贵州地区青贮饲料中天然乳酸菌分离鉴定及优良菌株筛选

为改善和提高青贮饲料的品质及筛选优良乳酸菌,本试验从贵州地区玉米青贮饲料中分离到175株天然乳酸菌,通过形态特征、生理生化和16S rRNA序列测定对乳酸菌多样性进行分析,再根据耐温、耐酸性试验筛选出优良乳酸菌,并对菌株的生长速率、乳酸产量进行测定。结果表明：分离出的乳酸菌有7种,其中主要菌种为植物乳杆菌（L.plantarum）,其余包括短乳杆菌（L.brevis）、副干酪乳杆菌（L.paracasei）、鼠李糖乳杆菌（L.rhamnosus）、戊糖片球菌（P.pentosaceus）、布氏乳杆菌（L.parabuchneri）和罗斯乳杆菌（L.rossiae）。从中初步筛选出10株生长速率较快、产酸能力较强的乳酸菌,有3株（DF-20、DS-45和FG-77）植物乳杆菌,2株（DS-69和FG-85）短乳杆菌,2株（DS-71和RH-135）戊糖片球菌,1株（DF-12）副干酪乳杆菌,1株（QZ-35）类布氏乳杆菌,1株（HP-166）鼠李糖乳杆菌。其中菌株DS-69和RH-135具有较强的高温适应性和酸性环境适应性,且产酸能力强,可作为良好乳酸菌添加剂应用于青贮饲料中。 [关键词] 玉米|青贮|乳酸菌|生长特性     

3．摄入益生茵能够降低二甲肼对大鼠结肠的基因毒性

印度学者研究了嗜酸乳杆菌和干酪乳杆菌对二甲肼（DMH）基因毒性的降低作用。他们将实验大鼠随机分为4组,第一组为DMH对照组：第二组为益生菌凝乳组（凝乳中加入嗜酸乳杆菌和干酪乳杆菌）,饲喂益生菌凝乳并进行DMH皮下注射：第三组为普通凝乳对照组,     

嗜酸乳杆菌、双歧杆菌和干酪乳杆菌的选择性计数

LC培养基只能计数干酪乳杆菌，MRS－水杨素（或山梨醇）培养基可以计数嗜酸乳杆菌和干酪乳杆菌，而MRS培养基可以计数嗜酸乳杆菌、干酪乳杆菌和双歧杆菌，于是通过减法原则就能从混合物中单独计数它们。另外，MRS－NNLP培养基也可用于选择性计数双歧杆菌。     

牦牛“奶渣”中乳酸菌的分离鉴定

本文对产自香格里拉的鲜＂奶渣＂和发酵＂奶渣＂中的乳酸菌进行分离鉴定。结果表明,从鲜＂奶渣＂中分离到27株乳酸菌,并鉴定出7种（亚种）,其中德氏乳杆菌、麦芽香乳杆菌、发酵乳杆菌和乳酸乳球菌4种菌被确定是鲜＂奶渣＂中的优势种群。从发酵＂奶渣＂中分离到40株乳酸菌,并鉴定出9个种（亚种）,其中德氏乳杆菌、麦芽香乳杆菌、发酵乳杆菌、嗜酸乳杆菌和乳酸乳球菌5种菌被确定是发酵＂奶渣＂中的优势种群。     

鸡源乳酸菌的分离鉴定

研究采用MRS培养基从健康AA肉鸡十二指肠、空肠、回肠及盲肠中分离获得11株乳酸菌,并对其进行形态观察、生理生化试验及16S r DNA序列同源性分析,最终确定所分离的菌株主要是5种乳酸杆菌(嗜酸乳杆菌R1、植物乳杆菌R2、鼠李糖乳杆菌R3、短乳杆菌R4和发酵乳杆菌R5)和4种肠球菌(盲肠肠球菌R9、粪肠球菌R8和R10、鸟肠球菌R7、屎肠球菌R6和R11)。     

