降胆固醇嗜酸乳杆菌的诱变选育

通过诱变获得具有降胆固醇功能的优良嗜酸乳杆菌新菌株。利用亚硝基胍(作用浓度为1 g/L)对嗜酸乳杆菌进行诱变。突变菌株测定其耐渗透压和抗胆盐能力后,在含有0.3 g/L胆固醇的培养基中培养48 h,测定降胆固醇率。挑选优良突变菌株制成酸奶并喂养高脂大鼠模型,28 d后测定血清及粪便胆固醇指标。嗜酸乳杆菌突变后获得的60个突变菌株中有8株具有良好的耐渗透压和抗胆盐能力,其中突变株Y48的清除胆固醇能力最高,清除率达到(61.44±1.8)%。Y48发酵酸奶喂养高脂大鼠模型28 d后与对照大鼠模型相比,血清中TC、TG明显减少(P<0.05),粪便TC明显增加。通过诱变获得了优良的嗜酸乳杆菌突变菌株,为今后获得优质降胆固醇乳酸制品提供良好的候选菌种。     

嗜酸乳杆菌黏附鸡胚肠黏膜上皮细胞能力的研究

目的建立肠黏膜上皮细胞模型和测定嗜酸乳杆菌的黏附能力。方法将嗜酸乳杆菌用胃蛋白酶和HCl-H2O低pH以及反复冻融、灭活等方法处理后测定其黏附能力。结果成功地建立了鸡胚肠黏膜上皮细胞模型;经胃蛋白酶和HCl-H2O低pH以及灭活处理后,嗜酸乳杆菌的黏附能力和对照组差异有显著性。反复冻融后的嗜酸乳杆菌黏附能力与对照组差异无显著性。结论胃蛋白酶和HCl-H2O pH2.0会使嗜酸乳杆菌黏附肠黏膜上皮细胞能力下降,热灭活可使嗜酸乳杆菌的黏附能力提高,反复冻融对嗜酸乳杆菌的黏附能力无明显影响。     

双歧杆菌四联活菌片中嗜酸乳杆菌的分离及生长动力学研究

目的以双歧杆菌四联活菌片为实验材料,利用酸化的MRS培养基筛选分离得到嗜酸乳杆菌,对其进行进一步的生长动力学研究,确定嗜酸乳杆菌的生长数学模型。方法通过浓度梯度稀释法利用改良MRS培养基对双歧杆菌四联活菌片中的嗜酸乳杆菌进行分离,利用分光光度仪和平板菌落计数两种不同的方法测定嗜酸乳杆菌在发酵过程中不同发酵时间的细胞浓度的动态变化,经软件处理后拟合出嗜酸乳杆菌细胞生长的Logistic数学模型。结果Logistic方程能很好地拟合嗜酸乳杆菌细胞生长的动态变化,并得到嗜酸乳杆菌在本实验条件下的数学模型,为进一步研究、利用嗜酸乳杆菌生长能力、产酸能力和产香能力等具有重要的理论指导意义。     

体外拮抗幽门螺杆菌的人嗜酸乳杆菌菌株的选育

目的 探讨人嗜酸乳杆菌对幽门螺杆菌（Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ,ＨＰ）毒力株的体外拮抗作用,筛选出对ＨＰ毒力株有明显拮抗作用的嗜酸乳杆菌菌株。方法 从健康人胃肠道中分离出５２株嗜酸乳杆菌可疑株,通过其培养特性,生理特性,生化反应及代谢产物测定等进行鉴定,获得２６株嗜酸乳杆菌。同时,从临床患者胃活检标本中分离出２３株ＨＰ菌株,用ＰＣＲ方法筛选出ｃａｇＡ阳性ＨＰ毒力株,然后,采用打孔法进行嗜酸乳杆菌培养上清拮抗ＨＰ毒力株的实验,以１％的乳酸作对照。结果 筛选出４株对ＨＰ毒力株有明显拮抗作用的嗜酸乳杆菌,这种拮抗作用不依赖嗜酸乳杆菌分泌的乳酸。结论 人嗜酸乳杆菌在体外对ＨＰ毒力株具有明显拮抗作用。该研究为应用微生态疗法治疗ＨＰ感染提供了理论基础。     

