产广谱细菌素乳酸菌的筛选和鉴定

以大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌和枯草芽孢杆菌为指示菌,从分离自四川传统发酵食品中的267株乳酸菌中,采用平板打孔法初筛、牛津杯法复筛（排除酸、过氧化氢干扰以及胰蛋白酶和木瓜蛋白酶处理）,筛选出1株分离自醪糟的具有较强抑菌作用的产广谱细菌素的乳杆菌菌株P158,结合形态学、生理生化特性和16S rDNA序列同源性分析,该菌株被鉴定为植物乳杆菌（Lactobacillus plantarum）。     

乳杆菌阴道分离株16S rDNA鉴定及优势菌株产H_2O_2和自凝集能力分析

目的从阴道分泌物标本分离鉴定乳杆菌,分析与健康相关优势菌种产过氧化氢(H2O2)和自凝集能力,揭示菌株特异性的潜在益生特性。方法利用MRS固体培养基从41例阴道分泌物标本中分离单菌落,纯培养后提取细菌基因组DNA,PCR扩增16S rDNA序列,依据序列一致性确定细菌种属。利用TMB-HRP-MRS培养基测定优势乳杆菌菌株产H2O2能力,并测定其自凝集能力。结果获得155个细菌分离株,分属乳杆菌属104株(67.1%)、肠球菌属21株(13.5%)、链球菌属28株(18.1%)、葡萄球菌属1株(0.6%)和双歧杆菌属1株(0.6%)。各菌属在无或有妇科临床症状患者标本中的构成比例差异有统计学意义(χ2=7.4918,P=0.0236),乳杆菌在前者检出率为83.3%,显著高于其在后者47.1%的检出率(χ2=4.4879,P=0.0341)。乳杆菌中卷曲乳杆菌分离株所占比例最高(67.3%,70/104)。来自不同标本的22株卷曲乳杆菌中77.3%菌株产H2O2,且这些菌株具有强弱不同的自凝集能力(27.5%~97.3%)。结论乳杆菌属特别是卷曲乳杆菌种是健康阴道环境中优势菌属,其产H2O2和自凝集能力存在明显菌株特异性,有2株卷曲乳杆菌此两特性均很强可供进一步研发。     

抑黄曲霉乳酸菌的筛选及菌种鉴定

目的筛选具有抑制黄曲霉(Aspergillus flavus)生长的乳酸菌。方法以各地泡菜、实验室自制泡菜、豆浆渣以及新鲜猪肠、鸡肠道内容物为材料,采用牛津杯法筛选所需菌株。对筛选出的菌株进行生理生化及16S rRNA基因序列同源性分析。结果分离得到756株乳酸菌,其中有6株菌株对黄曲霉的生长有明显的抑制作用。结论实验获得的6株产酸菌,3株为植物乳杆菌(Lactobacillus plantarum),2株为消化乳杆菌(L.alimentari-us),1株为亚利桑那乳杆菌(L.arizonenensis)。     

潜在多重耐药患者肠道内乳杆菌的分离与鉴定

目的从临床潜在多重耐药患者粪便中分离获得乳杆菌,完成种属鉴定并分析其临床意义。方法采集临床患者粪便,在MRS和含碳酸钙的LBS固体培养基上分离培养,挑取目的菌落反复分离获得纯化菌株,利用生化鉴定方法进行种属鉴定。结果 45份样本共得到7株乳杆菌,其中植物乳杆菌5株、鼠李糖乳杆菌2株。结论研究发现,分离到的植物乳杆菌和鼠李糖乳杆菌可能具有更强的耐受性与生物学活性。分离获得的植物乳杆菌和鼠李糖乳杆菌有待日后对其开展特性、耐受性及安全性等研究,以便对分离的菌株有更深入的认识。     

