基于耐酸性与抑菌性效果的益生菌候选菌株体外筛选

目的基于筛选耐酸性强和抑菌效果好的菌株为靶点,为益生菌研发提供优良的候选菌株。考察菌株耐酸性与抑菌性的相关性,为益生菌快速筛选探索新方法。方法选取62株不同乳杆菌、魏斯菌、粪肠球菌,在酸性程度不同的培养基中采用接种培养的方式研究受试菌株耐酸能力。用大肠埃希菌和金黄色葡萄球菌作为指示菌,采用牛津杯法研究受试菌株的抑菌能力。分析同种细菌的耐酸能力和抑菌能力的相关关系。结果在酸性环境下,受试菌株存活率超过50%的有22株,耐酸性较强的菌株有DMLA16017、DMLA16116、DMLA16117、DMLA16118和DMLA16131。受试菌株对病原菌的抑菌力较强的有DMLA16017、DMLA16009、DMLA16118、DMLA16002和DMLA15015。结论鼠李糖乳杆菌DMLA16017和发酵乳杆菌DMLA16118在耐酸性和抑菌性上表现出优良性状。可以通过受试菌株对酸性耐受能力指示受试菌株对病原菌抑制能力,实现对受试菌株的快速筛选。     

水产源芽胞杆菌的分离鉴定及生物学功能分析

筛选具有较好生物学功能的芽胞杆菌(Bacillus),用以改善池塘养殖过程饲料等有机物降解、抑制水体中的病原菌,对从健康养殖鱼虾塘水体中分离的菌株,进行生化试验和16S rDNA序列分子鉴定;通过产酶、耐酸、耐高温试验,抑菌试验以及安全性试验研究分离菌株的生物学功能。试验共筛选出8株细菌,经生化试验及16S rDNA序列的分子鉴定,确定菌株ZHX17、ZHX18、ZHX31为贝莱斯芽胞杆菌（Bacillus velezensis）,菌株ZHX14、ZHX15为地衣芽胞杆菌（Bacillus licheniformis）、菌株NSX4、NSX7、NSX9为枯草芽胞杆菌（Bacillus subtilis）。试验结果表明,8株芽胞杆菌均具有较强的耐酸、耐高温特性,其中3株贝莱斯芽胞杆菌具有较强的分泌淀粉酶、纤维素酶、蛋白酶的能力,抑菌效果好于其他5株芽胞杆菌。安全性试验结果表明,8株芽胞杆菌对草鱼、罗非鱼相对安全。8株芽胞杆菌同时具备分泌淀粉酶、纤维素酶和蛋白酶3种胞外酶的能力,其中贝莱斯芽胞杆菌具有较强的病原菌抑菌能力,可以作为病原拮抗益生菌做进一步研究。     

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。     

从人胃肠道源乳杆菌株中初步筛选耐酸耐胆盐优良株

目的筛选对酸和胆盐耐受性均较强的优良乳杆菌株。方法模拟人体胃酸环境（pH=3）和十二指肠高胆盐环境（胆盐含量=3‰）测定104株人胃肠道源乳杆菌在酸性及高胆盐环境下作用4h后的存活菌数,并与对照相比较。对结果进行统计学分析。结果16株菌在酸性环境及高胆盐环境下4h后的活菌下降对数值均小于2。筛选出3株具有较强耐酸耐胆盐能力的乳杆菌株。结论大多数人胃肠道源乳杆菌对酸和胆盐的耐受能力均不强,对酸的耐受性普遍强于对胆盐的耐受性。     

猪肠道乳杆菌的筛选及其生物学特性研究

目的从健康仔猪肠道中筛选到用于研制微生态制剂的优良乳杆菌株。方法本研究选用乳杆菌选择培养基LBS、MRS从健康仔猪肠道分离到67株乳杆菌,对其染色镜检、生化试验和耐酸、耐胆汁、耐高温、抑菌活性及动物安全性等生物学特性进行初步研究。结果从其中筛选到3株嗜酸乳杆菌,均能耐受pH3．0的酸度、0．8％～1．0％的牛胆盐和60℃的高温,其代谢产物对猪肠道致病性大肠埃希菌和猪沙门菌具有抑制作用,且对小白鼠无致病性。结论3株嗜酸乳杆菌符合益生菌的要求,可作为猪用益生素制剂的候选菌株。     

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。     

商品鸡盲肠内容物乳酸菌的分离与鉴定

实验以健康商品鸡盲肠为菌源,对附着其上的乳酸菌进行分离与鉴定,就所获得的菌株的耐酸耐胆汁、黏附特性、抑菌性进行初步研究。实验结果表明,从商品鸡盲肠中分离出6株乳酸菌,根据耐酸耐胆汁试验,病原菌生长抑制试验以及肠道黏膜粘附试验,筛选出2株优势菌株CX001和CX005,经乳杆菌属的生化鉴定,初步确定为短乳杆菌。     

