罗伊氏粘液乳杆菌K07的生物学特性及其基因分析

从新疆昭苏县传统发酵乳酪中分离出2株罗伊氏粘液乳杆菌（编号K07、K08）,对2株菌的生长和产酸性能、耐酸耐胆盐、模拟胃肠道的耐受、产罗伊氏菌素能力和抗氧化活性进行了评估;此外还测定了2株菌的抑菌性能并对K07菌株进行基因分析。结果表明,2株菌均在培养12 h后进入对数生长末期,发酵终点pH值为4.45。K07对酸、胆盐和模拟胃肠道的耐受及抗氧化能力强于K08。pH值2.50处理4 h,K07存活率为73.27%。0.15%胆盐胁迫下K07菌株OD600 nm 上升0.3个单位需5.50 h,模拟胃肠道试验K07的存活率为74.13%。两株菌的无细胞提取物抗氧化活性高于完整细胞,其中K07无细胞提取物对DPPH自由基和羟自由基的清除率为42.39%、15.69%,还原力17.96%。K07产罗伊氏菌素量为183.05 mmol/L显著高于K08,且K07抑菌性能较好,对白色念珠菌和无乳链球菌的抑菌圈直径为22.20 mm、18.26 mm。K07基因组全长1 950 440 bp,预测蛋白质编码基因2 067种,GC含量为38.69%。研究结果显示罗伊氏粘液乳杆菌K07具有良好的益生特性,为后续产业化应用提供理论依据。     

发酵鱼酱酸产GABA乳酸菌的分离筛选及发酵特性

从传统发酵鱼酱酸中筛选出产γ-氨基丁酸（γ-aminobutyric acid，GABA）的乳酸菌并分析其菌株发酵特性。通过薄层色谱法定性、Berthelot比色法定量获得产GABA菌株，并进行耐酸、耐胆盐、氨基酸脱羧酶活性、抑菌性、生长曲线及pH值、产酸速率等菌株发酵特性分析。结果表明：从分离自鱼酱酸不同发酵阶段的387 株乳酸菌中，获得15 株典型产GABA菌株，包括2 株食窦魏斯氏菌（Weissella cibaria）、1 株熊蜂魏斯氏菌（Weissella bombi）、11 株植物乳杆菌（Lactobacillus plantarum）和1 株戊糖乳杆菌（Lactobacillus pentosus）。15 株乳酸菌产GABA能力及发酵特性在主成分分析图上差异显著，其中食窦魏斯氏菌Y113产GABA量为0.239 mg/mL，高于其他菌株；植物乳杆菌Y279和Y64展现出较好的耐酸性、耐胆盐性、抑菌性、无氨基酸脱羧酶活性、生长速率及产酸速率快的特点。鉴于其优良的发酵特性、益生特性及产GABA能力，菌株Y279可作为鱼酱酸工业化、标准化生产的潜在优良菌株。     

抑制阴道加德纳菌乳酸菌的筛选及安全性研究

为筛选具有较强抑制阴道加德纳菌(Gardnerella vaginalis)特性的乳酸菌菌株，该研究通过双层琼脂扩散法测定菌株抑制阴道加德纳菌的能力，从筛菌样品中筛选能力较强的乳酸菌，通过溶血性试验、动物致病性试验评价其安全性，并进行了耐酸耐胆盐能力试验。结果表明，筛选菌株中罗伊氏粘液乳杆菌(Limosilactobacillus reuteri)HCS02-001抑菌能力最强，发酵上清液抑菌圈直径为18.23 mm，发酵上清液经过3倍浓缩后抑菌圈直径可达21.58 mm。菌株HCS02-001无溶血性，无致病性，在pH 2.0条件下培养17 h活菌数对数值为7.97，存活率为85.52%，在1.5%胆盐条件下培养17 h活菌数对数值为8.48，存活率为90.99%。结果显示罗伊氏粘液乳杆菌HCS02-001具有较强的抑制阴道加德纳菌特性，初步判定其具备安全性。     