内蒙古发酵酸乳制品中乳酸菌的分离鉴定

以采集自内蒙古锡林郭勒盟正蓝旗的2份发酵酸乳样品为研究对象,采用传统纯化培养技术、16S rDNA基因序列测定并构建系统发育树的方法,对样品中的乳酸菌进行了分离鉴定,以期解析发酵乳中乳酸菌的多样性,积累食品微生物资源。研究结果显示,共分离15株乳酸菌,被鉴定为3个属9个种,3个属分别为乳酸乳球菌属、明串珠菌属和乳杆菌属,9个种分别为乳酸乳球菌叶蝉亚种（Lactococcus lactis subsp. hordniae,3株）、乳酸乳球菌（Lactococcus lactis,1株）、乳酸乳球菌乳脂亚种（Lactococcus lactis subsp. cremoris,1株）、乳明串珠菌（Leuconostoc lactis,3株）、肠膜明串珠菌肠膜亚种（Leuconostoc mesenteroides subsp. mesenteroides,1株）、肠膜明串珠菌右旋葡聚糖亚种（Leuconostoc mesenteroides subsp. dextranicum,1株）、植物乳杆菌（Lactobacillus plantarum,1株）、副干酪乳杆菌（Lactobacillus paracasei,3株）和干酪乳杆菌（Lactobacillus casei,1株）。综上表明,该研究采集的酸乳样品中乳酸菌资源丰富,是内蒙古传统乳制品优良发酵剂的研发和产业化生产的潜在微生物资源。     

鸡肠黏膜乳酸菌的抗菌能力及其对抗菌素的药敏特性

从肉鸡空肠黏膜上分离到6株菌，均为革兰氏阳性菌，其中3株乳杆菌（R2、R4、R6），3株肠球菌（R1、R3、R5）。生理生化鉴定表明，R2为嗜酸乳杆菌；R4为德氏乳杆菌乳亚种；R6为德氏乳杆菌德氏亚种；R1、R3和R5为粪肠球菌。产乳酸能力分析表明，培养至12h时R2产乳酸速度最快，到24h时R4产生乳酸浓度最高，为69．59mmol／L。在抑菌试验中，R2、R4对鸡大肠杆菌O1、鸡伤寒沙门氏菌O9和金黄色葡萄球菌的抑制作用显著高于其它菌株（P〈0．05）；药敏试验结果表明，R2和R4对治疗性抗菌索青霉素、四环索、卡那霉素、链霉素、万古霉素有较强的耐受性；对添加剂用抗菌索二硝托胺、硫酸多粘菌素、氨苯胂酸、杆菌肽锌的耐受性较强。     

自然发酵牦牛酸奶中乳酸菌的分离鉴定

为系统揭示青海部分地区自然发酵牦牛乳中的微生物乳酸菌种群结构,丰富牦牛乳微生物菌种资源库数据,本试验通过纯培养方法对采集于青海省海北藏族自治州海晏县、黄南藏族自治州泽库县及同仁市3个不同地区当地居民自然发酵的牦牛酸奶分离得到10株乳酸菌,并利用生化鉴定试剂盒对该10株乳酸菌进行了菌种的鉴定,结果表明4株杆菌分离株和6株球菌分离株。其中6株归为嗜热链球菌,2株归为嗜酸乳杆菌,1株归为罗伊氏乳杆菌,1株归为干酪乳杆菌干酪亚种。其中,海晏县样品菌种为干酪乳杆菌干酪亚种和嗜热链球菌,同仁县样品中的菌种为嗜酸乳杆菌、嗜热链球菌和罗伊氏乳杆菌,泽库县样品中的菌种为嗜热链球菌。研究结果为青海地区牦牛乳酸菌资源数据库的丰富提供了依据。     

益生菌在养猪生产上的应用研究进展

益生菌（probiotics）作为一种安全无污染、无残留、无耐药性的新型微生物饲料添加剂,已被广泛应用于畜禽业并成为国内外养殖者关注的热点。各国对于应用生产的益生菌菌种规定不同。2009年,美国食品与药物管理局（FDA）和美国饲料控制官员协会（AAFCO）公布了可直接饲喂的46种微生物菌种名单,我国农业部2008年第1126号公告《饲料添加剂品种目录》中允许的饲料级微生物添加剂菌种仅有16种,它们包括：粪肠球菌、屎肠球菌、乳酸肠球菌、枯草芽孢杆菌、两歧双歧杆菌、地衣芽孢杆菌、嗜酸乳杆菌、乳酸乳杆菌、干酪乳杆菌、植物乳杆菌、戊糖片球菌、乳酸片球菌、酿酒酵母、产朊假丝酵母、沼泽红假单胞菌和保加利亚乳杆菌。而中华人民共和国农业部公告第2045号《饲料添加剂品种目录（2013）》中已将可直接添加微生物饲料添加剂增加到34种。目前应用于养猪业主要的两种益生菌制剂是乳酸菌类和芽孢杆菌类。     