婴幼儿粪便中双歧杆菌的分离及其菌株特性研究

为筛选具有潜在益生功能的双歧杆菌,本研究采用改良MRS培养基,从婴幼儿粪便中分离到5株菌株:AR499,AR667,AR668,AR669和AR610。通过16S r DNA测序分别鉴定为两歧双歧杆菌,短双歧杆菌,动物双歧杆菌,长双歧杆菌和假小链双歧杆菌。对其耐酸耐胆盐能力和黏附性能进行测试,结果表明,菌株AR499耐酸性较好,菌株AR610有较强的耐胆盐能力,菌株AR499的自动聚集能力和细胞表面疏水性都较高。实验表明,菌株AR499可作为一株耐受性和黏附性能较好的益生菌进一步深入研究,以期应用于优良双歧杆菌产品的开发。     

猪源乳酸菌的分离、鉴定及其生物学特性

【目的】将分离自猪肠道粘膜、食糜和粪便的乳酸菌,通过产乳酸能力、生长性能、耐酸和耐胆盐性能及抑菌能力评价,筛选适应养猪生产的潜在益生特性的菌株。【方法】共分离获得155株乳酸菌纯菌株,从中筛选出4株产酸能力较强的乳酸菌,结合生理生化试验及细菌16S rRNA测序鉴定其种属,评价候选乳酸菌的生长情况、耐酸、耐胆盐及抑菌特性。【结果】综合变色时间(8 h)、pH值(3.9)和乳酸含量(100 mmol/L),筛选出4株(L45、L47、L63和L79)候选菌株,经鉴定依次为罗伊氏乳杆菌、植物乳杆菌、约氏乳杆菌和粪肠球菌。该4株乳酸菌均可在体外快速生长;L47和L79能够耐受pH 2.5的酸性环境,L47能够耐受0.5%胆盐环境;各乳酸菌上清液与指示菌共培养,发现对E coli K88和沙门氏菌均产生了抑制作用,其中L47上清液对指示菌的抑制作用较强。【结论】L47具有较好的产酸性能与生长性能、可耐受猪胃酸和肠道胆盐环境,对E.coli K88和沙门氏菌具有较好的抑制作用,说明该乳酸菌具有潜在的益生特性。     

抑制龋齿生成菌(变异链球菌)的优势共培养菌株的筛选CSCD

目的比较10株益生菌的各项生理功能,筛选抑制龋齿生成菌(变异链球菌)的优势共培养菌株,为后期开发应用奠定基础。方法对组合菌株及单个菌株进行体外抑菌试验筛选,并进一步探索对变异链球菌的抑菌机制。结果10株益生菌菌株中,植物乳杆菌AI-66、乳双歧杆菌AI-01、干酪乳杆菌AI-12和婴儿双歧杆菌AI-20抑制变异链球菌较其他菌株有优势;鼠李糖乳杆菌AI-11与发酵乳杆菌AI-25、婴儿双歧杆菌AI-20与副干酪乳杆菌AI-62、乳双歧杆菌AI-01与干酪乳杆菌AI-12这3个共培养组合菌株较单个菌株对变异链球菌的抑制效果差异具有统计学意义(均P<0.01)。其可能的抑菌物质在低pH具有较好的抑菌效果,且对热不敏感,对胰蛋白酶敏感,可能是小分子多肽类物质。结论筛选出的3个共培养组合菌株在抑制龋齿生成菌(变异链球菌)效果上较单菌株有优势,可应用于食品、功能食品及膳食补充剂中,以降低龋齿、口臭等口腔疾病的风险。     

降血尿酸益生菌株的筛选和降血尿酸机理的探索

【背景】高尿酸血症是人体内血尿酸含量显著高于正常水平的代谢性疾病,利用益生菌降解食物中外源性嘌呤类成分成为治疗高尿酸血症的新方法。【目的】筛选具有降低血尿酸作用的益生菌,并探索其作用机制。【方法】利用HPLC从多株实验菌株中筛选降解核苷酸(腺苷酸、鸟苷酸)、核苷(腺苷、鸟苷)、嘌呤(黄嘌呤、次黄嘌呤、鸟嘌呤)、尿酸能力最强的益生菌。首次利用质谱定性与定量检测菌株降解核苷与核苷酸过程中代谢物的变化,结合菌株对高尿酸血症模型大鼠血尿酸水平的影响,初步探索其降低血尿酸的机理。【结果】首次筛选出具有较强降解核苷酸与核苷能力的干酪乳杆菌ZM15(CGMCC No.13980),高尿酸血症模型大鼠验证其具有降低血尿酸的作用。结果显示菌株ZM15在胞内降解核苷酸、核苷后,胞内、外均测到鸟嘌呤、黄嘌呤、次黄嘌呤,且胞内3种嘌呤含量显著高于正常菌体内含量(P0.01),尿酸和尿囊素在胞内、外均未发现。【结论】干酪乳杆菌ZM15具有较强的降解核苷酸、核苷的能力,推测其主要通过与肠道上皮细胞竞争吸收核苷酸与核苷,从而对高尿酸血症模型大鼠具有降血尿酸作用。     

