丹江镇辣椒红酸汤细菌菌群多样性分析及抗食源性致病菌乳酸菌筛选

以贵州丹江镇传统辣椒红酸汤为研究对象,采用Illumina MiSeq高通量测序技术进行细菌菌群多样性分析,结合微生物纯培养技术和双层平板法从中分离优势乳酸菌,通过分子生物学技术对其进行菌种鉴定,并筛选抗食源性致病菌乳酸菌。结果表明,辣椒红酸汤样品中的优势细菌门为厚壁菌门（Firmicutes）和变形菌门（Proteobacteria）;优势细菌属为乳杆菌属（Lactobacillus）。从辣椒红酸汤样品中共分离得到17株乳酸菌,经鉴定,11株为干酪乳杆菌（Lactobacillus casei）、3株为副干酪乳杆菌（Lactobacillus paracasei）、1株为副干酪乳杆菌亚种（Lactobacillus paracasei subsp.）、1株为植物乳杆菌（Lactobacillus plantarum）、1株为乳酸杆菌（Lactobacillus sp.）,其中4株乳酸菌具有较好的产酸和抑菌性能,且干酪乳杆菌ST7-14的抑菌能力最好,对蜡样芽孢杆菌（Bacillus cereus）ST2-1和阴沟肠杆菌（Enterobacter cloacae）ST2-6的抑菌圈直径分别为37.0 mm和31.5 mm。     

西藏地区牦牛发酵乳制品中乳酸菌的分离与鉴定

实验分析西藏牧民家庭制作的牦牛发酵乳制品中酸奶和奶渣的微生物数量,通过菌种选择分离,采用传统分类与16S rRNA基因序列测定的方法对分离的乳酸菌进行了鉴定。结果表明:3份发酵牦牛乳样品中乳酸菌数为1.04×104～2.10×106CFU/ml,酵母菌数为8.20×103～6.70×105CFU/ml;2份奶渣样品中乳酸菌和酵母菌为0.2×10～1.0×10CFU/g;共分离15株乳酸杆菌、4株乳酸球菌和3株酵母;乳酸菌中10株为Lactobacillus paracasei、1株Lactobacillu fermentum、2株Lactobacillus reuteri、1株Lactobacillus brevis、1株Lactobacillus delbrueckii subsp.bulgaricus、2株Pediococcus acidilactici、另有2株分别为1株Pediococcu和1株Lactococcus菌株,未鉴定到种;L.paracasei为牦牛发酵乳制品中乳酸菌的优势种(占总分离株的55.6%)。     

恩施市泡萝卜中乳酸菌的分离鉴定及其对品质的影响

对5份恩施地区泡萝卜中的乳酸菌进行了分离鉴定,同时对其分离株在以萝卜为原料的泡菜中的发酵特性进行了评价。结果表明:分离出18株乳酸菌菌株,分别为隶属于片球菌属(Pediococcus)的戊糖片球菌(P.pentosaceus)和隶属于乳酸杆菌属(Lactobacillus)的食品乳杆菌(L.alimentarius)、短乳杆菌(L.brevis)、副干酪乳杆菌(L.paracasei)、发酵乳杆菌(L.fermentum)和植物乳杆菌(L.plantarum),其中8株分离株为L.plantarum。通过质构分析发现,乳酸菌纯种发酵制备的多数泡萝卜样品硬度和脆性均明显高于自然发酵样品。通过电子鼻分析发现,W1C、W3C和W5C对多数乳酸菌纯种发酵泡萝卜水的响应值明显偏高。通过主成分分析发现,菌株L.paracasei HBUAS51063和L.plantarum HBUAS51053具有相对较佳的发酵特性。由此可见,恩施市泡萝卜中乳酸菌以L.plantarum为主,乳酸菌纯种发酵可提升多数泡萝卜的品质。     

发酵型核桃乳生产专用乳杆菌的选育

从发酵面包、白芥丝、泡菜、酸菜中分离到14株乳杆菌,经核桃乳发酵产酸特性初筛,确定菌株L5、L7、L8及L12适合核桃乳发酵。通过菌落形态、菌体形态和16S rRNA基因序列分析,确定4株菌分别为鼠李糖乳杆菌（Lactobacillus rhamnosus）、副干酪乳杆菌（Lactobacillus paracasei）、食窦魏斯氏菌（Weissella cibaria）干酪乳杆菌（Lactobacillus casei）。将上述菌株与实验室保存的嗜热链球菌SA进行核桃乳发酵实验,感官评分分别为78分、95分、83分和82分;4个发酵核桃乳中9种氨基酸含量均有不同程度提高,其中菌株L7发酵产品的氨基酸含量增幅最大,由0.67%增至1.60%。因此副干酪乳杆菌L7更适合发酵型核桃乳的生产。     