内蒙古通辽市牧区传统乳制品中乳杆菌的分离鉴定

试验从内蒙古通辽市科左后旗和扎鲁特旗牧区自产的乳样中分离到29株乳杆前,并对其进行了鉴定。结果显示．优势菌株植物乳杆菌21株,玉米乳杆菌、干酪乳杆菌、肠孔杆菌、短乳杆菌和嗜酸乳杆菌各1株,另有3株没有鉴定到种。     

开菲尔粒菌相构成的研究

开菲尔乳的风味和特性与开菲尔粒的菌相组成密切相关,对来自波兰的开菲尔粒中的微生物菌相进行分离鉴定表明,该开菲尔粒主要由干酪乳杆菌(9株)、乳酸乳球菌乳酸亚种(6株)、肠膜明串珠菌右旋匍聚糖亚种(3株)、酿酒酵母(2株)以及巨大克鲁维氏酵母(5株)构成.     

Sequencing of variable regions of the 16S rRNA gene for identification of lactic acid bacteria isolated from the intestinal microbiota of healthy salmonids

The aim of this study was to identify lactic acid bacteria (LAB) using polymerase chain reaction (PCR) amplification of variable regions of the 16S rRNA gene. Thirteen LAB strains were isolated from the intestinal microbiota of healthy salmonids. A approximately 500-bp region of the highly conserved 16S rRNA gene was PCR-amplified and following this, a portion of the amplicon (272-bp) including the V1 and V2 variable regions was sequenced. The sequence containing both the V1 and V2 region provided strong evidence for the identification of LAB. The LAB strains were identified as Carnobacterium maltaromaticum, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, and Leuconostoc mesenteroides. The method described was found to be a very simple, rapid, specific, and low-cost tool for the identification of unknown strains of LAB.     

小花棘豆与玉米混贮微生物特性及脱除苦马豆素乳酸菌的筛选

旨在探讨小花棘豆与玉米按不同比例混贮对微生物特性及乳酸菌多样性影响,挖掘具有脱除苦马豆素活性的乳酸菌,为小花棘豆青贮饲料的开发利用提供理论参考。以小花棘豆和全株玉米为原料,按不同比例10:0(T₁)、9:1(T₂)、8:2(T₃)7:3(T₄)、6:4(T₅)、5:5(T₆)、4:6(T₇)、0:10(T₈)混合青贮,室温发酵60 d,检测青贮前、后微生物种类、数量变化,鉴定分离出的乳酸菌,并测定其对苦马豆素的脱除率。试验结果:1)原料中乳酸菌数量随着玉米混入量的增加逐渐升高,青贮饲料各处理间乳酸菌数量无显著差异;原料中的肠细菌各处理间无显著差异,经过青贮发酵后肠细菌均未检测到;好氧细菌数量在原料和青贮饲料中均随着玉米比例的增加逐渐减少;酵母菌数量在各处理间均无显著差异;原料中的霉菌数量随着玉米混入比例增加逐渐增加,而青贮饲料中的霉菌数量均在检出线以下。2)从小花棘豆与玉米混贮的原料中共分离得到乳酸菌菌株4株,经鉴定属于Lactococcus lactis subsp. hordniae、Lactococcus lactis subsp. lactis和Lactococcus taiwanensis 3种;从青贮饲料中分离出乳酸菌菌株9株,经鉴定属于Lactobacillus plantarum subsp. plantarum、Lactobacillus brevis、Lactobacillus pentosus和Lactobacillus amylovorus 4种,当玉米混入比例升高时,原料中乳酸菌多样性有所增加,而青贮饲料中乳酸菌多样性有所下降。3)不同乳酸菌菌株对苦马豆素的脱除率均高于85%,其中菌株JD6E、JD4D、JD10D、JD2E和JD1F对苦马豆素的脱除率高达100%。经过热处理后,菌株JD10D和JD2E对苦马豆素的脱除率分别为100%和97.76%,而其他菌株对苦马豆素的脱除率下降31.76%~100%,其中菌株JD6D和JD1E经过热处理后对苦马豆素的脱除率分别下降到2.17%和0。结果表明,小花棘豆与玉米混贮可以增加青贮原料中乳酸菌的数量及多样性,降低青贮饲料和原料中好氧细菌的数量,有助于提高青贮成功率;乳酸菌对苦马豆素具有良好的脱除效果,菌株JD10D和JD2E对苦马豆素的吸附脱除作用最强,而菌株JD6D和JD1E对苦马豆素的降解作用最强,均可用作小花棘豆青贮脱除苦马豆素的发酵菌株。     

