猪源乳酸菌的分离、鉴定及其生物学特性

【目的】将分离自猪肠道粘膜、食糜和粪便的乳酸菌,通过产乳酸能力、生长性能、耐酸和耐胆盐性能及抑菌能力评价,筛选适应养猪生产的潜在益生特性的菌株。【方法】共分离获得155株乳酸菌纯菌株,从中筛选出4株产酸能力较强的乳酸菌,结合生理生化试验及细菌16S rRNA测序鉴定其种属,评价候选乳酸菌的生长情况、耐酸、耐胆盐及抑菌特性。【结果】综合变色时间(8 h)、pH值(3.9)和乳酸含量(100 mmol/L),筛选出4株(L45、L47、L63和L79)候选菌株,经鉴定依次为罗伊氏乳杆菌、植物乳杆菌、约氏乳杆菌和粪肠球菌。该4株乳酸菌均可在体外快速生长;L47和L79能够耐受pH 2.5的酸性环境,L47能够耐受0.5%胆盐环境;各乳酸菌上清液与指示菌共培养,发现对E coli K88和沙门氏菌均产生了抑制作用,其中L47上清液对指示菌的抑制作用较强。【结论】L47具有较好的产酸性能与生长性能、可耐受猪胃酸和肠道胆盐环境,对E.coli K88和沙门氏菌具有较好的抑制作用,说明该乳酸菌具有潜在的益生特性。     

高效降解草酸乳酸菌的筛选

尿路结石70%~80%主要由草酸钙结晶构成。人体内的草酸一般通过肠道内微生物降解,经由粪便排出或在泌尿道吸收由尿液排出。本研究对市场上商品化的发酵乳制品、饮料和药品中的乳酸菌进行分离,得到37株菌,包括嗜热链球菌、保加利亚乳杆菌、干酪乳杆菌、鼠李糖乳杆菌、植物乳杆菌、嗜酸乳杆菌、动物双歧杆菌、长双歧杆菌、粪肠球菌、屎肠球菌和乳球菌,并检测这些菌株降解草酸的能力。结果提示,乳酸菌在体外能够有效的降低培养物中的草酸浓度,并筛选出了具有高效降解草酸能力的乳酸菌菌株。有望成为尿石症预防的新措施。     

新疆发酵乳品中乳酸菌的分离鉴定及其耐药性

目的对新疆传统发酵乳品中乳酸菌进行分离鉴定并检测其耐药性。方法利用传统形态学鉴定法和生化鉴定等方法对新疆发酵乳中乳酸菌进行鉴定,采用纸片扩散法对分离鉴定的菌进行耐药性分析。结果从新疆发酵乳品中共分离出8株乳酸菌,经鉴定分别为瑞士乳杆菌(Lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)、马乳酒样乳杆菌(Lactobacillus kefianofaciens)、乳酸乳球菌(Lactococcus lactis)、副干酪乳杆菌(Lactobacillus paracasei)、副干酪乳杆菌类坚韧亚种(Lactobacillus paracasei subsp.tolerans)、哈尔滨乳杆菌(Lactobacillus harbinensis)、希氏乳杆菌(Lactobacillus hilgardii),并且发现8株乳酸菌对万古霉素、庆大霉素、阿莫西林、多西环素、环丙沙星、阿奇霉素、头孢他啶、头孢孟多具有一定敏感性。结论新疆发酵乳品中以乳杆菌居多,对常见抗生素具有一定的敏感性。     

林麝肠道乳酸菌及肠杆菌多态性分析及耐药性研究

为研究林麝肠道中乳酸菌和肠杆菌基因型及表型多样性并对其耐药性进行分析,本研究从10头健康成年林麝粪便样品中分离到22株乳酸菌和16株肠杆菌,通过16S rDNA进化分析和生理生化测定对分离菌株进行了鉴定,采用脉冲场凝胶电泳(PFGE)技术分析了分离菌株的基因型并利用药敏纸片法进行了分离菌株的耐药性研究。本研究共分离到了11种细菌,分别为大肠杆菌Escherichia coli、阴沟肠杆菌Enterobacter cloaca、河生肠杆菌E.amnigenus、肺炎克雷伯氏菌Klebsiella pneumoniae、蒙氏肠球菌Enterococcus mundtii、耐久肠球菌E.durans、明串球菌Leuconostoc fallax、植物乳杆菌Lactobacillus plantarum、嗜酸乳杆菌L.acidipiscis、屎肠球菌E.faecium和食窦魏斯氏菌Weissella cibaria。其中大肠杆菌(n=11,28.9%)是林麝肠道中的优势菌群,肠球菌(n=10,26.3%)是林麝肠道中的优势乳酸菌群。PFGE分型结果表明肠杆菌分为7个基因型,乳酸菌分为9个基因型。本研究首次分析了林麝肠道中可培养细菌的基因型及表型并且对其耐药性进行了测定,结果表明林麝肠道中的原生菌群多态性明显,并且对目前常用的抗生素敏感,未检测到耐药菌株。     