红薯淀粉废水中乳酸菌的筛选及其对食源性致病菌的抑制作用

从采集的红薯淀粉废水中分离出3株乳酸菌SPDJ3、SPEJ7 和SPEJ9。通过培养特征、形态特征,生理生化特征及基于16S rDNA序列的系统发育分析的检测,菌株SPDJ3和SPEJ7被鉴定为植物乳杆菌（Lactobacillus plantarum）,菌株SPEJ9被鉴定为副干酪乳杆菌（Lactobacillus paracasei）。3株乳酸菌发酵上清液对供试8株食源性致病菌均有抑菌作用,菌株SPDJ3对福氏志贺氏菌（Shigella flexneri）抑制效果较强,抑菌圈直径为（30.37±0.15） mm,菌株SPEJ7和SPEJ9对蜡样芽胞杆菌（Bacillus cereus）抑制效果较强,抑菌圈直径分别为（29.43±0.21） mm和（28.27±0.25） mm。     

生物保鲜乳酸菌的筛选及其细菌素特性研究

利用牛津杯法从传统泡菜和腊肠中分离的80株乳酸菌中筛选出10株具有较高抑菌活性的乳酸菌.通过排除酸性产物有机酸、过氧化氢干扰及抑菌谱试验,LAB5和LAB55仍表现广谱的抑菌活性;2菌株发酵上清液经蛋白酶K、木瓜蛋白酶和胰蛋白酶处理后抑菌活性均有降低,其中LAB5对木瓜蛋白酶敏感,因此可以初步判定这2株乳酸菌产生的抑菌物质为细菌素.2种细菌素具有良好的热稳定性,pH值越低抑菌能力越强;2株菌表现良好的产酸能力,经生理生化和16S rDNA鉴定为植物乳杆菌.     

对甘薯淀粉具有絮凝活性的乳酸菌的分离鉴定及其特性研究

从自然发酵甘薯酸浆中分离筛选到一株对甘薯淀粉颗粒具有高絮凝活性的乳酸菌L1,经鉴定该菌为副干酪乳杆菌副干酪亚种(Lactobacillus paracasei subsp. paracasei)。该菌株产絮凝剂的适宜pH值为6.5～8.5,适宜培养温度为25～35℃。葡萄糖、蔗糖、麦芽糖及乳糖均可作为该菌株的良好碳源,其适宜氮源为酵母粉或牛肉膏。通过对絮凝活性分布的研究,确定菌株所产的絮凝剂主要存在于菌体上。     

浓香型窖泥中高产细菌素乳酸菌的鉴定及特性

以双层琼脂扩散法从浓香型窖泥中筛选出1株(LP16)具有高抑菌活性的菌株,研究其代谢产物在窖泥中的作用。结果表明:经形态观察、生理生化试验和16S rRNA鉴定出菌株LP16为副干酪乳杆菌(Lactobacillus paracasei);其发酵上清液在排除有机酸、过氧化氢干扰后仍有明显抑菌效果,而经胰蛋白酶、蛋白酶K、木瓜蛋白酶和胃蛋白酶处理后残余抑菌率分别为40.43%、43.48%、47.83%和51.30%,抑菌活性部分丧失,说明抑菌成分包含蛋白属性的细菌素;菌株LP16所产细菌素分别经高温、低pH(pH<5)、紫外线照射、表面活性剂处理均能保持抑菌活性,且在pH2~10范围内具有pH耐受性;抑菌谱较广,对大肠杆菌、枯草芽孢杆菌以及部分乳酸菌有明显的抑制作用,但对霉菌和酵母菌无抑制作用。因此在浓香型白酒生产中该菌产生的细菌素不仅能抑制病原菌的生长,也能控制乳酸的过多生成。     