具有抑制腐败菌能力的罗伊氏乳杆菌筛选及在酸乳生产中的应用

以青霉菌作为酸乳腐败菌筛选具有抑制该菌能力的罗伊氏乳杆菌,并通过排除实验确定优势菌株的主要抑菌物质,再将优势菌株作为辅助发酵剂用于酸乳发酵,并对酸乳进行理化性质和感官品质的评定以及优势菌株在酸乳中生物防腐作用的探讨。结果表明,罗伊氏乳杆菌CICC6121是具有抑制青霉菌能力的优势菌株,其无细胞上清液中抑制青霉菌的主要活性成分为罗伊氏乳杆菌素;罗伊氏乳杆菌CICC6121在酸奶发酵过程中不会对保加利亚乳杆菌和嗜热链球菌产生拮抗作用,且所生产的酸乳与未接种CICC6121时的酸乳在理化性质和感官品质方面几乎无差别,在21 d的低温贮藏过程中,能够抑制酸乳中青霉菌的生长,减少因污染而造成酸乳的腐败变质。     

具有降胆固醇功能的罗伊氏乳杆菌的筛选及其益生作用

为获得具有较强降胆固醇功能的益生菌株,试验从内蒙古呼伦贝尔满洲里通达牧场制作的奶豆腐中筛选分离菌株,并采用邻苯二甲醛法测定菌株降胆固醇的能力,从中选出降胆固醇能力强的菌株,通过形态学鉴定、生理生化及16S rDNA对菌株进行鉴定,并测定了其耐受酸、胆盐和胃肠液的能力。结果筛选分离出5株具有降胆固醇功能的菌株,其中一株菌对胆固醇降解能力最强,降解率达50.60%,经鉴定此株菌为罗伊氏乳杆菌（Lactobacillus reuteri）,将其命名为HCS02-001（CGMCC No. 19746）;经对酸、胆盐及胃肠液的耐受试验,该株菌在pH 3.0和pH 2.0的环境中培养17 h后存活率分别为86.80%和85.52%;在0.30%、0.50%的胆盐浓度环境中培养17 h后存活率分别为97.42%和95.17%;在人工胃液中作用3 h,存活率为96.74%;在人工肠液中作用4 h,存活率高达98.41%。该研究筛选出了一株具有较好降胆固醇功能的罗伊氏乳杆菌（Lactobacillus reuteri）,其具有较强的耐受酸、胆盐环境和胃肠液的能力,对关于降胆固醇功能方面的产品研究开发具有重要意义。     

自然发酵三樱椒中乳酸菌的分离及鉴定

为获得适用于三樱椒发酵的乳酸菌,该研究采用钙溶圈法从自然发酵三樱椒中分离乳酸菌,通过形态观察和分子生物学技术对其进行菌种鉴定,并对其生长、产酸、耐酸特性进行分析。结果表明,从自然发酵三樱椒中共分离纯化得到19株乳酸菌,经鉴定,16株为植物乳杆菌（Lactobacillus plantarum）,3株为赫轮魏斯氏菌（Weissella hellenica）。培养初期,赫轮魏斯氏菌生长比植物乳杆菌快,但培养12 h后植物乳杆菌比赫轮魏斯氏菌生长速度更快;植物乳杆菌产酸能力优于赫轮魏斯氏菌,培养21 h后,植物乳杆菌发酵液的pH值稳定在3.7,赫轮魏斯氏菌发酵液pH值稳定在4.3,其中植物乳杆菌PLP-1产酸能力最好;植物乳杆菌的耐酸性优于赫轮魏斯氏菌,在pH值＜4.0的培养条件下赫伦魏斯氏菌生长受到严重抑制,而植物乳杆菌可以在pH值为3.0的培养基中生长,其中植物乳杆菌PLP-1耐酸性最好。     

木薯渣中乳酸菌的分离鉴定及其产酸能力分析

该研究利用传统培养法从自然发酵木薯渣中分离得到9株菌,通过菌株形态、生理生化特征及16S rDNA基因序列分析方法对菌株进行鉴定。结果显示,这9株菌均为乳杆菌属（Lactobacillus sp.）,其中菌株Y5、Y13、M14为干酪乳杆菌（Lactobacillus casei）,菌株Y6、Y12、M12为植物乳杆菌（Lactobacillus plantarum）,菌株Y7为丘状乳杆菌（Lactobacillus collinoides）,菌株M9为副干酪乳杆菌（Lactobacillus paracasei）,菌株M11为类布氏乳杆菌（Lactobacillus parabuchneri）;产酸及耐酸试验结果表明这5种菌均具有较强的产酸能力及耐酸性,发酵6 h后发酵液中pH值可达4.5以下,其中菌株Y12和M14的产酸能力最强,在15 h后其pH值均在3.6以下,可用于木薯渣或其他青贮饲料发酵剂的研制。     