长寿老人源产细菌素乳酸菌的筛选与分子生物学鉴定

本研究以124株我国广西巴马百岁以上长寿老人源乳酸菌菌株为试材,采用双层琼脂平板扩散法筛选产细菌素的优良菌株。在排除有机酸、H2O2等的干扰后,菌株B02、B03、B04、B07、B11、B25、B24和B78的发酵上清液对受试的大肠埃希菌、金黄色葡萄球菌等5株指示菌都表现出很强的抑制作用;进一步硫酸铵沉淀、透析及浓缩处理后,其抑菌活性显著增强,同时蛋白酶敏感性试验显示其具有蛋白质性质,这些结果共同确定其为乳酸菌细菌素。最后,通过16S rRNA序列分析鉴定后确定B02为副干酪乳杆菌;B03为植物乳杆菌;B04为动物双歧杆菌;B07为干酪乳杆菌;B11为德氏乳杆菌保加利亚亚种;B24为鼠李糖乳杆菌;B25为粪肠球菌;B78为植物乳杆菌。     

Viili乳制品中干酪乳杆菌的分离鉴定

从引进Viili乳制品中筛选、鉴定出3种优良乳酸菌。采用平板分离法从Viili乳制品中分离3株乳酸菌,通过表型1、6S rRNA的PCR扩增、克隆、测序鉴定。3株乳酸菌的表型鉴定结果符合伯杰细菌鉴定手册中乳杆菌属的干酪乳杆菌,16S rRNA序列同源性分析结果表明3株分离菌与干酪乳杆菌的同源性分别为99.93%、100.00%9、9.78%,从Viili乳制品分离到3株干酪乳杆菌。     

葡萄藤叶青贮中乳酸菌的分离与鉴定

为了筛选出性状优良的乳酸菌菌株,本试验以实验室纯培养方式从葡萄藤叶自然发酵的青贮中分离鉴定乳酸菌。经过菌落形态、细胞形态、生理生化鉴定和16S r DNA基因序列以及系统发育树的分析,从葡萄藤叶青贮中分离出5株乳酸菌,其中菌株P-1. 2和P-2. 2为戊糖乳杆菌(Lactobacillus pentosus),菌株P-1. 7为戊糖片球菌(Pediococcus pentosaceus),菌株P-2. 1为粪肠球菌(Enterococcus faecium),菌株P-2. 3为短乳杆菌(Lactobacillus brevis)。测定产酸速率和生长速率,发现菌株P-2. 1的产酸能力和生长性能最好。     

五株乳酸菌和三株芽孢杆菌的生物学特性和功能

【背景】乳酸菌和芽孢杆菌是应用于生产最多的益生菌,但不同菌株间的生长特性均不相同,因此了解菌株的生物学特性具有重要意义。【目的】研究菌株的生物学特性,能合理地开发和利用菌株,以保证菌株生产应用的安全性。【方法】活化后鉴定5株乳酸菌和3株芽孢杆菌并对其形态进行观察,探究菌株的生长曲线、产酸能力及最适生长条件,测定菌株的抑菌活性和产酶性能,同时探究菌株的益生性和安全性。【结果】五株乳酸菌分别编号鉴定为干酪乳杆菌R1、副干酪乳杆菌R2、香肠乳杆菌R3、福莱乳杆菌R4和唾液乳杆菌R5;3株芽孢杆菌分别编号命名为贝莱斯芽孢杆菌Y1、枯草芽孢杆菌Y2和地衣芽孢杆菌Y3。八株菌形态结构均不相同但都为杆状,均在2–10 h为对数生长期,18–24 h为稳定期,培养24 h时乳酸菌和芽孢杆菌的活菌数均保持在10⁹和10⁸ CFU/mL,最适生长温度为37.0℃。乳酸菌具有较强的产酸能力和抑菌活性,芽孢杆菌有较强的产酶性,在人工胃液中都有较强的耐受性。八株菌都无溶血活性、无毒力基因、对抗生素都保持中度敏感以上;其中唾液乳杆菌有四环素耐药基因,但对四环素抗性为中度敏感。【结论】八株菌生长繁殖速度快,乳酸菌产酸能力和抑菌活性较强,芽孢杆菌具有较强的产酶性能,在体外具有较好的益生性和安全性,可应用于生产实践。     