新疆酸马奶中乳酸菌的多态性分析及发酵性能研究

以新疆牧民传统家庭自制酸马奶样品为研究对象,考察乳酸菌的多态性及筛选优良性状的乳酸菌菌株。通过16S r RNA基因序列分析和生理生化试验等方法对分离出的菌株进行鉴定,并进行发酵性能测试。结果表明,本实验共分离出19株菌株,包括乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)(8株)、粪肠球菌(Enterococcus faecalis)(2株)、屎肠球菌(Enterococcus faecium)(2株)、嗜热链球菌(Streptococcus thermophilus)(1株)、干酪乳杆菌(Lactobacillus casei)(3株)、植物乳杆菌(Lactobacillus plantarum)(2株)和徳氏乳杆菌乳酸亚种(Lactobacillus delbrueckii subsp.Lactis)(1株)。其中,干酪乳杆菌JDB1.1505是一株产酸和产黏性能都比较突出的乳酸菌,发酵脱脂乳的滴定酸度达到了130.1°T,黏度为1420.1m Pa·s,具有良好的乳品发酵应用潜能。     

副干酪乳酸杆菌所产抑菌物质的特性研究

为了得到具有应用潜力的抑菌活性菌株,对分离自内蒙古牧区家庭手工制作的酸马奶中具有抑菌活性的乳酸杆菌采用双层琼脂平板扩散法进行筛选,并对有抑菌能力的乳酸菌进行分子生物学鉴定,同时对其所产抑菌物质的特性进行研究,筛选出的8株乳酸杆菌均对枯草芽孢杆菌(Bacillus subtilis)有抑制作用,选择其中一株乳杆菌Q-1-4(Lactobacillus paracasei)进行鉴定认定该菌株为副干酪乳杆菌。排除有机酸和过氧化氢干扰后,L.paracasei Q-1-4发酵离心后的无细胞上清液(Cell-free fermentation supernatant,CFS)对B.subtilis仍然有抑制作用,经胰蛋白酶、蛋白酶k、胃蛋白酶和木瓜蛋白酶处理后抑菌活性下降,说明所产生的抑菌物质中应含有蛋白类。并测出其产生的抑菌物质酸性条件下对枯草芽孢杆菌有抑制作用且对热稳定,对紫外线不敏感;与SDS和EDTA共同作用效果好于单一作用,而其它的表面活性剂、有机溶剂、金属离子对其无明显影响;具有一定的储藏稳定性且较广抑菌谱。     

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.     

乳杆菌发酵对黑米多酚含量及组成的影响

以植物乳杆菌（Lactobacillus plantarum 115,LP）、嗜酸乳杆菌（Lactobacillus acidophilus 14,LA）、干酪乳杆菌（Lactobacillus casei GB,LC）、保加利亚乳杆菌（Lactobacillus bulgaricus 134,LB）、副干酪乳杆菌（Lactobacillus paracasei 37,LPC）和鼠李糖乳杆菌（Lactobacillus rhamnosus GG,LGG）为发酵菌种,研究乳杆菌发酵对黑米游离酚和结合酚的含量及组成的影响。结果表明,6株乳杆菌发酵均显著提高了黑米游离酚的含量,但不同乳杆菌发酵后提高的程度具有显著差异,其中提高最显著的是LB。不同乳杆菌发酵后黑米中9种游离态和结合态单体酚的含量变化存在显著差异,其中,6株乳杆菌发酵后黑米中游离态的香草酸和对羟基苯甲酸的含量均显著降低（P<0.05）,LP菌株除外游离态咖啡酸的含量均显著提高（P<0.05）,而6株乳杆菌发酵后结合态阿魏酸含量均显著降低（P<0.05）。该研究为黑米的高效利用和天然活性产品的开发提供参考。     

东北酸菜优良乳酸菌筛选及其抑菌特性研究

该实验从东北酸菜中分离纯化并鉴定出40种乳酸菌,主要为植物乳杆菌（Lactobacillus plantarum）、融合魏斯氏菌（Weissella confuse）、副干酪乳杆菌（Lactobacillus paracasei）、干酪乳杆菌（Lactobacillus casei）、食窦魏斯氏菌（Weissella cibaria）。通过测定乳酸菌乳酸产量及其抑制大肠杆菌（Escherichia coli）的能力,探究乳酸菌的产酸量与其抑菌作用之间相互关系。结果表明,菌株C1、E8、A5产酸能力较强,发酵12 h后发酵上清液中乳酸质量浓度＞12 g/L,抑菌圈直径＞12 mm,对大肠杆菌抑制能力强。表明东北酸菜中乳酸菌的产酸量与抑菌作用存在紧密的联系。     