云南建水酸浆豆腐中乳酸菌的生长特性

为了筛选出优良的乳酸菌进行发酵制作酸浆,对分离自云南建水豆腐酸浆中的五株乳酸菌(SYG01、SYG02、SYG03、SYG04、SYG05)的生长曲线、产酸能力、耐酸能力和耐渗透压能力进行了比较。生长曲线实验表明在相同培养时间内,菌株SYG02繁殖能力最强,其次是菌株SYG03,而菌株SYG05、SYG04、SYG01的生长速率明显较慢;在产酸方面SYG02产酸能力最强,速度最快,SYG03、SYG05和SYG04次之,SYG01最弱。在耐酸方面,在pH4.0的环境下5株菌生长良好,在pH3.0的酸性环境下5株菌虽然能够存活,但活菌数的数量级仅在102~104 CFU/mL,其中菌株SYG02和SYG03比其它三株菌株表现出较好的耐酸能力。在耐渗透压方面,SYG02在8%(w/V)的NaCl中依然表现出较强的耐受性,活菌数的数量级达到107 CFU/mL,其余4株菌在NaCl含量大于6%(w/V)时生长受到明显抑制。并以发酵黄浆水的pH及产酸量为指标对菌株的产酸能力进行比较,发现混合菌株的产酸能力高于单菌株,其中菌株SYG02和菌株SYG03组合发酵的效果最好,在发酵72 h后黄浆水的pH为3.52,产酸量达到6.46 g/L。从而得出菌株SYG02具有良好的生长、产酸和耐渗透压能力,具有良好的应用潜力,对酸浆豆腐的工业化生产有重要的意义。     

罗伊氏乳杆菌J1胃肠道耐受性评价及其对免疫相关基因调控的体外研究

以分离自传统发酵乳制品中的罗伊氏乳杆菌(Lactobacillus reuteri,L.reuteri)J1为研究对象,对其黏附性能、生长特性、对胃肠道环境的耐受能力以及该菌株作用于Caco-2细胞不同时间后免疫相关基因表达量的动态变化进行评价。结果显示,罗伊氏乳杆菌J1具有良好的黏附性,黏附率达87.93%,具有定植于肠道中的潜力。该菌株在4 h进入对数期,14 h进入稳定期。在肠道耐受性评价中,菌株在pH为2.0和3.0条件下处理3 h后,其存活率分别为86.77%、97.12%;在胆盐质量浓度分别为0.3、0.5和1.0 g/100 mL的培养基中处理4 h后,其存活率分别为105.00%、82.60%和82.18%;经模拟人工胃液处理3 h后转入人工肠液继续培养8 h,存活率达82.71%;该菌株在7 g/100 mL的盐溶液中处理24 h后,其存活率为80.36%。此外,108CFU/mL的罗伊氏乳杆菌J1可刺激Caco-2细胞中免疫相关因子IL-1β的表达,抑制促炎因子IL-6和趋化因子IL-8的表达。综上表明,罗伊氏乳杆菌J1具有定植于肠道上皮细胞的潜力,耐受胃肠道环境,并发挥一定的免疫调节作用。     

新疆特色食品中优良乳酸菌的筛选及分离鉴定

该研究利用传统培养法从自然发酵的新疆特色食品中分离筛选出3株菌,通过菌株形态观察、生理生化试验及16S rDNA序列分析对菌株进行鉴定。结果表明,菌株L-6、L-7和L-8分别被鉴定为肠膜明串珠菌（Leuconostoc mesenteroides）、植物乳杆菌（Lactobacillus plantarum）及短乳杆菌（Lactobacillus brevis）。产酸试验结果表明,这3株菌均具有较强的产酸能力,发酵12 h后发酵液pH值可达4.0以下,其中菌株L-7产酸能力最强,在15 h后其发酵液pH值在3.5以下,可用于辣椒酱或其他发酵食品发酵剂的研制。     

In vitro testing of commercial and potential probiotic lactic acid bacteria

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.     

罗伊氏乳杆菌的益生特性及安全性分析

本研究对一位128岁老人源罗伊氏乳杆菌进行了体外益生特性和安全性分析。采用模拟胃肠液对49株罗伊氏乳杆菌进行耐受筛选,得到了3株存活率都大于90.00%的菌株,分别是LT018、LT037、LT046。对上述3株罗伊氏乳杆菌进行表面特性、抑菌活性和安全性分析,结果表明,LT018具有较好的粘附繁殖能力(疏水性为54.65%,凝集性为67.20%),菌株的表面疏水性和凝集性之间存在负相关(R=-0.869);非酸性抑菌活性物质在酸性条件下才发挥作用,它们的上清液对大肠埃希氏菌、金黄色葡萄球菌、蜡样芽胞杆菌、伤寒沙门氏菌和福氏志贺氏菌均有较明显的抑制作用,其中对金黄色葡萄球菌的抑制效果最佳,抑菌圈直径均大于15.00 mm;它们都不具有产生物胺有害物质的活性,除氯霉素外,对其他4种临床常用抗生素(红霉素、四环素、庆大霉素、万古霉素)敏感。综合上述结果,认为这些菌株具有良好的开发潜力。     