青贮饲料中优良乳酸菌的分离鉴定

试验从12种不同来源的青贮饲料中分离到25株乳酸菌,依据伯杰氏系统细菌学手册的标准和描述,把25株乳酸菌都鉴定到了属的水平,有17株乳酸菌鉴定到种的水平,其中:玉米乳杆菌2株、布氏乳杆菌4株、短乳杆菌4株、戊糖乳杆菌3株、干酪乳杆菌1株、粪肠球菌1株、坚强肠球菌2株。其余的8株乳酸菌根据本研究的结果还无法确定其种的归属。测定同时已鉴定的17株乳酸菌的产酸速率,结果发现,Sld2-1、Ssd3、Shw3-7和Ssm3-1都表现出较强的产酸能力。     

苜蓿青贮优势菌种筛选及应用效果

本试验旨在筛选自然发酵苜蓿青贮的优势菌种,应用于高水分苜蓿青贮的调制,研究不同微生物及组合对高水分苜蓿青贮品质的影响。试验先调制自然发酵苜蓿,青贮30 d后,从中筛选优势菌种并进行鉴定。根据优势菌种筛选结果,组合了9组发酵菌种用于苜蓿青贮的调制,分别为:以植物乳杆菌与乳酸片球菌为发酵菌种,活菌数比例分别为1∶1、1∶2和2∶1,编号为1、2、3组;以植物乳杆菌、枯草芽孢杆菌和乳酸片球菌为发酵菌种,活菌数比例分别为2∶1∶1、1∶1∶1和4∶1∶1,编号为4、5、6组;以植物乳杆菌、解淀粉芽孢杆菌和乳酸片球菌为发酵菌种,活菌数比例分别为2∶1∶1、1∶1∶1和4∶1∶1,编号为7、8、9组;以不加任何菌种的自然苜蓿青贮作为对照。每组3个重复,发酵60 d后,对苜蓿青贮进行感官评定,并测定氨态氮/总氮(NH3-N/TN)、总酸含量和pH。结果表明:用MRS、LB和NA 3种培养基从自然发酵苜蓿青贮中筛选到微生物菌种31株,对其中18株进行了分子鉴定,主要菌种为植物乳杆菌9株、乳酸片球菌1株、粪肠球菌2株、各种芽孢杆菌6株。感官评分结果表明乳酸菌(植物乳杆菌和乳酸片球菌)比例大的各组评分等级显著高于对照组和添加了添加枯草芽孢杆菌的各组(P<0.05)。对于NH3-N/TN、总酸含量和pH等指标,乳酸菌比例较低的2、5、8和对照组与其余各组差异显著(P<0.05)。综上所述,植物乳杆菌和乳酸片球菌是苜蓿青贮主要菌种,用于苜蓿青贮有利于青贮品质的提高。枯草芽孢杆菌会影响苜蓿青贮的气味,解淀粉芽孢杆菌为菌种会改善苜蓿青贮风味,但比例太高会增加NH3-N含量。     

In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against fish pathogens

The present study describes the screening of five lactic acid bacteria (LAB) for use as probiotics based on their competitive adhesion and production of antagonistic substances against some fish pathogens. A reduction of adhesion of all pathogenic strains tested was obtained with three of the LAB strains (Lactococcus lactis subsp. lactis CLFP100, Lactococcus lactis subsp. cremoris CLFP102 and Lactobacillus curvatus CLFP150). With the exception of fish pathogens Flavobacterium psychrophilum and Renibacterium salmoninarum that were not inhibited by LAB strains, production of antagonistic compounds by all tested LAB was observed against at least one of the indicator strains. Based on mucus adhesion, competitive exclusion, and suppression of fish pathogen growth, the selected LAB strains can be considered for future challenge experiments in fish as a very promising alternative to the use of chemotherapeutic agents.     