Preliminary selection study of potential probiotic bacteria from aquacultural area in Tunisia

In order to test the ability to produce antibacterial substances within marine bacteria, prior to select potential probiotics for use in shellfish farming, we targeted a large collection of bacterial isolates (132 strains), brought from the clamRuditapes decussatus and 37 reference strains. First, we proceeded to their biochemical identification and the screening of antibiotic resistance profiles. Else, we investigated their inhibitory activityin vitro against several fish and shellfish pathogens, using two methods: the double-layer agar and the direct simultaneous antagonism methods. The results showed high frequencies of inhibitory producing bacteria (IPB) within the isolates. These bacteria (25%) were aerobic mesophylic bacteria belonging to various bacterial groups: 33.7% oxidase-positive Gram-negative bacteria, 7.4%Enterobacteriaceae and 28% lactic acid bacteria. Besides this group, nine strains produced strong inhibition effect. These bacteria belonged to:Aeromonas hydrophila, Aeromonas sobria, Pseudomonas cepacia, Vibrio sp,Serratia liquefaciens andLactobacillus rhamnosus. They were active against pathogenic bacteria belonging to the genera:Aeromonas, Pseudomonas andVibrio. These potential probiotics were submitted to further investigations prior to their introduction in larval shellfish farming.     

Selecting lactic acid bacteria for their safety and functionality by use of a mouse colitis model

Studies showed that specific probiotics might provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening of new probiotics is needed to study possible adverse interactions with the host, particularly when intended for administration to individuals with certain health risks. In this context, the objective of this study was to investigate the role of three lactobacilli (LAB) on intestinal inflammation and bacterial translocation using variations of the mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis. We first compared the in vitro ability of LAB to survive gastrointestinal tract (GIT) conditions and their ability to persist in the GIT of mice following daily oral administration. As a control, we included a nonprobiotic Lactobacillus paracasei strain, previously isolated from an endocarditis patient. Feeding high doses of LAB strains to healthy and to TNBS-treated mice did not induce any detrimental effect or abnormal translocation of the bacteria. Oral administration of Lactobacillus salivarius Ls-33 had a significant preventive effect on colitis in mice, while Lactobacillus plantarum Lp-115 and Lactobacillus acidophilus NCFM did not. None of the three selected LAB strains translocated to extraintestinal organs of TNBS-treated mice. In contrast, L. paracasei exacerbated colitis under severe inflammatory conditions and translocated to extraintestinal organs. This study showed that evaluations of the safety and functionality of new probiotics are recommended. We conclude that not all lactobacilli have similar effects on intestinal inflammation and that selected probiotics such as L. salivarius Ls-33 may be considered in the prevention or treatment of intestinal inflammation.     

Screening and Characterization of Purine Nucleoside Degrading Lactic Acid Bacteria Isolated from Chinese Sauerkraut and Evaluation of the Serum Uric Acid Lowering Effect in Hyperuricemic Rats

Hyperuricemia is well known as the cause of gout. In recent years, it has also been recognized as a risk factor for arteriosclerosis, cerebrovascular and cardiovascular diseases, and nephropathy in diabetic patients. Foods high in purine compounds are more potent in exacerbating hyperuricemia. Therefore, the development of probiotics that efficiently degrade purine compounds is a promising potential therapy for the prevention of hyperuricemia. In this study, fifty-five lactic acid bacteria isolated from Chinese sauerkraut were evaluated for the ability to degrade inosine and guanosine, the two key intermediates in purine metabolism. After a preliminary screening based on HPLC, three candidate strains with the highest nucleoside degrading rates were selected for further characterization. The tested biological characteristics of candidate strains included acid tolerance, bile tolerance, anti-pathogenic bacteria activity, cell adhesion ability, resistance to antibiotics and the ability to produce hydrogen peroxide. Among the selected strains, DM9218 showed the best probiotic potential compared with other strains despite its poor bile resistance. Analysis of 16S rRNA sequences showed that DM9218 has the highest similarity (99%) to Lactobacillus plantarum WCFS1. The acclimated strain DM9218-A showed better resistance to 0.3% bile salt, and its survival in gastrointestinal tract of rats was proven by PCR-DGGE. Furthermore, the effects of DM9218-A in a hyperuricemia rat model were evaluated. The level of serum uric acid in hyperuricemic rat can be efficiently reduced by the intragastric administration of DM9218-A (P<0.05). The preventive treatment of DM9218-A caused a greater reduction in serum uric acid concentration in hyperuricemic rats than the later treatment (P<0.05). Our results suggest that DM9218-A may be a promising candidate as an adjunctive treatment in patients with hyperuricemia during the onset period of disease. DM9218-A also has potential as a probiotic in the prevention of hyperuricemia in the normal population.     