具有降胆固醇功能益生菌的筛选研究

目的通过体外实验从发酵制品及人体肠道内分离筛选出安全高效的降胆固醇乳酸菌,并研究其生理生化特性,为今后功能性乳制品的开发提供优良菌种。方法应用MRS．胆固醇培养基液体发酵法进行目的菌株筛选,并测定其耐酸及耐胆盐等能力。结果筛选出4株具有降胆固醇能力的乳酸菌,其降解率分别为DM8403163．1％、DM8403470．3％、DM850381．1％、DM8605663．1％;同时4株乳酸菌均表现出不同程度的耐酸及耐胆盐能力,其中DM86056能力最强,其他3株乳酸菌次之;经鉴定,DM84031为保加利亚乳杆菌、DM84034为嗜热链球菌、DM8503为詹氏乳杆菌、DN86056为屎肠球菌。结论经上述实验综合比较,DM86056降胆固醇能力比较强且耐酸及耐胆盐等能力也较强,因此有待于将其应用于动物体内进行深入研究。     

浆水中降胆固醇乳酸菌的筛选及其功能特性

摘要:【目的】获取高降胆固醇菌种并明确其功能特性。【方法】以浆水为实验材料,利用高降胆固醇培养基筛选出降胆固醇的乳酸菌,并研究高降胆固醇菌株的耐酸、耐盐等功能特性,而后采用生理生化特性鉴定和16S rDNA分子生物学鉴定结合的方法鉴定高降胆固醇菌株的种属。【结果】所分离的乳酸菌都有一定的降胆固醇能力,其中有4株菌对培养物中胆固醇的降解率大于75%,经鉴定发现有乳酸乳球菌乳亚种(Lactococcus lactis subsp． lactis)1株,乳酪短杆菌(Brevibacterium casei)2株,棉籽糖乳球菌(Lactococcus raffinolactis)1株。【结论】从浆水中筛选出4 株高降胆固醇乳酸菌,且其功能性质良好,有进一步开发利用价值。     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

苜蓿青贮用乳酸菌复合系Al2的组成多样性

苜蓿是高蛋白的饲料作物,青贮是保存青草过冬的主要手段,但苜蓿是难青贮的作物,添加乳酸菌制剂是解决苜蓿青贮难的有效措施。本研究室通过连续的限制性培养筛选到苜蓿青贮用乳酸菌复合系Al2,用变性梯度凝胶电泳(DGGE)、平板分离及建立16S rDNA克隆文库3种手段相结合分析Al2的组成多样性,确认Al2复合菌系由7株菌组成,全部属于Lactobacillus。其中3株菌Al2-1i,Al2-2i,Al2-3i通过平板分离得到,分别属于L.plantarum(99.9%)、L.kimchii(99.4%)、L.farciminis(100%)。在Al2复合菌系的16S rDNA克隆文库中,上述7种菌的组成比例分别为55.21%、19.79%、14.58%、3.13%、3.13%、3.13%、1.03%。     

Monitoring Lactobacillus plantarum in grass silages with the aid of 16S rDNA-based quantitative real-time PCR assays

Ensiling plant material with the aid of lactic acid bacteria (LAB) is a common agricultural practice for conserving forages independently of the time point of harvest. Despite ensiling being a natural process, it can be improved by the treatment of the harvested forage with starter cultures before storage. Within this context, Lactobacillus plantarum (L. plantarum) is the most frequently used LAB in commercially available starter cultures. In order to enable the monitoring of the population dynamics of L. plantarum in silage, methods for species-specific detection based on the 16S ribosomal DNA (rDNA) sequence were developed by applying a quantitative real-time polymerase chain reaction (QRT-PCR) approach. The QRT-PCR assay was also applied to estimate the development of the L. plantarum population within experimental grass silages. In addition, a multiplex QRT-PCR assay was developed to estimate the amount of L. plantarum 16S rDNA in relation to total bacterial 16S rDNA. This multiplex QRT-PCR assay was applied to monitor the influence of different silage additives on the L. plantarum population.     

Bioconversion of oleuropein to hydroxytyrosol by lactic acid bacteria

The aim of this work is to study the conversion of oleuropein-a polyphenol present in olives and olive oil by-products-into hydroxytyrosol, a polyphenol with antioxidant and antibacterial properties. The hydrolysis reaction is performed by lactic acid bacteria. Six bacterial strains (Lactobacillus plantarum 6907, Lactobacillus paracasei 9192, Lactobacillus casei, Bifidobacterium lactis BO, Enterococcus faecium 32, Lactobacillus LAFTI 10) were tested under aerobic and anaerobic conditions. The oleuropein degradation and hydroxytyrosol formation were monitored by HPLC. Results showed that oleuropein could be successfully converted into hydroxytyrosol. The most effective strain was Lactobacillus plantarum 6907, with a reaction yield of hydroxytyrosol of about 30 %. Different reaction mechanisms were observed for different microorganisms; a different yield was observed for Lactobacillus paracasei 9192 under aerobic or anaerobic conditions and an intermediate metabolite (oleuropein aglycone) was detected for Lactobacillus paracasei 9192 and Lactobacillus plantarum 6907 only. This study could have significant applications, as this reaction can be used to increase the value of olive oil by-products and/or to improve the taste of unripe olives.     

