and Streptococcus thermophilus and compares their characteristics. Enzymes from both strains are serine-peptidases. They both have a molecular weight of about 165,000 daltons, an isoelectric point near 4.5, and are constituted of two subunits. The pH optimum of the enzyme isolated from L. lactis is 7.0, whereas the enzyme from S. thermophilus possesses a broad pH optimum between 6.5 and 8.2 with glycyl-L-prolyl-aminomethylcoumarin as substrate. Below pH 5, both enzymes are unstable; however, that from S. thermophilus is more rapidly denatured. The enzyme from S. thermophilus is more sensitive to heat than the corresponding enzyme from L. lactis. Enzymes from the both strains have different specificities towards various substrates and are differently effected by metals, chelators, and other inhibitors. The importance of this enzyme for the metabolism of lactic acid bacteria is discussed.     

Interactions between Lactococcus lactis and Streptococcus thermophilus strains in Cheddar cheese processing conditions

The growth of pure and mixed cultures of Lactococcus lactis and Streptococcus thermophilus under simulated Cheddar cheese manufacture was examined. Cell-free wheys (CFW) of the cultures were prepared for analysis by automated spectrophotometry (AS). The maximal growth rate of the lactococci in S. thermophilus R0083 CFW was 13% higher than that noted in their own CFW and three lactococci also gave higher biomass levels (OD_max). During simulated Cheddar cheese fermentations with four paired cultures, one L. lactis strain grew 20% less when paired with S. thermophilus R0083, and an increase in colony forming units (cfu) was found with one other lactococcal strain. Viable counts of S. thermophilus in mixed cultures varied by less than 0.1 log cfu mL^?1. The AS data on OD_max in CFW were useful in predicting the evolution of cfu in the fermented mixed cultures. As a function of strain, the presence of S. thermophilus in a Cheddar fermentation process can enable extended growth of the lactococci.     

The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1 °C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.     

Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus

Swiss mice, fed for 8 consecutive d with 50 micrograms/d of viable cultures of Lactobacillus acidophilus and Streptococcus thermophilus, showed significant variation in their immune system. In order to study this phenomenon assays for macrophage and lymphocyte function were carried out. Both lactic acid bacteria enhanced significantly the enzymatic and phagocytic activity of peritoneal macrophages as checked against the controls and also accelerated the phagocytic function of the reticuloendothelial system as revealed by the carbon clearance test. On the 2nd d (100 micrograms), L. acidophilus reached a peak of K = .271, which remained high. Streptococcus thermophilus was effective only on the 2nd d and then decreased. The lymphocytic activity studied by immunoglobulin secreting cells was assayed by Jerne's method of plaque-forming cells (PFC). This activity also was increased by the two microorganisms. Streptococcus thermophilus proved more effective than L. acidophilus. Lactobacillus acidophilus and S. thermophilus activated macrophages and lymphocytes and produced the same increase in the immune response of mice whether administered orally or intraperitoneally.     

保加利亚乳杆菌与嗜热链球菌共生机理的研究进展

乳酸菌在乳中的共生机制是非常复杂的网络体系。通过对蛋白质代谢、核苷酸碱基代谢、氧化性应激以及碳源物质代谢等方面的研究,对保加利亚乳杆菌与嗜热链球菌共生机理的研究进展进行详细的阐述。旨在为发酵剂菌体的筛选及应用提供参考。     

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.     

乳酸乳球菌细胞壁蛋白酶的分离纯化

本研究确定了分离纯化乳酸乳球菌细胞壁蛋白酶(CEP)的最佳技术路线.用裂解液(50mmol/L Tris-HCI,2mmol/L EDTA-Na2,100mmol/L NaCl,0.5%Tritonx-100,1mg/ml溶菌酶,pH8.5)悬浮菌体(20ml/g),37℃下保温3h,离心后取上清液即为粗酶液.粗酶液通过45%硫酸铵沉淀,DEAE-Sephadcx A-25和Sephacryl-S-300 HR两步层析,可以得到纯化的细胞壁蛋白酶.蛋白酶提纯倍数达到74.048%,最后回收率为14.865%,PAGE电泳检测为一条带,SDS-PAGE检测蛋白酶为单体结构,分子量大约为53kD.用纯化后CEP酶解乳清蛋白,酶解液ACE抑制率为45%.     

菌株性能及球杆菌比例对酸奶品质的影响

分析测定了从某直投式发酵剂中分离的两株嗜热链球菌（Streptococcus thermophilus）S.t S1、S.t R1和一株保加利亚乳杆菌（Lactobacillus delbrueckii subsp.bulgaricus）L.bL1的发酵性能,并将球菌与杆菌以不同比例组合发酵,比较酸奶发酵时间、粘度、乳清析出、后酸化和感官评价差异。结果表明,嗜热链球菌S.tS1生长速度略慢于S.tR1,酸化速度相当,但S.tS1的胞外多糖产量和发酵乳粘度明显高于S.tR1,保加利亚乳杆菌L.bL1的上述性能均比两株球菌差。S.tS1与L.bL1菌株以100：1的比例进行组合发酵时,粘度达0.549Pa·s;乳清析出仅2.5mL/10g;4℃冷藏保存15d后,酸度上升12°T,酸奶产品各项指标优于其他菌株组合。菌株性能和组合方式对酸奶产品的品质具有显著的影响。     

