5 L发酵罐中粪肠球菌发酵工艺参数优化

本试验旨在通过单因素试验及Box-Behnken响应面试验设计法优化饲用粪肠球菌EXW 27发酵工艺,探索搅拌速度、中和剂、pH、接种量、初始糖含量、通气方式以及补料工艺等对菌体在发酵罐内生长的影响。优化菌体小试发酵工艺为:将种子液以3%(V/V)接种于装液量为70%的5 L小罐,发酵温度37℃,搅拌转速165 r/min,通空气保压0.02 Mpa,自动流加12.5%的氨水控制pH在5.5,当pH高于5.5流加葡萄糖补料发酵18 h左右结束,在此最佳条件下所得发酵液的活菌浓度约为3.799×10^10 cfu/mL,比优化前提高了近3倍,为以后工业化生产提供参考。     

饲用粪肠球菌培养基及发酵条件的优化研究

为提高饲用粪肠球菌发酵活菌数,通过单因素试验和正交优化试验对粪肠球菌的培养基和发酵条件进行优化.确定最佳发酵培养基为蛋白胨3.2％,酵母粉1％,葡萄糖2.8％,吐温-80 1 ml/L,柠檬酸三铵0.2％,七水硫酸镁0.02％,一水硫酸锰0.005％,碳酸钙1.0％.最佳发酵条件为培养基初始pH(7.1+0.1),接种量2.5％～5.0％,温度34～37℃,150 r/min振荡培养.在最佳培养基和发酵条件下,粪肠球菌活菌数最高可达8.5×109 cfu/ml.     

粪肠球菌产伏尔加霉素E5的生物学特性研究

为了确定分离自健康猪胃肠道粪肠球菌产生的细菌素伏尔加霉素E5(enterocin E5)的生物学特性,本研究检测了该细菌素对热、酸碱、酶类的耐受性及其抗菌谱,enterocin E5经过121℃高压处理10min对指示菌抑菌活性没有影响;细菌素于pH 2～12作用后,对大肠埃希菌仍有抑菌活性,最强的pH抑菌活性范围为pH 4～6;enterocin E5对胰蛋白酶和蛋白酶K处理敏感,过氧化氢酶、脂肪酶和淀粉酶对其抑菌活性没有影响;enterocin E5对21株动物源病原微生物具有抑制作用,革兰阴性病原菌包括大肠埃希菌、鸡白痢沙门菌、鸡伤寒沙门菌、多杀性巴氏杆菌等,特别对产生多重抗药性的3株鸡伤寒沙门菌具有明显的抑菌活性。抑制革兰阳性金黄色葡萄球菌、马腺疫链球菌、猪链球菌,对炭疽芽胞杆菌弱毒株和蜡样芽胞杆菌产生显著的抑菌活性。结果表明,enterocin E5对酸碱是比较稳定的,具有很宽泛的pH耐受范围,属于热稳定IIa亚型细菌素,能够作为一种抗菌物质防控动物源细菌病。     

粪肠球菌R-WL发酵培养基的优化

本试验旨在优化培养基提高粪肠球菌(Enterococcus faecalis)R-WL发酵液的菌体密度,为高效、优质的微生态制剂产品研制奠定基础。首先通过生长曲线的测定、培养条件和培养基成分的单因素试验,确定菌种RWL的最适培养条件为:时间14 h、pH 8.5、温度37℃、接种量3%,最适碳源为乳糖,浓度为10 g/L,最适氮源为蛋白胨+酵母膏,浓度为30 g/L。然后通过Plackett-Burman试验分析培养基中对粪肠球菌发酵影响最重要的主要因素为蛋白胨、乙酸钠和柠檬酸二铵,再通过最陡爬坡试验获得主要因素的最适范围,最后通过Box-Behnken试验和响应面分析得到主要因素的最适添加量。结果表明:最优发酵培养基配方为:乳糖10 g/L、酵母膏15 g/L、蛋白胨12.18 g/L、乙酸钠4.5 g/L、柠檬酸二铵4.5 g/L、硫酸镁0.58 g/L、硫酸锰0.25 g/L、磷酸氢二钾2 g/L、轻质碳酸钙3 g/L,初始pH 8.5。通过试验验证,优化后发酵培养基的发酵菌液活菌数可达(6.44±0.10)×109cfu/mL,比原MRS培养基活菌数提高了71.28%。     

肠球菌产enterocin基因的检测

肠球菌属(Enterococci)是乳酸菌中对营养要求不高、兼性厌氧、容易培养、分布广泛的一类革兰阳性球菌.肠球菌广泛应用于乳制品加工、蔬菜及肉制品的发酵工艺中.肠球菌在生长繁殖过程中除产生乳酸、乙酸等有机酸外,还能产生多种具有抑菌或杀菌活性的细菌素,在抑制多种动物源病原微生物和食物病原菌、腐败菌等方面具有重要作用.     