16S rRNA PCR鉴定嗜酸乳杆菌鸡源分离株

用自行设计的嗜酸乳杆菌16S rRNA的特异性引物和建立的PCR方法对初步鉴定为嗜酸乳杆菌的鸡源分离株进行检测。PCR检测结果表明分离株和嗜酸乳杆菌参考株扩增出大小一致的目的片段,从分子水平对鸡源分离菌进行了鉴定。     

乳杆菌耐胆汁、降解结合胆盐和同化胆固醇能力的研究

对8株植物乳杆菌的胆汁耐受力、降解结合胆盐能力以及同化胆固醇能力进行了研究。不同的菌株在添加了牛胆汁的MRS中生长速度具有明显差异,同化胆固醇能力也明显不同,而降解结合胆盐的能力没有明显区别。分析发现,菌株的胆汁耐受力和降解结合胆盐能力,胆汁耐受力和同化胆固醇能力,以及降解结合胆盐能力和同化胆固醇能力之间都没有明显的相关性。     

嗜酸乳杆菌LA85对小鼠免疫机能的研究

目的：研究嗜酸乳杆菌LA85对小鼠免疫机能的影响。方法：将160只雄性小鼠随机分为4组,即对照组、低、中、高三个剂量组。采用灌胃的方式给予实验小鼠不同剂量的嗜酸乳杆菌LA85。通过对小鼠平均体重、脾脏和胸腺免疫指数、细胞免疫、体液免疫、单核-吞噬性细胞和自然杀伤细胞（NK细胞）活性的评价,探讨嗜酸乳杆菌LA85对小鼠免疫机能的影响机制。结果：与对照组相比,嗜酸乳杆菌LA85低、中、高剂量组能够显著提升小鼠的胸腺指数,嗜酸乳杆菌LA85高剂量组能够显著提升小鼠的脾脏指数。同时能够显著提高小鼠的淋巴细胞增殖分化能力、半数溶血值、巨噬细胞的吞噬能力、NK细胞的细胞活性;嗜酸乳杆菌对小鼠的体重没有影响。结论：给予小鼠口服嗜酸乳杆菌LA85可以通过改善细胞免疫和体液免疫功能,从而提高免疫力。     

嗜酸乳杆菌LA-G80的毒理学研究

目的对嗜酸乳杆菌的毒性进行研究。方法采用大、小鼠急性毒性试验、Ames试验、小鼠骨髓细胞微核试验、小鼠精子畸形试验和大鼠30 d喂养等对嗜酸乳杆菌进行安全性试验研究。结果急性经口毒性试验表明,大、小鼠灌胃给予嗜酸乳杆菌,最大耐受剂量雌雄两性别均大于20.0 g/kg体重,Ames试验、微核试验和精子畸形试验结果均为阴性。大鼠30 d喂养试验结果表明各项指标均未见明显毒性反应。结论在本次实验条件下,嗜酸乳杆菌未见遗传毒性。由此可初步判定,使用嗜酸乳杆菌是安全可靠的。     

诱导选育嗜酸乳杆菌的鉴定

对亚硝基胍(NTG)诱变获得的嗜酸乳杆菌菌株的抗酸、抗胆盐及多项酶活力进行测定。亚硝基胍诱变的嗜酸乳杆菌培养于模拟胃肠环境的pH 3.5酸性环境90 min后,再培养于中性0.2%胆盐环境中,对其生长情况进行评估。对抗酸抗胆盐菌株的各项酶活性进行评估。嗜酸乳杆菌突变后筛选的2株菌株都具有良好的抗酸和抗胆盐能力,其中突变株Y48在模拟胃肠环境中生长良好,并具有良好的酶活性。诱变获得的优良嗜酸乳杆菌株,具有良好的抗酸抗胆盐及酶活各项性能,为开发优质的嗜酸乳杆菌新产品提供了条件。     

