乳杆菌代谢产物对化脓性链球菌的抑制作用

【目的】分析乳杆菌代谢产物对化脓性链球菌的抑制作用。【方法】基于双层平板打孔法,通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用;然后分别采用高效液相色谱法和4-氨酰安替比林法检测乳杆菌代谢产物中的有机酸和H2O2含量;最后,检测乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。【结果】对化脓性链球菌的抑菌效果以植物乳杆菌KLDS1.0667最好,副干酪乳杆菌KLDS1.0342-1次之,瑞士乳杆菌KLDS1.0203抑菌效果最差;乳酸和乙酸产量KLDS1.0667>KLDS1.0342-1>KLDS1.0203;H2O2产量KLDS1.0203>KLDS1.0667>KLDS1.0342-1。在抑菌试验中,乳杆菌的发酵上清液经去除H2O2处理后抑菌圈直径都减小;将发酵上清液的p H调至7.0后均检测不到抑菌圈。结果表明,乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和H2O2,其中乳酸是产生抑菌作用的最主要物质。乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)分别为1.28、0.64和0.008 g/L,对化脓性链球菌的最小杀菌浓度(MBC)分别为5.12、2.56和0.032 g/L。【结论】乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用,主要抑菌物质为有机酸和H2O2。     

不同因素下双歧杆菌BB-12对变形链球菌拮抗作用的体外研究

目的探讨不同因素下双歧杆菌BB-12对变形链球菌的抑制作用。方法采用液体培养法分别培养双歧杆菌BB-12与变形链球菌,利用紫外分光光度计调节具有相同吸光度值的菌液,观察不同因素下双歧杆菌BB-12对变形链球菌的拮抗作用,使用扫描电镜观察早期变形链球菌生物膜的变化。结果双歧杆菌BB-12在不同因素作用下对变形链球菌均具有拮抗作用,双歧杆菌BB-12能够抑制早期变形链球菌生物膜的形成。结论双歧杆菌BB-12对变形链球菌有拮抗作用。     

双歧杆菌发酵果蔬汁的体外生物拮抗作用研究

目的研究双歧杆菌发酵果蔬汁的体外生物拮抗作用及影响因素。方法采用纸片扩散法(K-B法)、琼脂打孔法和试管稀释法(MIC)研究双歧杆菌发酵果蔬汁对常见病原菌的体外拮抗作用,并讨论pH、加热对拮抗作用的影响。结果发酵果蔬汁及其处理物(加热、不同pH)均具有一定的抑菌作用。结论双歧杆菌发酵果蔬汁对常见病原菌有一定的拮抗作用。     

罗伊乳杆菌增殖培养基中碳源氮源的优化

目的提高罗伊乳杆菌的发酵活菌数,以提高发酵产率,降低生产成本。方法采用光电比浊法与活菌计数法,通过单因素试验和正交设计方法对罗伊乳杆菌的增殖培养基的碳源和氮源进行优化,并且绘制优化前后的生长曲线。结果罗伊乳杆菌增殖培养基中最佳的碳源、氮源种类与浓度为葡萄糖2.1%、蔗糖3.0%、牛肉膏1.5%、酵母粉1.4%,最佳的培养时间为10h。结论通过优化罗伊乳杆菌的增殖培养基,提高了发酵产率,降低了生产成本。     

抑制龋齿生成菌(变异链球菌)的优势共培养菌株的筛选CSCD

目的比较10株益生菌的各项生理功能,筛选抑制龋齿生成菌(变异链球菌)的优势共培养菌株,为后期开发应用奠定基础。方法对组合菌株及单个菌株进行体外抑菌试验筛选,并进一步探索对变异链球菌的抑菌机制。结果10株益生菌菌株中,植物乳杆菌AI-66、乳双歧杆菌AI-01、干酪乳杆菌AI-12和婴儿双歧杆菌AI-20抑制变异链球菌较其他菌株有优势;鼠李糖乳杆菌AI-11与发酵乳杆菌AI-25、婴儿双歧杆菌AI-20与副干酪乳杆菌AI-62、乳双歧杆菌AI-01与干酪乳杆菌AI-12这3个共培养组合菌株较单个菌株对变异链球菌的抑制效果差异具有统计学意义(均P<0.01)。其可能的抑菌物质在低pH具有较好的抑菌效果,且对热不敏感,对胰蛋白酶敏感,可能是小分子多肽类物质。结论筛选出的3个共培养组合菌株在抑制龋齿生成菌(变异链球菌)效果上较单菌株有优势,可应用于食品、功能食品及膳食补充剂中,以降低龋齿、口臭等口腔疾病的风险。     