Isolation and characterization of Lactobacillus species having potential for use as probiotic cultures for dogs

ABSTRACT:  The need to control pathogenic microorganisms in the intestinal tract of dogs is a growing concern. There is interest in using probiotics such as species of Lactobacillus to help control canine intestinal infections. For successful use as a probiotic, the bacterial species should be of canine intestinal origin since these species exhibit host specificity. Serial dilutions of freshly voided dog feces were plated on Lactobacillus selection (LBS) agar to isolate the cultures. Isolates were identified based on Gram stain, catalase test, and fermentation patterns using API 50 CH kits. All potential isolates were compared for bile resistance based on relative ability to grow in broth containing 0.3% Oxgall, the ability to inhibit Salmonella Typhimurium in associative broth cultures, and the production of reuterin. Of the lactobacilli isolated, Lactobacillus reuteri was the dominant species. However, some cultures of L. acidophilus also were isolated. We found variations among the isolates of L. reuteri and L. acidophilus with respect to bile tolerance. In general, isolates of L. reuteri appeared to be more bile resistant than were isolates of L. acidophilus . There were also variations in the ability to inhibit growth of S. Typhimurium. Some isolates of L. reuteri produced reuterin while others did not.     

In vitro evaluation of Lactobacillus gasseri strains of infant origin on adhesion and aggregation of specific pathogens

Numerous Lactobacillus species are members of the normal healthy human intestinal microbiota, and members of the Lactobacillus family predominate among the current marketed probiotic strains. Most of the current commercial probiotic strains have not been selected for specific applications but rather have been chosen based on their technological properties. Often the ability of such strains to temporarily colonize the gastrointestinal tract may be lacking, and the interactions with intestinal microbiota are few. Furthermore, the competitive exclusion properties of potential probiotic bacteria are strain specific and vary greatly. Thus, it is highly desirable that new candidate probiotic isolates originate from the healthy target population. In this study, seven newly isolated strains of Lactobacillus gasseri originating from feces of a healthy newborn child were evaluated for their ability to adhere to intestinal mucus, to autoaggregate and coaggregate with the model pathogens Cronobacter sakazakii (ATCC 29544) and Clostridium difficile (1296). All the bacterial strains, single or in combination, in viable and nonviable forms, were able to autoaggregate. The coaggregation with C. sakazakii or C. difficile was higher (P < 0.05) in nonviable than in the viable forms. Single L. gasseri strains showed similar adhesion abilities to intestinal colon mucus. The seven L. gasseri strains when combined were also able to significantly compete with, displace, and inhibit the adhesion of C. sakazakii and C. difficile in the mucus model. This study demonstrates that the studied L. gasseri strains fulfill the basic adhesion and aggregation properties for probiotics and could be considered for potential future use in children.     

婴儿源鼠李糖乳杆菌的筛选及其促肠道类器官生长的研究

本研究以分离得到的5株来源于一月龄婴儿的鼠李糖乳杆菌（Lactobacillus rhamnosus）与2株副干酪乳杆菌（Lactobacillus paracasei）作为实验菌,以鼠李糖乳杆菌GG（Lactobacillus rhamnosus GG,LGG）作为对照菌株,通过耐酸实验、耐胆盐实验、疏水性实验、自动聚集能力测定和细胞黏附等实验对这些菌株的消化道耐受性进行评价。进一步选择在耐受性实验中综合评价效果最好的鼠李糖乳杆菌SW-02与肠道类器官进行共培养,用倒置相差显微镜观察类器官形态、生长状况,计算出芽率,利用EdU染色评估类器官中细胞增殖情况,利用实时荧光定量PCR检测增殖标志基因Ki67、肠道干细胞标志物Lgr5和紧密连接蛋白Zo-1的mRNA水平,利用酶联免疫吸附试验测定粘蛋白MUC2含量。结果证实:与其他菌株相比,鼠李糖乳杆菌SW-02有较强的耐酸性;SW-02、SW-03和SW-X的耐胆盐能力相对较强;LGG、SW-01、SW-02、SW-04和TX-02对二甲苯的疏水性高;对于自动聚集能力,SW-01最强,SW-02次之;对HT-29细胞的黏附性实验显示,菌株SW-01、SW-02和SW-03具有较高的黏附能力;将SW-02与类器官共培养时,SW-02显著（P<0.05）提高了类器官的出芽率和出芽个数,促进类器官的生长;与对照组相比,SW-02组类器官的Ki67、Lgr5和Zo-1的mRNA水平均显著增加,MUC2分泌量显著增加（P<0.05）。综上,鼠李糖乳杆菌SW-02具有较好的消化道耐受性和促进肠道类器官生长的能力,可以作为今后开发具有益生性产品的潜在菌株。     