嗜酸乳杆菌与嗜热链球菌共发酵互生机理的研究

目的:对嗜酸乳杆菌和嗜热链球菌共发酵时两者的互生作用机理进行研究。方法:以质量浓度100gL脱脂乳为培养基,将嗜酸乳杆菌主要代谢产物—氨基酸,如赖氨酸、精氨酸、缬氨酸分别以0.0083mgml、0.0036mgml、0.0053mgml的量加入含嗜热链球菌脱脂乳培养基中,40℃培养,测定凝乳时间;将嗜热链球菌的代谢产物-乳酸、甲酸分别为0.1332mgml和0.075mgml的量加入含嗜酸乳杆菌脱脂乳培养基中,37℃培养,测定其发酵乳的凝乳时间。结果:嗜热链球菌发酵乳凝乳时间由12h缩短到4h,pH为4.52～4.62,吉尔涅尔度为54.23～64.74°T;嗜酸乳杆菌发酵乳的凝乳时间由16h缩短为5h,pH为4.61～4.65;且嗜酸乳杆菌在CO2环境中发酵时,发酵时间明显缩短。结论:嗜酸乳杆菌和嗜热链球菌共发酵时具有互生关系。     

Complete genome sequencing of Lactobacillus acidophilus 30SC, isolated from swine intestine

Lactobacillus acidophilus 30SC has been isolated from swine intestines and considered a probiotic strain for dairy products because of its ability to assimilate cholesterol and produce bacteriocins. Here, we report the complete genome sequence of Lactobacillus acidophilus 30SC (2,078,001 bp) exhibiting strong acid resistance and enhanced bile tolerance.     

Effects of Lactobacillus acidophilus 43121 and a mixture of Lactobacillus casei and Bifidobacterium longum on the serum cholesterol level and fecal sterol excretion in hypercholesterolemia-induced pigs

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.     

嗜酸乳杆菌NCFM对Caco-2细胞基因表达谱的影响

摘要：【目的】嗜酸乳杆菌NCFM作为一株具有良好益生功能的模式菌株,采用基因芯片技术对其作用后的宿主细胞基因的变化情况进行分析,在生物、食品等领域具有较大研究价值。【方法】将嗜酸乳杆菌NCFM和Caco-2细胞共同培养2h,提取Caco-2细胞的总RNA,并将RNA反转录成cDNA,与Human Genome U133 Plus 2.0 Array基因表达谱芯片杂交,杂交后进行图像扫描和数据分析,并采用Real-time RT PCR方法对差异表达的基因进行验证。【结果】采用基因芯片方法检测了Caco-2细胞经嗜酸乳杆菌NCFM作用2h后的基因表达变化,发现差异表达基因为508个,其中有473个基因上调,35个基因下调,初步推测Caco-2细胞能诱导多个基因的表达,以发挥嗜酸乳杆菌NCFM的益生功能。并且经Real-time RT PCR验证,表达差异显著的3个免疫调节相关基因CCL2、PTX3和TNFRSF9的确在嗜酸乳杆菌NCFM作用期间高表达。【结论】以上这些结果促进了对嗜酸乳杆菌NCFM益生功能的认识,也为揭示该乳酸菌的作用机理提供了理论基础。     

Optimization of cholesterol removal, growth and fermentation patterns of Lactobacillus acidophilus ATCC 4962 in the presence of mannitol, fructo-oligosaccharide and inulin: a response surface methodology approach

AIMS: To optimize cholesterol removal by Lactobacillus acidophilus ATCC 4962 in the presence of prebiotics, and study the growth and fermentation patterns of the prebiotics. METHODS AND RESULTS: Lactobacillus acidophilus ATCC 4962 was screened in the presence of six prebiotics, namely sorbitol, mannitol, maltodextrin, hi-amylose maize, fructo-oligosaccharide (FOS) and inulin in order to determine the best combination for highest level of cholesterol removal. The first-order model showed that the combination of inoculum size, mannitol, FOS and inulin was best for removal of cholesterol. The second-order polynomial regression model estimated the optimum condition of the factors for cholesterol removal by L. acidophilus ATCC 4962 to be 2.64% w/v inoculum size, 4.13% w/v mannitol, 3.29% w/v FOS and 5.81% w/v inulin. Analyses of growth, mean doubling time and short-chain fatty acid (SCFA) production using quadratic models indicated that cholesterol removal and the production of SCFA were growth associated. CONCLUSIONS: Optimum cholesterol removal was obtained from the fermentation of L. acidophilus ATCC 4962 in the presence of mannitol, FOS and inulin. Cholesterol removal and the production of SCFA appeared to be growth associated and highly influenced by the prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Response surface methodology proved reliable in developing the model, optimizing factors and analysing interaction effects. The results provide better understanding on the interactions between probiotic and prebiotics for the removal of cholesterol.     