益生菌拮抗阪崎肠杆菌的初步研究

目的研究鼠李糖乳杆菌和植物乳杆菌等8种常见益生菌对阪崎肠杆菌的拮抗作用。方法采用牛津杯法测定益生菌耗尽上清对阪崎肠杆菌的抑菌圈,获得对阪崎肠杆菌具有较强抑菌能力的鼠李糖乳杆菌和植物乳杆菌;采用混合培养法对2株益生菌与阪崎肠杆菌的拮抗竞争能力进行测试。结果 8种益生菌耗尽上清均能抑制阪崎肠杆菌,其抑菌能力具有热稳定性且依赖于酸性pH环境。阪崎肠杆菌(107CFU/mL)与鼠李糖乳杆菌(108CFU/mL或109CFU/mL)共孵育至24 h,其活菌量开始逐渐下降,至120 h孵育结束下降到105CFU/mL;菌量比为1:10的阪崎肠杆菌与植物乳杆菌共孵育至24 h,其活菌量开始逐渐下降,菌量比为1:100时则提前至8 h,至120 h孵育结束活菌量均下降到102CFU/mL。结论鼠李糖乳杆菌和植物乳杆菌均能有效地竞争拮抗阪崎肠杆菌。     

有抑菌能力乳杆菌的筛选 及其抑菌物质的初步探究

目的筛选具有抑菌能力的乳杆菌菌株并对其抑菌物质进行初步探究。方法采用琼脂扩散法进行抑菌试验,筛选出具有抑菌能力的乳杆菌菌株;通过热稳定性试验检测抑菌物质耐高温的能力;通过有机酸排除与过氧化氢排除试验检测这两种物质对抑菌作用是否有影响;用胃蛋白酶、胰蛋白酶和蛋白酶K消化处理各株乳杆菌无细胞发酵液后进行抑菌试验,判断抑菌物质是否为蛋白多肽类物质。结果 2株副干酪乳杆菌与1株保加利亚乳杆菌对大肠埃希菌、铜绿假单胞菌、伤寒沙门菌和痢疾志贺菌有抑菌效果。这3株乳杆菌无细胞发酵液中的抑菌物质经过高温处理后仍具有抑菌能力,但抑菌能力与处理前相比显著降低(P0.05);有机酸对照组未产生明显的抑菌圈;过氧化氢排除后的无细胞发酵液的抑菌能力未受影响;经过蛋白酶作用3株乳杆菌无细胞发酵液的抑菌能力显著降低(P0.05)或消失。生物被膜态副干酪乳杆菌2的抑菌能力与浮游态相近,其无细胞发酵液中的抑菌物质可以完全耐受100℃处理与胃蛋白酶的消化作用,可部分耐受胰蛋白酶的消化作用。结论副干酪乳杆菌1、副干酪乳杆菌2和保加利亚乳杆菌具有良好的抑菌能力,它们产生的主要抑菌物质为蛋白多肽类,此物质具有较好的耐高温能力与耐蛋白酶能力;被膜态副干酪乳杆菌产生的抑菌物质表现出了更强的抗胁迫能力与稳定性。     

具有拮抗无乳链球菌能力乳杆菌的筛选及抑菌特性

目的从罗非鱼(Nile tilapia)肠道中筛选出具有抑菌作用的乳杆菌,测定其对无乳链球菌(Streptococcus agalactiae)的抑菌效果,分析乳杆菌抑制无乳链球菌的有效成分,并利用分子生物学手段对筛选的乳杆菌菌种进行鉴定。方法采用双层平板法对具有抑制无乳链球菌的乳杆菌进行筛选,牛津杯法对抑菌效果进行测定,酶蛋白敏感性测定、热处理、有机酸处理等方法分析抑菌活性物质有效成分,16S rDNA分子标记对乳杆菌进行鉴定。结果从罗非鱼肠道中筛选出14株乳杆菌,其中菌株RS2对无乳链球菌具有明显的抑菌效果;不同蛋白酶种类、pH处理对乳杆菌无细胞培养液均有不同的影响,经80℃处理的乳杆菌无细胞培养液,其抑菌效果未显著改变(t=0.169 2,P=0.873 8)。此外,此株乳杆菌对猪霍乱沙门菌(Salmonella choleraesuis)、大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和志贺菌(Shigella sp.)等病原菌具有良好的抑制作用。经鉴定,该乳杆菌为植物乳杆菌(Lactobacillus plantarum)。结论从罗非鱼肠道中分离得到的植物乳杆菌菌株RS2对无乳链球菌等致病菌具有一定的抑制作用,推断其抑菌有效成分为细菌素类物质。此项研究对开发抗生素替代产品,提高食品的品质具有一定价值。     