神农架川金丝猴源肠道粪肠球菌的分离与鉴定

通过培养特征、形态观察、抑菌性实验,从神农架健康野生金丝猴肠道分离到8株抑菌效果明显的菌株;生理生化鉴定和16S rRNA基因序列同源性分析,该8株菌为粪肠球菌。毒力因子检测和动物急性毒性实验筛选出2株安全性良好的菌株,研究两菌株对胃肠道环境（低pH值、高胆盐）的耐受特性和生长情况。结果表明,粪肠球菌dlt7a和dlt7b株繁殖能力很强,无迟缓期,具有较强的耐受胃酸及肠道高胆盐环境的能力,且安全性好,具有较好的益生特性,可作为金丝猴微生态制剂的候选菌株。     

Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli

This study attempted to isolate lactobacilli strains from healthy vaginal ecosystem to search for a new effective antibacterial probiotic strain. The strains were identified and characterized for their probiotic properties including bile salt and acid tolerance, growth at acidic pH, their ability to utilize protein, starch, and lipid, the production of hydrogen peroxide and bacteriocin as well as their antibiotic resistance patterns. The antibacterial activity of the culture supernatant of these strains were tested against a wide range of Gram-positive and Gram-negative pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Salmonella typhi, and Salmonella typhimurium. None of the strains inhibited the growth of Gram-negative bacteria. Contrastly, the culture supernatant of strain L 22, identified as Lactobacillus reuteri, significantly inhibited all of the clinical isolates of methicillin-resistant S. aureus (MRSA). The antibacterial effect of the selected strain L 22 was further investigated. In the presence of L 22, the bacterial growth was assessed in vitro by viable bacterial counting. The numbers of viable cells were significantly lower in L 22-containing broth than those in the control by 6h. This finding clearly demonstrates that strain L 22 can produce an anti-MRSA effect. The antibacterial ability of the strain L 22 was fundamentally attributed to their bacteriocin production which can cause both cell inhibition and cell death.     

The Inhibition Effect of Lactobacilli Against Growth and Biofilm Formation of <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>

The emergence of antibiotic-resistant and food-spoilage microorganisms has renewed efforts to identify safe and natural alternative agents of antibiotics such as probiotics. The aim of this study was the isolation of lactobacilli as potential probiotics from local dairy products with broad antibacterial and anti-biofilm activities against antibiotic-resistant strains of Pseudomonas aeruginosa and determination of their inhibition mechanism. Antibiotic susceptibility and classification of acquired resistance profiles of 80 P. aeruginosa strains were determined based on Centers for Disease Control and Prevention (CDC) new definition as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) followed by antibacterial assessment of lactobacilli against them by different methods. Among the 80 P. aeruginosa strains, 1 (1.3%), 50 (62.5%), and 78 (97.5%) were PDR, XDR, and MDR, respectively, and effective antibiotics against them were fosfomycin and polymyxins. Among 57 isolated lactobacillus strains, two strains which were identified as Lactobacillus fermentum using biochemical and 16S rDNA methods showed broad inhibition/killing and anti-biofilm effects against all P. aeruginosa strains. They formed strong biofilms and had bile salts and low pH tolerance. Although investigation of inhibition mechanism of these strains showed no bacteriocin production, results obtained by high-performance liquid chromatography (HPLC) analysis indicated that their inhibitory effect was the result of production of three main organic acids including lactic acid, acetic acid, and formic acid. Considering the broad activity of these two L. fermentum strains, they can potentially be used in bio-control of drug-resistant strains of P. aeruginosa.     

2株动物微生态制剂菌的分离鉴定及其生物学特性初步研究

目的从饲料中进行微生物的分离与培养,筛选动物微生态制剂候选菌株。方法利用变性梯度凝胶电泳(DGGE)筛选得到的7株微生物区分为2种菌,经测序确定其为热带假丝酵母和植物乳杆菌;检测了不同pH、温度、胆盐、金属铜和需/厌氧对其生长的影响。结果当pH小于2.5或铜离子高于150 ppm时,植物乳杆菌无法生长,而热带假丝酵母数量略有下降;胆盐和金属离子对2种菌影响较小,42℃培养条件下相对于30℃培养时热带假丝酵母数量下降了6个数量级。结论筛选得到的2株菌具有应用于动物微生态制剂的潜力,为动物微生态制剂候选菌筛选和评价提供了基础数据。     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.