Assimilation of cholesterol by Lactobacillus acidophilus.

Considerable variation was found among strains of Lactobacillus acidophilus isolated from the fecal flora of pigs with regard to the ability to grow well in the presence of bile and to assimilate cholesterol from a laboratory growth medium. The uptake of cholesterol occurred only when the culture(s) was growing in the presence of bile under anaerobic conditions. Consumption of L. acidophilus RP32, which was selected for its ability to grow well in the presence of bile and to assimilate cholesterol from the laboratory medium, significantly inhibited increases in serum cholesterol levels of pigs (P less than 0.05) fed a high-cholesterol diet. Consumption of L. acidophilus P47, which was selected for its ability to grow in the presence of bile and lack of ability to remove cholesterol from the growth medium, failed to have a similar effect. This indicates that certain strains of L. acidophilus act directly on cholesterol in the gastrointestinal tract, and thus may be beneficial in reducing serum cholesterol levels.     

Assimilation of cholesterol by Lactobacillus acidophilus

Considerable variation was found among strains of Lactobacillus acidophilus isolated from the fecal flora of pigs with regard to the ability to grow well in the presence of bile and to assimilate cholesterol from a laboratory growth medium. The uptake of cholesterol occurred only when the culture(s) was growing in the presence of bile under anaerobic conditions. Consumption of L. acidophilus RP32, which was selected for its ability to grow well in the presence of bile and to assimilate cholesterol from the laboratory medium, significantly inhibited increases in serum cholesterol levels of pigs (P less than 0.05) fed a high-cholesterol diet. Consumption of L. acidophilus P47, which was selected for its ability to grow in the presence of bile and lack of ability to remove cholesterol from the growth medium, failed to have a similar effect. This indicates that certain strains of L. acidophilus act directly on cholesterol in the gastrointestinal tract, and thus may be beneficial in reducing serum cholesterol levels.     

几株乳酸菌益生潜力及降胆固醇的研究

采用体外试验方法研究7株乳酸菌和双歧杆菌对人体消化道不良环境的抵抗能力、在肠道上皮的黏附定植能力及其降胆固醇潜力。结果表明:受试菌对CCL-187细胞粘附能力存在显著差异(P<0.05),粪肠球菌M1粘附能力最强;各菌对胃液和胰液都有较强耐受能力,在模拟胃液和胰液中分别培养3h和4h后仍能保证95%以上存活;在含胆汁培养基中受到不同程度抑制,但都能存活;受试菌在生长过程中能够使环境中胆固醇降低从25%～54%不等,粪肠球菌A30和A31和嗜酸乳杆菌A878和PB1能使胆固醇降低40%以上,是良好的降胆固醇备选菌株。     

Effect of Lactobacillus isolates on the adhesion of pathogens to chicken intestinal mucus in vitro

AIMS: The aims of this study were to investigate in vitro the effects of Lactobacillus isolates from a chicken on adhesion of pathogenic Salmonella and Escherichia coli to chicken intestinal mucus obtained from different intestinal regions. METHODS AND RESULTS: Bacteria were labelled by using methyl-1,2-[(3)H]-thymidine. The bacterial adhesion was assessed by measuring the radioactivity of bacteria adhered to the mucus. The results showed that the abilities of Lactobacillus spp. to bind to the same intestinal mucus were higher than those of pathogenic Salmonella and E. coli. Pretreatment of intestinal mucus with Lactobacillus fermentum and Lactobacillus acidophilus, alone or in combination, reduced the adhesion of the tested pathogens, but the reductive extent of pathogenic adhesion by Lactobacillus spp. in combination was relatively high. CONCLUSIONS: The tested bacteria had different adhesions to mucus glycoproteins isolated from different intestinal regions of chicken. Lactobacillus acidophilus and Lact. fermentum in combination revealed a better ability to inhibit attachments of Salmonella and E. coli to chicken intestinal mucus than Lactobacillus sp. alone. SIGNIFICANCE AND IMPACT OF THE STUDY: A mixture of intestinal Lactobacillus spp. from a chicken may play a protective role in excluding pathogenic Salmonella and E. coli from the intestine of chicken.