番茄青枯病拮抗菌筛选鉴定及其发酵条件初探

从健康番茄根系采样,筛选出4株对番茄青枯病有较强拮抗作用的菌株,在NA培养基上抑菌圈直径>9 mm。其中拮抗菌株YB6抑菌活性最强且拮抗效果稳定,通过形态学观察及部分生理生化特征测定,初步确定为节杆菌属。通过单因素试验进行了发酵条件初步研究,得到适宜的发酵条件为:发酵时间3 d,培养温度30°C,初始pH值9.0,接种量3%,转速100 r/min,碳源蔗糖,氮源酵母浸膏。通过初步优化后拮抗菌株抑菌活性明显增强,最终对青枯病菌SST-Y和G2M1.70抑菌圈直径与NB培养基相比增加了76.72%和81.14%,差异显著。     

罗伊氏乳杆菌的益生功能

罗伊氏乳杆菌是目前已报道的几乎可存在于所有脊椎动物和哺乳动物肠道内的乳酸杆菌,是具有益生功效的肠道益生菌.通过对罗伊氏乳杆菌良好的肠道定植能力和其产生的罗伊氏菌素的介绍,阐明其可能的益生作用机理.重点论述了罗伊氏乳杆菌促进人类和动物健康功能的研究进展,并探讨了今后罗伊乳杆菌益生菌制剂的工业化发展趋势.     

Effect of LGG yoghurt on Streptococcus mutans and Lactobacillus spp. salivary counts in children

The aim of this study was to establish effect of 14 day consumption of commercially available yoghurt containing Lactobacillus rhamnosus ATCC53103 - LGG (Bioaktiv LGG, Dukat, Croatia) on Streptococcus mutans and Lactobacillus spp. salivary counts in children. Twenty five patients, 6-10 yr old participated in the study. At the inclusion in the study caries risk for every patient was evaluated. The saliva samples were tested with chair side kits for saliva buffer capacity (CRT buffer, Vivadent, Schaan, Liechtenstein), S. Mutans and Lactobacillus counts (CRT bacteria test, Vivadent, Schaan, Liechtenstein). Seven, 14 and 30d after yoghurt consumption saliva samples were tested again with CRT buffer and CRT bacteria tests. Obtained data were analyzed using chi2 and Kruskal-Wallis tests. Results showed significant increase in saliva buffer capacity 30d after yoghurt consumption. S. Mutans salivary counts were significantly decreased after 30d. Significant differences in Lactobacillus counts were not observed. It could be concluded that daily consumption of yoghurt containing LGG have an inhibitory effect on oral pathogenic bacteria and may be beneficial in caries prevention.     

Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with beta-(2-->1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10(7)) with beta-(2-->1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Effect of Nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 Growing in Pure or Mixed Culture on Human Teeth in an Artificial Mouth

Streptococcus mutans and Streptococcus mitior were both capable of colonizing the surfaces of human teeth in an artificial mouth. Although fermentable carbohydrate was not essential for colonization, highest numbers were recovered after 5 d if 5–0°_***0 (w/v) sucrose had been available intermittently. When grown together in mixed culture the interaction of the two species was affected profoundly by the available nutrients. In the presence of synthetic saliva alone, Strep. mitior was strongly antagonistic to Strep. mutans. When a nutrient broth containing sucrose was also provided intermittently there was slight inhibition of Strep. mutans accompanied by an increase in the proportion of Strep. mitior in the plaque, although the former was the dominant organism under these conditions. When 0–5% (w/v) glucose replaced the sucrose, mutual antagonism occurred and fewer organisms were recovered than if only synthetic saliva had been available. One reason why a high-sucrose diet encourages colonization by Strep. mutans may be that insoluble extracellular polysaccharide confers a competitive advantage upon it in the face of antagonistic agents such as the hydrogen peroxide produced by Strep. mitior.     