阴道中益生特性乳杆菌的分离与功能评价

目的分离健康女性阴道中的乳杆菌并鉴定其益生特性,为开发治疗妇科疾病的复方益生菌制剂提供新型菌株。方法采集健康女性阴道分泌物并分离筛选乳杆菌,通过16SrDNA序列分析鉴定乳杆菌分离株,并对其产酸性能、产H2O2能力、抑菌能力、产生物膜能力进行检测。结果从50名健康女性阴道内共分离出179株乳杆菌,其中卷曲乳杆菌101株、詹氏乳杆菌42株、格氏乳杆菌26株、植物乳杆菌5株、唾液乳杆菌3株以及干酪乳杆菌2株。179株乳杆菌中有146株具有产酸能力,发酵液pH值的最低的5株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、詹氏乳杆菌J87,植物乳杆菌J75以及格氏乳杆菌J35,其pH分别为4.20、4.23、4.24、4.26及4.36;产H2O2弱阳性菌株有87株、阳性有37株、强阳性有9株,这9株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、卷曲乳杆菌J20、詹氏乳杆菌J87,詹氏乳杆菌J90、詹氏乳杆菌J15、格氏乳杆菌J11、植物乳杆菌J75、植物乳杆菌J69以及植物乳杆菌J40;能拮抗大肠埃希菌的菌株有115株、拮抗金黄色葡萄球菌的有84株、拮抗白假丝酵母的有52株;经统计,对三者同时有拮抗作用且作用最强的只有6株,分别为卷曲乳杆菌J3、卷曲乳杆菌J50、卷曲乳杆菌J62、詹氏乳杆菌J87、詹氏乳杆菌J16和格氏乳杆菌J66;不同乳杆菌产生物膜能力数值范围在1.0~5.4,卷曲乳杆菌、詹氏乳杆菌、干酪乳杆菌的生物被膜形成能力显著高于其他三种菌(P0.05)。在全部179株菌中,卷曲乳杆菌J3和詹氏乳杆菌J87既具有强的产酸能力和产过氧化氢能力,又有较强抑菌活性,同时产生物膜能力也最强。结论卷曲乳杆菌J3和詹氏乳杆菌J87具有优良的生物学特性,有望成为用于治疗妇科疾病微生态制剂的备选菌株。     

模拟人体胃肠道环境筛选益生乳杆菌

【目的】筛选具有益生特性的乳杆菌作为保健型酸奶的候选菌株。【方法】从健康人肠道和奶豆腐中分离筛选出耐受人工胃液的乳杆菌,对其进行体外益生特性(人工胃肠液耐受性、胆盐耐受性、抑菌活性及胆固醇降解能力)研究。【结果】从在乳杆菌分离培养基上有溶钙圈的41株菌株中筛选出5株耐酸、耐人工胃液较强的菌株,经16S rR NA基因测序鉴定,其中3株为乳杆菌,分别命名为植物乳杆菌Lp MT-3、植物乳杆菌Lp MT-5和唾液乳杆菌LsA F-7。在人工胃液中3株菌的耐受力均强于商品化的对照菌株LGG(鼠李糖乳杆菌GG);转入肠液4 h后直至26 h,Lp MT-5存活率基本稳定在45%左右,仅次于LGG。胆盐浓度为0.10%时,3株乳杆菌的耐胆盐能力均强于LGG;胆盐浓度为0.20%时,Lp MT-3和LsA F-7仍能存活。3株乳杆菌均具有抑菌活性,对粪肠球菌的抑制最明显,其次是金黄色葡萄球菌,对大肠杆菌、沙门氏菌的抑制作用较差。3株乳杆菌对胆固醇的清除效力依次为Lp MT-3LpM T-5Ls AF-7;清除率依次为Ls AF-7Lp MT-3LpM T-5。【结论】筛选出3株适应人体胃肠液环境、耐胆盐、抑菌及降胆固醇活力强的乳杆菌,可作为进一步开发新的益生菌产品和保健型酸奶的菌株。     

蒙古国地区酸乳中乳酸菌的鉴定及耐酸菌株筛选

本研究对采集自蒙古国地区牧民家庭中17份发酵乳样品中的乳杆菌进行了分离、鉴定、生物学特性和耐酸性研究。共分离出45株乳杆菌。通过形态观察、生理生化试验、糖发酵试验及16S rDNA序列分析等研究将这些菌株鉴定为Lactobacillum fermentum(L. fermentum)31 株, L. helveticus 12株, L. plantarum 1株 和L. casei 1株, 所以认为L. fermentum是蒙古国地区传统酸乳中的优势菌群。经pH 为3.0 的人工胃液耐受性试验复筛后发现, 存活率在80%以上的仅1株, IMAU20085的存活率高达81.44%。菌株的分离鉴定以及高耐酸性菌株的筛选, 对我国益生菌资源的保藏和开发有重要的意义, 对我国未来益生菌的开发具有重要价值。     

健康仔猪肠道乳杆菌黑龙江地方株的鉴定与种属分析

为了从健康仔猪肠道中分离筛选用于研制微生态制剂的乳酸杆菌菌株,选择黑龙江省大庆、哈尔滨及宝泉岭地区部分猪场,采集12-60日龄健康仔猪肠道粪样64份。选用MRS乳酸杆菌专用培养基分离培养乳酸杆菌,通过分离株的形态特征和培养特性筛选出革兰阳性,厌氧,无芽胞杆菌48株。再通过生化试验鉴定和PCR种属分析,确定18株为乳酸杆菌。其中,罗伊乳杆菌7株,嗜酸乳杆菌5株,约氏乳杆菌2株,短乳杆菌1株,干酪乳杆菌假植物亚种1株,植物乳杆菌1株,詹氏乳杆菌1株。     