不同发酵特性的嗜热链球菌与保加利亚乳杆菌共发酵的特性

为探究具有不同优良性状的嗜热链球菌株与保加利亚乳杆菌株共发酵的特性,用产酸快的嗜热链球菌StCH-1菌株和产黏好的嗜热链球菌StTA040菌株与保加利亚乳杆菌Lb0925B菌株组合,测定其组合菌株的发酵性能。通过实验发现组合菌株发酵速率均比单菌株发酵速率快,其中含有嗜热链球菌StCH-1的组合菌株发酵速率较快,而含有嗜热链球菌StTA040的组合菌株的胞外多糖产量较高,发酵乳的黏度较高,持水力较强;三组分发酵剂的发酵速率快,发酵乳在贮藏期间黏度高,持水力强,通过感官分析得出三组分发酵剂制得的发酵乳的口感和风味最佳。     

镉胁迫下泡菜乳酸乳球菌的形态变化

通过扫描电镜和透射电镜分别观察不同质量浓度水平的Cd2+对泡菜乳酸乳球菌（Lactococcus lactis subsp.lactis）细胞的影响,扫描电镜结果显示：Cd2+质量浓度在0、10 mg/L时,泡菜乳酸乳球菌呈椭圆形、表面光滑、菌体生长繁殖旺盛,随着Cd2+质量浓度的增加菌体细胞表面产生白色颗粒状物质、菌体细胞存活数量大幅下降（OD600 nm值由1.336下降到0.515）。当添加200 mg/L Cd2+时,几乎没有见到明显的菌体、显示有少量棱形晶状物。透射电镜结果显示：当Cd2+质量浓度为0～50 mg/L时泡菜乳酸乳球菌结构完整、细胞内容物分布均、菌体生长较为正常,当菌体暴露于100、200 mg/L Cd2+时菌体细胞出现异常现象,如细胞破裂、内容物从薄膜穿孔中释放、质壁分离等。两类电镜结果均表明：在低质量浓度Cd2+（≤50 mg/L）胁迫下,对泡菜乳酸乳球菌的生长几乎不产生影响,添加Cd2+质量浓度上升到100、200 mg/L时泡菜乳酸乳球菌正常生长受到抑制。     

嗜酸乳杆菌和乳酸链球菌混合培养及产物分析

嗜酸乳杆乳菌(LA)和酸链球菌(SL)在33℃培养48 h的发酵液中抑菌(金黄色葡萄球菌ATCC25923)效果最好;2菌混培20h后生物量和体系总产酸量远大于各自纯培养,乳酸含量一直上升,到24 h达23.175 mg/L,乙酸含量一直稳定在12 mg/L左右,变化不大,乳酸与抑菌关系密切。LA和SL两菌混培接种比例的抑菌优先顺序为(1:1)、(2:1)、(1:2)。     

Contribution of Lactococcus lactis subsp lactis IFPL 359 and Lactobacillus casei subsp casei IFPL 731 to the Proteolysis of Caprine Curd Slurries

ABSTRACT: Proteolysis was studied in low-fat caprine curd slurries pre-incubated (24 h at 30 °C) with an extra dose of rennet, 10.0 or 22.0 μg g^-1 (LR and HR slurries, respectively), and treated at 70 °C for 15 min prior to incubation with sonicated cells of Lactococcus lactis subsp lactis IFPL 359 alone or combined with Lactobacillus casei subsp casei IFPL 731. The addition of lactobacilli to slurries containing lactococci increased the conversion of water-soluble nitrogen to nonprotein nitrogen and amine nitrogen. Major differences were found for some free amino acids, such as glutamic acid and proline, and hydrolysis of most hydrophobic peptides produced by lactococci, which are associated with bitterness in cheese.     

Serological identification of Streptococcus salivarius subsp. thermophilus

Streptococcus salivarius subsp. thermophilus (Str. thermophilus) has no group-specific antigen. HCl extracts of 551 strains of streptococci isolated from cheese or yogurt (123 Str. thermophilus and 428 Enterococcus faecalis - Ent. faecium spp.) were examined with three type-specific antisera prepared against representatives of Str. thermophilus and also with Lancefield group D antisera. All typical (80) or atypical (43) strains of Str. thermophilus reacted with at least one type-specific antiserum and none of the enterococci reacted. Only two strains reacted with both type-specific and group antisera.     

乳酸乳球菌胞外多糖磷酸化的工艺优化

采用单因素和正交试验,对乳酸乳球菌胞外多糖的磷酸化工艺进行研究,探讨磷酸盐用量、反应温度、反应时间、反应pH值对乳酸乳球菌胞外多糖最终PO43-接枝量的影响。所得的乳酸乳菌球菌胞外多糖磷酸化的工艺优化条件为：胞外多糖与磷酸盐质量比为6:1、温度90℃、时间4h、pH6.0,此条件下所得PO43-的接枝量为1.639mg/g。