疮痂链霉菌拮抗菌株BU396的分离鉴定与抗菌性质分析

【背景】随着马铃薯种植面积的扩大,疮痂病的发生也日益严重,且化学防治存在环境污染等诸多弊端,因此利用安全和高效的生物防治手段对该病害进行防治成为研究热点。【目的】对疮痂链霉菌(Streptomyces scabies)拮抗菌株进行分离、鉴定和功能基因分析,对抗菌物进行分离及抗菌特性的研究。【方法】从马铃薯疮痂病的病害土壤中分离、筛选并鉴定得到拮抗菌株,采用PCR法对菌株进行抗菌物合成基因检测。通过硫酸铵沉淀法分离得到抗菌物,进行抗菌谱和稳定性检测,并通过盆栽试验进一步验证该菌株的生防效果。【结果】通过抗菌试验筛选得到拮抗细菌BU396,根据形态学、生理生化性质和分子生物学鉴定结果,确定其为贝莱斯芽孢杆菌(Bacillus velezensis)。功能基因分析表明BU396中含有表面活性素等4种抗菌物的合成相关基因。采用硫酸铵沉淀法对其培养液上清进行分离,当硫酸铵的饱和度为75%时,抗菌物在沉淀中析出。抗菌谱试验结果显示该抗菌物对多种动植物病原菌均具有抗菌效果。稳定性试验表明该抗菌物耐热,不易被酶解,对多种金属离子不敏感,具有宽泛的pH活性范围,盆栽试验结果显示菌株BU396处理能够明显降低马铃薯疮痂病的发病率。【结论】分离并鉴定了一株对疮痂链霉菌具有显著拮抗作用的贝莱斯芽孢杆菌BU396。该菌株含有多种抗菌物合成的相关基因,其抗菌物具有广谱的抗菌活性和良好的稳定性,对马铃薯疮痂病的生防效果显著。本研究为马铃薯疮痂病的生物防治及后续抗菌物质的深入研究奠定了基础。     

Salivacin 140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140 active against pathogenic bacteria

K. ARIHARA, S. OGIHARA, T. MUKAI, M. ITOH AND Y. KONDO. 1996. Fifteen of 353 environmental isolates of lactic acid bacteria consistently showed activity against Listeria monocytogenes, Streptococcus mutans, Actinomyces viscosus , and/or Propionibacterium acnes . Strain T140, isolated from the surface of Japanese pampas grass leaves and identified as Lactobacillus salivarius subsp. salicinius , also had activity against several Lactobacillus species, Staphylococcus aureus and Yersinia enterocolitica . Since the antagonistic factor(s) produced by T140 was sensitive to a proteolytic enzyme, it was concluded that a bacteriocin (named salivacin 140) was involved in the inhibition activity. Strain T140 required a high initial pH (7.5–8.5) in agar plates for bacteriocin production.     

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.     

Application of Probiotics in Folate Bio-Fortification of Yoghurt

Folate deficiency is a public health concern affecting all age groups worldwide. The available evidence reveals that adding probiotic bacteria to the yoghurt starter cultures during yoghurt production process under fermentation conditions increases the folate content of yoghurt. The present study was conducted to measure two folate derivatives, i.e., 5-methyltetrahydrofolate and 5-formyltetrahydrofolate, in bio-fortified yoghurt samples including (1) yoghurt containing Streptococcus thermophilus and Lactobacillus bulgaricus, (2) probiotic yoghurt containing Lactobacillus acidophilus LA-5 and Bifidobacterium lactis BB-12, (3) probiotic yoghurt containing native strains of Lactobacillus plantarum 15HN, (4) probiotic yoghurt containing native strains of Lactococcus lactis 44Lac, and (5) probiotic yoghurt containing commercial strains of Lactobacillus plantarum LAT BY PL. During storage at 4 °C for 21 days, the highest levels of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate, which were statistically significant, were detected in the yoghurt made using Lact. plantarum 15HN. Moreover, the highest total folate concentration (1487 ± 96.42 μg/L) was specified in the yoghurt containing Lact. plantarum 15HN on the 7th day. It can be conjectured that this product can be suggested as a proper alternative to synthetic folic acid and may not have the side effects of using synthetic folic acid overdoses.

     

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).