中国传统乳制品中优良乳酸菌的筛选及复合菌株发酵的初步研究

该试验针对中国少数民族传统乳制品,分离纯化得到56株乳酸菌。其中7株厌氧菌,11株乳球菌,38株乳杆菌。通过传代试验、凝乳时间、酸奶酸度及凝乳时风味的感官评价,选出其中4株性能较佳的乳酸菌：杆菌A、B,球菌C、D。球杆菌1∶1进行复合菌发酵性能研究,结果表明复合菌在发酵过程中,产酸速度比单菌株发酵剂快,能在5h内达到凝乳,且后酸化程度较弱,4℃保藏21d后酸度维持在120°T左右,符合工业化发酵剂的要求。通过对4种复合菌发酵剂的后酸化能力、产双乙酰能力、蛋白水解能力以及发酵乳的感官效果比较,最后筛选出AD复合菌发酵牛乳的效果最佳,具有成为生产发酵剂的潜力。     

Lectin-like binding of lactobacilli considered for their use in probiotical preparations for animal use

Three gut lactobacilli from piglets (Lactobacillus plantarum L 5, Lactobacillus paracasei L 81, Lactobacillus fermentum L 670) and Lactobacillus casei subsp. pseudoplantarum L.c.) from a calf were examined by microtitre plate binding assay for their lectin-like binding activity after their cultivation on Rogosa agar and in MRS broth. Three ECM (extracellular matrix) molecules (fetuin, porcine fibronectin and porcine mucin) were selected for this assay. Additionally, the effect of heparin on the binding of these three ECM molecules by Lactobacillus strains in microtitre plates was tested. Moreover, haemagglutination tests with pig, cattle, sheep, and hen erythrocytes were performed. However, none of the four Lactobacillus strains examined did react with any of the erythrocytes tested. The differences between individual strains were observed in their binding to immobilised ECM molecules. The best adherent was the Lactobacillus plantarum L5, however, the other three strains showed also good ECM binding. With regard to an influence of cultivation medium on lectin-like binding activity, binding of all ECM molecules was expressed in Lactobacillus paracasei L 81 to significantly higher degree (P < 0.001) after cultivation on Rogosa agar than in MRS broth. Similarly, strains Lactobacillus fermentum L 670 and Lactobacillus casei subsp. pseudoplantarum L.c. displayed significantly higher (P < 0.001) binding of fibronectin and mucin after growth on Rogosa agar in comparison with MRS broth cultivation. However, no significant (on fetuin and fibronectin binding) or opposite effect (on mucin binding) of cultivation medium was observed in Lactobacillus plantarum L 5 strain. The influence of cultivation medium on fetuin binding by Lactobacillus fermentum L 670 was also not significant while Lactobacillus casei subsp. pseudoplantarum L.c. bound fetuin significantly better (P < 0.01) after growth on Rogosa agar. Heparin pretreatment increased the binding of the ECM molecules by the Lactobacillus fermentum L 670 strain significantly (P < 0.001 or P < 0.05) with the exception of porcine fibronectin when the strain was cultivated in MRS broth. This result is important especially in the connection with the previous observations that heparin decreased ECM binding of enteropathogens as staphylococci or clinical enterococcal isolates. Following up on some earlier strain characteristics, these results indicate that the selected lactobacilli are probably suitable for probiotic purposes.     

Antibiotic resistance in Lactococcus species from bovine milk: presence of a mutated multidrug transporter mdt(A) gene in susceptible Lactococcus garvieae strains

A total of 72 Lactococcus strains (41 Lactococcus lactis and 31 Lactococcus garvieae) isolated from bovine milk were tested for susceptibility to 17 antibiotics and screened for the presence of antibiotic resistance genes using a microarray. Resistance to tetracycline, clindamycin, erythromycin, streptomycin, nitrofurantoin were found. The tetracycline-resistant L. garvieae and L. lactis harbored tet(M) and tet(S). L. lactis that were resistant to clindamycin were also resistant to erythromycin and possessed the erm(B) gene. The multidrug transporter mdt(A), originally described in L. lactis, was detected for the first time in L. garvieae and does not confer decreased susceptibility to erythromycin nor tetracycline in this species. Mdt(A) of L. garvieae contains one mutation in each antiporter motif C, which is known to play an essential role in drug efflux antiporters. This suggests that the mutations found in the C-motifs of Mdt(A) from L. garvieae may be responsible for susceptibility. The study revealed the presence of antibiotic resistance genes in non-pathogenic and pathogenic lactococci from bovine milk, including a mutated multidrug transporter in L. garvieae.