一株分离自口腔的鼠李糖乳杆菌及其特性

目的分离筛选出在维护口腔微生态平衡方面具有潜在益生特性的乳杆菌菌株。方法从健康志愿者的口腔样本中分离乳杆菌,采用生理生化和16S rDNA分子测序进行菌株鉴定,并检测其抑菌能力、凝集能力、表面疏水性以及对溶菌酶耐受性。结果筛选出1株鼠李糖乳杆菌LR863,对变形链球菌、戈登链球菌、牙龈卟啉单胞菌、具核梭杆菌和伴放线放线杆菌具有抑菌作用,经蛋白酶处理后其抑菌活性降低。同时鼠李糖乳杆菌LR863有较强的自凝集能力并对上述5株指示菌有共凝集效果,对二甲苯、氯仿和乙酸乙酯的疏水率依次为76.91%、87.46%和41.88%,能耐受2.0 mg/mL浓度的溶菌酶。结论鼠李糖乳杆菌LR863具有优良生物学特性,可作为口腔保健产品的候选益生菌株。     

EZAL-MY96乳酸菌种菌株分离鉴定

目的：对引进法国罗地亚EZAL-MY96直投式酸奶发酵剂(DVS)进行菌株分离鉴定。方法：利用半选择性培养基进行发酵剂菌株分离,并采用糖发酵实验和RAPD指纹图谱进行鉴定。结果：EZAL-MY96中球菌的平板菌落与光镜显微观察、糖发酵实验和RAPD指纹图谱均只有1种情况,而杆菌均有2种情况。结论：从EZAL-MY96菌种中共分离到3株菌,其中1株鉴定为嗜热链球菌,1株为德氏乳杆菌保加利亚亚种,另1株为未知的乳杆菌。     

Robusta coffee beans post-harvest microflora: Lactobacillus plantarum sp. as potential antagonist of Aspergillus carbonarius

Coffee contamination by ochratoxigenic fungi affects both coffee quality as well as coffee price with harmful consequences on the economy of the coffee exporting countries for whom which is their main source of income. Fungal strains were isolated from coffee beans and identified as black Aspergilli. Ochratoxigenic moulds like Aspergillus carbonarius were screened and selected for detailed studies. Also lactic acid bacteria (LAB) were isolated from silage coffee pulp and their antifungal activity was tested on dual-culture agar plate. Ten of the isolated LAB demonstrated antifungal effect against A. carbonarius. API 50 CH and APIZYM were used to perform phenotypic identification. 16S rDNA sequencing was made to confirm the results.     

A differential medium for lactic acid-producing bacteria in a mixed culture

Aims:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue (mMRS-BPB) was tested as a medium for counting and differentiation of each lactic acid-producing bacterium (LAB), especially in a mixed culture.
Methods and Results:  Type strains of 10 LAB species ( Lactobacillus acidophilus , L. brevis , L. bulgaricus , L. gasseri , L. paracasei , L. plantarum , L. reuteri , Weissella confusa , Bifidobacterium bifidum and B. infantis ) and five commercial yogurts were inoculated on plate count agar with bromocresol purple, mMRS, and mMRS-BPB. Each type strain showed more clearly formed colonies on the three media under anaerobic conditions than under aerobic conditions. Especially each type strain produced colonies with specific characteristics of each species on mMRS-BPB. Commercial yogurts produced the largest number of colonies with various shapes and colours on mMRS-BPB.
Conclusions:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue under anaerobic conditions is appropriate for counting and differentiating each LAB in a mixed culture.
Significance and Impact of the Study:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue will be useful in isolation and enumeration of each LAB from fermented foods as well as intestinal microflora.     

EGCG经AKT1-Mdm-2-p53途径抑制卵巢癌HO-8910细胞的增殖

目的：初步探讨EGCG对卵巢癌HO-8910细胞增殖的抑制作用及其机制。方法：通过绘制细胞生长曲线、平皿克隆和软琼脂集落形成实验观察EGCG对HO-8910细胞增殖的抑制作用;Westem—blotting检测AKT1、Mdm-2与p53蛋白的表达。结果：（1）细胞生长曲线、平皿克隆和软琼脂集落形成实验结果显示,EGCG可有效抑制HO-8910细胞的增殖（n=3,P〈0．05）。（2）Western—blotting检测结果显示,EGCG处理后AKT1与Mdm-2蛋白表达均降低,而p53蛋白表达升高（P〈0．05）。结论：EGCG通过抑制HO-8910细胞中AKT1与Mdm-2蛋白表达,促使p53蛋白表达而发挥其对细胞增殖的抑制作用。     

