高抑菌活性乳酸杆菌的筛选及性质

从自酿酸奶中分离得到1株高抑菌活性菌株,经16S rDNA测序后鉴定为Lactobacillus sp.FSZ。以大肠杆菌、金黄色葡萄球菌为指示菌,取得良好抑菌效果。经组分分析及蛋白酶降解,抑菌活性物质确定为蛋白物质,推测其由一些高分子的蛋白类物质和低分子的多肽类物质组成。抑菌活性物质在酸性条件下显示出良好的抑菌活性,发酵液经60℃处理30 min后,活性基本没有下降,经100℃处理30 min仍保留83.9%的活性,表现出良好的热稳定性。     

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.     

<Emphasis Type="SmallCaps">L</Emphasis>(+)-Lactic Acid Production Using <Emphasis Type="Italic">Lactobacillus Casei</Emphasis> in Solid-State Fermentation

Lactobacillus casei was grown at 37 °C on sugarcane bagasse (5 g) soaked with cassava starch hydrolysate (final moistening volume 34 ml) containing 3 g reducing sugar in a solid-state condition. The maximum yield of l-lactic acid after various process optimisations was 2.9 g/5 g initial substrate corresponding to 97% conversion of sugar to lactic acid with initial substrate moisture of 72%.     

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

Purification and molecular characterization of antibacterial compounds produced by Lactobacillus murinus strain L1

AIMS: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1. METHODS AND RESULTS: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine. CONCLUSIONS: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.     

Growth of Lactobacillus plantarum in media containing hydrolysates of fish viscera

AIMS: To compare growth of Lactobacillus plantarum on media containing hydrolysates (peptones) from cod viscera with growth on commercial media. METHODS AND RESULTS: Growth of Lact. plantarum on various fish peptones and commercial peptones/extracts was evaluated using both a Bioscreen apparatus (microtiter plates, no pH control) and fermentors (with pH control). Generally, the performance of the fish peptones was good and only beaten by the performance of yeast extract. Replacement of the 22 g l(-1) complex nitrogen source in standard MRS medium with only 5 g l(-1) fish peptone reduced the biomass yield with only 10%, whereas replacement with a mixture of 2.5 g l(-1) fish peptone and 2.5 g l(-1) yeast extract increased the biomass yield by 10%. CONCLUSIONS: Peptones derived from cod viscera support excellent growth of Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: We show that peptones derived from cod viscera are promising constituents of growth media for fastidious food bacteria such as lactobacilli. Media containing these peptones show excellent performance while problems associated with the use of meat-derived peptones (BSE, kosher status) or plant-derived peptones (genetically modified organisms) are avoided.     

Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spray-drying

AIMS: The aim of this study was to optimize survival of Lactobacillus delbrueckii subsp. bulgaricus during spray-drying and subsequent storage through optimizing the pH of growth conditions. METHODS AND RESULTS: Cell concentrates previously grown without or with pH controlled were spray-dried and stored at 20 degrees C and heat treated at 57 degrees C. Cells grown under noncontrolled pH were more resistant to both drying and heating than cells grown under controlled pH but no significant differences were observed during storage. The intracellular proteins profile of cells grown under both conditions was studied by two-dimensional SDS-polyacrylamide gel electrophoresis. Eight proteins were identified using automated mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data acquisition. Of the identified proteins, only cochaperonin GroES corresponded to a known heat shock protein (HSP). The other proteins identified are proteins involved in glycolysis. For cells grown under noncontrolled pH the expression of the Hsp70, GroES and GroEL, measured by Western blotting, was enhanced. CONCLUSIONS: The higher resistance of cells grown under noncontrolled pH correlates with the enhanced production of heat shock proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of L. bulgaricus under controlled pH (commonly used by the starter cultures production industry) results in cells more sensitive to stresses frequently encountered by the cells during starter cultures preparation/storage/utilization.     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

TUNEL法检测乳酸杆菌对小鼠宫颈鳞癌的凋亡诱导作用

目的应用脱氧核苷酸转移酶介导的dUTP缺口末端标记法———TUNEL法,检测乳酸杆菌(Lactoba-cillus,LB)对小鼠宫颈鳞癌的凋亡诱导作用。方法建立615近交系小鼠U14移植瘤动物模型,并随机分组,TUNEL法检测各组细胞凋亡指数(apoptosis index,AI)。结果乳酸杆菌实验组的AI为(11.25±4.06)%,与对照组的差异有非常显著性(P<0.01),与抗癌药顺铂组的差异有显著性(P<0.05)。结论乳酸杆菌可诱导U14移植瘤细胞凋亡,这可能是其抑癌机制之一。     

1株植物乳杆菌生物学特性的研究

研究1株植物乳杆菌(N3)的生物学特性,实验结果表明该菌能耐受80~85℃的高温和0.20kg/cm2蒸汽压力;直接分解玉米淀粉的乳酸产率为7.05%(36 h)和8.19%(48 h);能耐受pH为4.5的酸性环境;在人工胃液中的活菌数为4.1×106CFU/g;对金霉素、土霉素、痢特灵和氟哌酸等抗生素的敏感性强,而对防霉剂和脱霉素不敏感。植物乳杆菌(N3)是1株优良的益生素生产菌。