Effect of NaCl-tolerant lactic acid bacteria and NaCl on the fermentation characteristics and aerobic stability of silage

NaCl-tolerant lactic acid bacteria (LAB) strains LC-10 ( Lactobacillus casei ) and LP-15 ( Lact. plantarum ) and NaCl were used as additives to sorghun ( Sorghum bicolor ). Numbers of LAB were significantly ( P < 0·05) higher in all the additive-treated silages than in the control silage at an early stage of ensiling. During the fermentation process, addition of NaCl or LAB effectively inhibited the growth of aerobic bacteria and clostridia, but not yeasts. All the additive-treated silages had significantly ( P < 0·05) lower pH, ammonia nitrogen content, dry matter loss and gas production but significantly ( P < 0·05) higher lactic acid content and residual water soluble carbohydrates compared with the control silage. The improvement in silage quality was in the order : LAB > NaCl > control. Yeast counts were high in all additive-based silages and they increased during the exposure of the silages to air. As a result, these silages suffered aerobic deterioration, whereas the control silage was stable. The results confirmed that the NaCl or LAB improved fermentation quality but did not prevent aerobic deterioration of the silage.     

接种植物乳杆菌(Lactobacillus plantarum)对小规模饲料稻青贮品质的影响

【目的】研究接种植物乳杆菌对小规模饲料稻品质的影响。【方法】以自然发酵的样品为对照,接种不同来源植物乳酸菌发酵饲料稻,发酵30 d后对饲料稻的感官进行评价;通过选择性平板对饲料稻青贮中的不同微生物进行计数;并采用V-Score评价法对发酵品质进行评定。【结果】相对自然发酵的样品而言,接种植物乳杆菌的青贮样品感官评分等级达到优良;乳酸菌为优势菌株,引起腐败变质的好氧菌、霉菌、大肠杆菌等受到抑制;接种发酵的样品中乳酸含量明显增加,氨态氮的产生量为对照的1/2左右,V-Score评分为满分。【结论】供试的植物乳杆菌,尤其是从青饲料和青贮材料中分离的菌株能有效改善饲料稻青贮的品质,可考虑用作青贮饲料稻发酵剂。     

Degradation of fructans by epiphytic and inoculated lactic acid bacteria and by plant enzymes during ensilage of normal and sterile hybrid ryegrass

Degradation of grass fructans by epiphytic or inoculated lactic acid bacteria during ensilage was examined using both normal and sterile hybrid ryegrass. It was clear that even in the absence of bacteria fructan degradation occurred, but at a significantly slower rate than in normal grass which had not been inoculated with lactic acid bacteria. Fructan degradation in sterile herbage suggests that plant fructan hydrolases were partially responsible for this process in all herbages, irrespective of treatment. Inoculation of sterile herbage with a strain of Lactobacillus plantarum known to lack the ability to degrade grass fructans resulted in a slower rate of fructan breakdown than when inoculated with Lactobacillus casei subsp. paracasei , a confirmed fructan degrader. In the later stages of the fermentation of uninoculated normal herbage when water-soluble carbohydrate appeared to be limiting, lactic acid was fermented to acetic acid. However, this fermentation pathway was not observed in either of the inoculated silages. The results suggest that silage inoculant bacteria possessing fructan hydrolase activity may have potential for improving silage fermentation, particularly when late cut, low sugar grass containing a high proportion of fructans is ensiled.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Application of antimicrobial-producing lactic acid bacteria to control pathogens in ready-to-use vegetables

Five psychrotrophic strains of lactic acid bacteria (Lactobacillus casei, Lact. plantarum and Pediococcus spp.) were isolated from 22 samples of commercial salads. These strains were shown to inhibit Aeromonas hydrophila, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus on MRS agar, in salads and in juice prepared from vegetable salads. Lactobacillus casei IMPCLC34 was most effective in reducing total mesophilic bacteria and the coliform group; Aer. hydrophila, Salm. typhimurium and Staph. aureus disappeared after 6 d of storage, while the counts for L. monocytogenes remained constant. The potential application of antimicrobial-producing lactic acid bacteria as biopreservatives of ready-to-use vegetables is suggested.