冷鲜牛肉中蜂房哈夫尼亚菌(Hafnia alvei)的分离与鉴定

在出口冷鲜牛肉的沙门氏菌检验过程中,从HE琼脂平板上分离到1株蜂房哈夫尼亚菌,该菌三糖铁斜面产碱,底层产酸,不产生H2S,不产气,革兰阴性。经BBL Crystal微生物半自动检测仪检测,同时结合伯杰细菌鉴定手册的生化试验结果鉴定为蜂房哈夫尼亚菌(Hafnia alvei)。     

一株类芽孢杆菌的分离鉴定及其抗革兰氏阴性菌活性测定

【背景】随着碳青霉烯类和多粘菌素类可转移耐药基因的发现及扩散,多重耐药革兰氏阴性细菌感染更加难以治疗。【目的】筛选有效拮抗革兰氏阴性菌的菌株,为新型抗生素的发掘奠定基础。【方法】利用胰蛋白胨大豆琼脂培养基筛选土壤源细菌,通过16S rRNA基因序列鉴定其种属;通过全基因组测序,antiSMASH比对分析菌株产抗生素潜能,双层琼脂平板法验证其抗菌活性;通过甲醇萃取其次级代谢产物,高效液相色谱串联质谱(HPLC-MS/MS)进行次级代谢产物分析。【结果】从北京周边土壤样品中分离到一株类芽孢杆菌CAU136 (Paenibacillus pabuli CAU136),经过生物信息学分析和antiSMASH比对,表明该菌株有较强合成次级代谢产物的潜能,双层琼脂平板法验证其能抑制多株革兰氏阴性菌生长,HPLC-MS/MS检测结果显示其可能分泌多粘菌素E。【结论】类芽孢杆菌CAU136可能分泌多粘菌素E,能有效拮抗革兰氏阴性菌。     

Inhibitory effect of different chicken-derived lactic acid bacteria isolates on drug resistant Salmonella SE47 isolated from eggs

Lactic Acid Bacteria (LAB) regulate and maintain the stability of healthy microbial flora, inhibit the adhesion of pathogenic bacteria and promote the colonization of beneficial micro-organisms. The drug resistance and pathogenicity of Salmonella enteritis SE47 isolated from retail eggs were investigated. Meanwhile, Enterococcus faecalis L76 and Lactobacillus salivarius LAB35 were isolated from intestine of chicken. With SE47 as indicator bacteria, the diameters of L76 and LAB35 inhibition zones were 12 mm and 8·5 mm, respectively, by agar inhibition circle method, which indicated that both of them had inhibitory effect on Salmonella, and L76 had better antibacterial effect; two chicken-derived lactic acid bacteria isolates and Salmonella SE47 were incubated with Caco-2. The adhesion index of L76 was 17·5%, which was much higher than that of LAB35 (10·21%) and SE47 (4·89%), this experiment shows that the higher the bacteriostatic effect of potential probiotics, the stronger the adhesion ability; then Caco-2 cells were incubated with different bacteria, and the survival of Caco-2 cells was observed by flow cytometry. Compared with Salmonella SE47, the results showed that lactic acid bacteria isolates could effectively protect Caco-2 cells; finally, after different bacteria incubated Caco-2 cells, according to the cytokine detection kit, the RNA of Caco-2 cells was extracted and transcribed into cDNA, then detected by fluorescence quantitative PCR, the results showed that L76 could protect Caco-2 cells from the invasion of Salmonella SE47, with less cell membrane rupture and lower expression of MIF and TNF genes. Therefore, the lactic acid bacteria isolates can effectively inhibit the adhesion of Salmonella and protect the integrity of intestinal barrier.     

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml^-1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

EGCG经AKT1-Mdm-2-p53途径抑制卵巢癌HO-891O细胞的增殖

初步探讨EGCG对卵巢癌HO-8910细胞增殖的抑制作用及其机制.方法:通过绘制细胞生长曲线、平皿克隆和软琼脂集落形成实验观察EGCG对HO-8910细胞增殖的抑制作用;Western-blotting检测AKT1、Mdm-2与p53蛋白的表达.结果:(1)细胞生长曲线、平皿克隆和软琼脂集落形成实验结果显示,EGCG可有效抑制HO-8910细胞的增殖(n=3,P＜0.05).(2)Westemblotting检测结果显示,EGCG处理后AKT1与Mdm-2蛋白表达均降低,而p53蛋白表达升高(P＜0.05).结论:EGCG通过抑制HO-8910细胞中AKT1与Mdm-2蛋白表达,促使p53蛋白表达而发挥其对细胞增殖的抑制作用.