巴马长寿老人源乳酸杆菌的筛选及其益生特性

为获得巴马长寿老人肠道乳杆菌并研究其益生特性,采用改良MRS+Ca CO3固体培养基与改良MRS+X-gal固体培养基从10位广西巴马百岁老人粪便中筛选出19株乳杆菌,再通过生理生化实验、糖发酵实验、模拟胃肠转运实验、疏水性实验和自凝集实验,筛选出消化道逆环境耐受性强和粘附力好的菌株3株(GU-L3、GU-P132和GU-G23),进一步对三株菌进行了16Sr DNA序列鉴定,结果发现GU-L3为干酪乳杆菌(Lactobacillus casei);GU-P132为发酵乳杆菌(Lactobacillus fermentum);GU-G23为格氏乳杆菌(Lactobacillus gasseri)。三个菌株经模拟胃肠液转运实验后,存活率皆达到107 CFU/m L以上,自凝集率分别为12.43±0.03%、78.88±0.01%和35.92±0.02%,疏水率分别为6.38±0.02%、81.77±0.02%和10.58±0.05%,粘附性也高于其他菌株。经与国内外有关报道相比,综合认为这三株来自巴马老人肠道的乳酸杆菌益生特性明显,具有较好的开发潜力。     

四川泡菜中潜在益生性植物乳杆菌的筛选及安全性评价

为筛选具有潜在益生作用和安全性的植物乳杆菌,以四川泡菜中分离的114株植物乳杆菌为出发菌株,进行耐酸性、耐胆盐能力、耐模拟胃肠液能力、自聚能力、致病菌共聚性、抗生素耐药性和溶血性研究。经过pH2.0酸性条件、0.2%浓度胆盐培养后,初步筛选出13株耐受能力较好的菌株。对13株菌进行模拟胃肠液、聚集能力、耐药性和溶血性试验。结果表明,13株菌模拟胃肠液耐受存活率均高于75%,自聚集能力23%~52%,与致病菌单增李斯特菌和大肠埃希氏菌的共聚集能力分别为10%~29%和16%~37%。在药敏和最小抑菌浓度（MIC）试验中,潜力菌株对10种常见抗生素的耐药性表现基本一致,对β-内酰胺类、酰胺醇类抗生素较敏感,对氨基糖苷类、喹诺酮类、糖肽类、大环内酯类和四环素类抗生素有较强耐受性;13株菌均无溶血现象,说明具有一定的安全性。因此,筛选得到的13株植物乳杆菌均有潜在的益生作用和较高安全性,研究结果为益生菌的开发提供了菌种资源。     

Assessment of adhesion properties of novel probiotic strains to human intestinal mucus

Potential new probiotic strains Lactobacillus brevis PELI, L. reuteri ING1, L. rhamnosus VTT E-800 and L. rhamnosus LC-705 were assessed for their adhesion properties using the human intestinal mucus model. The effect on the adhesion of exposure to acid and pepsin and to milk were tested to simulate gastric and food processing conditions, and the effect of different growth media on adhesion was tested. The properties of the four strains were compared to the well-investigated probiotic L. rhamnosus strain GG. Three of the tested strains showed significant adhesion properties in the mucus model, while L. brevis PELI had intermediate adhesion and L. rhamnosus LC-705 adhered poorly. Pretreatment with different milks decreased the adhesion and low pH and pepsin treatment reduced the adhesion of all tested strains except L. rhamnosus LC-705. No competitive exclusion of pathogenic Salmonella typhimurium or Escherichia coli SfaII was observed. The results indicate that major differences exist between tested proposed probiotic strains. The growth media and the food matrix significantly affect the adhesive ability of the tested strains. This has previously not been taken into account when selecting novel probiotic strains.