副干酪乳杆菌Q-1-4的筛选鉴定及抗菌物质特性分析

为获得应用潜力较高的抗菌菌株,对内蒙古地区传统乳制品中分离的8株乳酸杆菌进行抗菌菌株筛选,获得了一株具有广谱抗菌活性的菌株(编号为Q-1-4)。经常规生理生化及分子生物学实验,鉴定该菌为副干酪乳杆菌(Lactobacillus paracasei)。通过实验排除有机酸和过氧化氢干扰后,L.paracasei Q-1-4发酵离心后的无细胞发酵上清液(cell-free fermentation supernatant,CFS)对鼠伤寒沙门氏菌Salmonella typhimurium CMCC50115仍具有抑制作用,且经蛋白酶处理后抗菌活性下降,说明L.paracasei Q-1-4所产抗菌物质中含有蛋白类物质。其在pH 2.0~5.5范围内对鼠伤寒沙门氏菌有抑制作用,且在此pH值范围内该抗菌物质对热稳定,对紫外线不敏感;与十二烷基硫酸钠、乙二胺四乙酸共同作用效果好于单一作用,而其他的表面活性剂、有机溶剂、金属离子对其无明显影响;该抗菌物质具有一定的贮藏稳定性且抑菌谱较广。     

抗食源性致病菌益生菌的筛选及其抑菌活性评价

采用交叉划线法、径向划线法、琼脂扩散法及液体共培养法评价各益生菌菌株及其代谢产物对4 株具代表性食源性致病菌的抑菌作用,并从20?株益生菌中筛选出同时对单核细胞性李斯特菌和金黄色葡萄球菌（革兰氏阳性致病菌）及大肠杆菌和鼠伤寒沙门菌（革兰氏阴性致病菌）具有良好抑菌活性的益生菌菌株。将筛选出的益生菌添加到羟丙基纤维素、丙三醇及魔芋粉的高分子复合材料中制备生物活性抑菌膜,并对抑菌膜中益生菌的存活情况及抑菌膜的抗菌效果进行评价。交叉划线法及径向划线法实验结果一致：乳杆菌属和肠球菌属的菌株,特别是Lactobacillus paracasei CICC 20241菌株对4?株目标致病菌均具有较高的抗菌活性;琼脂扩散法实验表明：L. paracasei CICC 20241菌株的培养液、上清液和菌悬液均具有较好的抑菌效果;液体共培养法实验表明：乳酸菌L. paracasei CICC 20241先于致病菌接种时表现出更高的生长活性和拮抗活性。此外,本实验制备的抑菌膜是L. paracasei CICC 20241菌株的良好载体,适宜该菌株的生长存活并对目标致病菌表现出持续的抗菌活性,对于开发鲜食食品的包装材料具有较大的应用潜力。     

湘西传统酸鱼中乳酸菌的分离及特性研究

对从湘西传统酸鱼4个不同发酵阶段分离的196株乳酸菌种类进行了研究,分离出的乳酸菌主要包括植物乳杆菌、戊糖片球菌、食品乳杆菌、明串珠菌、肠球菌及干酪乳杆菌等,并且这些乳酸菌在发酵过程中呈现出一种动态的变化进程。同时对分离到的乳酸菌进行了一系列的生理生化试验;产酸速率实验,耐食盐、耐亚硝盐实验,耐酸及耐胆盐实验,氨基酸脱羧酶实验以及抑菌实验,选育出具有较好的发酵剂潜力的3株植物乳杆菌和1株戊糖片球菌。     

Phenotypic and molecular assessment of antimicrobial resistance in Lactobacillus paracasei strains of food origin

Antimicrobial resistance data in food-associated lactic acid bacteria (LAB) such as lactobacilli are mostly based on nonstandardized methodologies and/or have been obtained for only a limited number of strains. This susceptibility study included a diverse collection of 115 isolates mainly of food origin originally identified as Lactobacillus paracasei or Lactobacillus casei. Upon reidentification and removal of potential replicate isolates using repetitive DNA element PCR fingerprinting, 65 genotypically unique L. paracasei strains and the L. casei type strain were selected for broth microdilution and Etest assays using the LAB susceptibility test medium. In both methodologies, strains appeared uniformly susceptible to ampicillin and clindamycin but exhibited natural resistance to streptomycin and gentamicin. Three L. paracasei strains from cheese displayed acquired resistance to tetracycline (MIC > or = 32 microg/ml) and/or to erythromycin (MIC >16 microg/ml), which was linked to the presence of a tet(M) or tet(W) gene and/or an erm(B) gene, respectively. Partial sequencing revealed that the tet(M) genes found in two of these strains belonged to two tet(M) sequence homology groups previously found in enterococci. Collectively, phenotypic and genotypic data allowed us to propose tentative epidemiological cutoffs for L. paracasei and L. casei for differentiating susceptible strains from those strains harboring one or more acquired resistance factors.     

Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation

Autochthonous strains of lactic acid bacteria (LAB) have been isolated from traditionally homemade cheeses collected from specific ecological localities across Serbia and Montenegro. Genetic and biochemical analysis of this LAB revealed that they produce bacteriocins, proteinases and exopolysaccharides. LAB produces a variety of antimicrobial substances with potential importance for food fermentation and preservation. Apart from the metabolic end products, some strains also secrete antimicrobial substances known as bacteriocins. Among the natural isolates of LAB from homemade cheeses, bacteriocin producers were found in both lactococci and lactobacilli. Lactococcus lactis subsp. lactis BGMN1-5 was found to produce three narrow spectrum class II heat-stable bacteriocins. In addition to bacteriocin production, BGMN1-5 synthesized a cell envelope-associated proteinase (CEP) and shows an aggregation phenotype. Another isolate, L. lactis subsp. lactis BGSM1-19 produces low molecular mass (7 kDa) bacteriocin SM19 that showed antimicrobial activity against Staphylococcus aureus, Micrococcus flavus and partially against Salmonella paratyphi. Production of bacteriocin reaches a plateau after 8 h of BGSM1-19 growth. Bacteriocin SM19 retained activity within the wide pH range from 1 to 12 and after the treatment at 100 degrees C for 15 min. Among collection of lactobacilli, the isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8 produces heat-stable bacteriocin SJ (approx. 5 kDa) polypeptide. It retained activity after treatment for 1 h at 100 degrees C, and in the pH range from 2 to 11. In addition to isolates from cheeses, bacteriocin-producing human oral lactobacilli were detected. Most of them showed antimicrobial activity against streptococci, staphylococci and micrococci, but not against Candida. Isolate BGHO1 that showed the highest antimicrobial activity was determined as L. paracasei. Interestingly, Lactobacillus helveticus BGRA43, which was isolated from the human intestine showed strong activity against Clostridium sporogenes, but it was not possible to detect any bacteriocin production in this isolate by using standard procedures. Further analysis of antimicrobial activity revealed that BGRA43 has a relatively broad spectrum. Lactobacilli resistant to nisin were also detected among natural isolates. They produce bacteriocins, which have no activity against nisin producing lactococci.     

白酒窖泥中乳酸菌分离鉴定及其发酵产挥发性风味物质比较

从白酒窖泥中分离得到11株乳酸菌,经16S rDNA基因序列同源性分析,鉴定为1株短乳杆菌、1株鼠李糖乳杆菌、2株干酪乳杆菌和7株铅黄肠球菌。分析了11株菌MRS培养基发酵产乳酸性能,结果表明:乳酸产量与菌群生长呈正相关,鼠李糖乳杆菌L9、干酪乳杆菌L10与L11发酵液乳酸含量较高,分别为15.74、15.58、14.74 g/L。4株代表菌相同条件下发酵高粱培养液产挥发性风味组分,共有物质13种,包含了白酒中重要香气成分:乙酸、己酸、乙酸苯乙酯和苯乙醇,且乙酸、己酸相对含量较高。各菌产可挥发性组分差异明显,相对含量较高的化合物种类为:短乳杆菌L5产烷烃类化合物(27.91%),铅黄肠球菌L8产醇类化合物(43.14%),鼠李糖乳杆菌L9产酯类(7.35%)、酮类(3.61%)和吡嗪类(3.3%)化合物,干酪乳杆菌L11产酸类(27.98%)和芳香类(5.96%)化合物。仅有短乳杆菌L5产四甲基吡嗪,短乳杆菌L5和铅黄肠球菌L8可明显产乙醇,鼠李糖乳杆菌L9和干酪乳杆菌L11产3-羟基-2-丁酮。这些物质对白酒风味和口感有重要影响。研究显示,窖泥中各种乳酸菌特征不一,对白酒酿造有不同影响。本研究旨在为进一步挖掘白酒酿造功能微生物、拓展乳酸菌的应用提供理论依据。