基于响应面法优化饲用粪肠球菌发酵培养基

为了实现粪肠球菌(Enterococcus faecalis)E_(XW)27的高密度培养,对健康仔猪肠道分离的粪肠球菌E_(XW)27发酵培养基进行响应面优化,以MRS为基础培养基,活菌浓度为评价指标,对发酵培养基中碳氮源、微量元素、缓冲盐体系以及促生长因子等通过单因素试验和响应面试验优化高密度发酵培养的培养基配方,确定最佳活菌浓度时的培养基组成。结果显示:蔗糖57.3 g/L、蛋白胨45 g/L、磷酸氢二钾3.75 g/L、柠檬酸铵3.5 g/L、乙酸钠6.25 g/L、硫酸镁1.5 g/L、硫酸锰0.45 g/L、组氨酸1.4 g/L、维生素C 0.01 g/L、碳酸钙10 g/L。发酵液最高活菌数达到1.03×10~(10) CFU/mL,是相同条件下MRS培养基中活菌数的10.61倍,菌体浓度显著提高,具有实际应用价值。     

猪源粪肠球菌的基因型及耐药性分析

为了解上海地区规模化猪场中粪肠球菌的耐药性和基因型,2013年3月-2014年5月从上海地区10个规模化养猪场收集到分离鉴定的粪肠球菌133株,采用微量肉汤稀释法对12种抗菌药物进行药敏试验,采用PCR技术分别检测分离株中β-内酰胺类抗生素耐药相关基因(TEM)、氨基糖苷类抗生素耐药相关基因[aac(6’)/aph(2")和aph(3’)-Ⅲ和ant(6)-Ⅰ]、四环素耐药相关基因(tetM)、红霉素耐药相关基因(ermB和mefA)和万古霉素耐药相关基因(vanA和vanB)。结果显示,在133株粪肠球菌中,耐药基因ermB、tetM、TEM、ant(6)-Ⅰ、aac(6’)/aph2"、aph(3’)-Ⅲ、vanA的检出率分别为:95.5%(127/133)、91.0%(121/133)、63.2%(84/133)、45.9%(61/133)、42.1%(56/133)、24.1%(32/133)、3.0%(4/133);未检出mefA和vanB基因的菌株。分离菌株对12种抗菌药物的耐药性较为严重,且以6~9耐为主要耐药株,占81.2%。试验表明,上海地区猪源粪肠球菌多重耐药现象严重,携带抗菌药物相关耐药基因是导致分离株耐药的主要原因。     

乳酸粪肠球菌的筛选及富硒效果的试验研究

采用厌氧罐培养,对乳酸粪肠球菌进行筛选、富硒效果及富硒条件的研究,结果乳酸粪肠球菌具有较强的耐受硒和富集硒的能力,其富集硒的效果受培养发酵液中的硒添加量、接种量和发酵时间的影响.获得乳酸粪肠球菌最佳发酵产量和富硒量的条件,考虑到试验中的菌体红色因素,确定硒添加量35 mg/L、接种量10%、发酵时间28 h、pH4.5~6.3,是比较理想的乳酸粪肠球菌富硒发酵增殖培养条件.其发酵产量和最佳富硒量为:菌体产量404.10 mg/100ml,干物质产量683.14 mg/100 ml,菌体富硒量248.56 μg/100 ml,干物质富硒量393.21 μg/100 ml,发酵液环境硒量31.07 mg/L,干物质非有机硒量147.65 μg/100 ml.     

新疆北疆地区猪源粪肠球菌的耐药性分析

为了解新疆北疆地区猪源粪肠球菌的耐药性及相关耐药基因型的分布情况,本试验采用K-B（Kirby-Baller）琼脂扩散法检测了49株猪源粪肠球菌对8种抗菌药物的敏感性,并采用PCR法对9种相关耐药基因进行检测并测序,测序结果与GenBank中的相应基因序列比对。药敏试验结果显示,分离菌对链霉素耐药率最高,其次为青霉素和红霉素,对呋喃妥因、氨苄西林高度敏感。PCR检测结果显示,β-内酰胺类耐药基因tem的检出率最高,为93.88%,其次是四环素类耐药基因tetM,为85.71%,喹诺酮类基因gyrA和parC检出率均为42.86%,氨基糖苷类耐药基因aph（3'）-Ⅲ、aac（6'）/aph2″和ant（6'）-Ⅰ的检出率分别为36.73%、16.33%和16.33%,未检出mefA和ermB基因。本试验从表型与基因型分析发现,北疆地区猪源粪肠球菌的多重耐药现象非常严重,且其耐药表型与基因型并不完全一致。     

粪肠球菌生长曲线的测定

用分光光度仪和活菌计数两种不同的方法对粪肠球菌生长曲线进行测定,结果表明,在细菌对数生长期以前,两种方法所得结果基本一致,但在衰亡期,两种方法具有差异,活菌记数法更能如实反映细菌的生长情况.     

鲤鱼源粪肠球菌的体外抑菌试验研究

为证明益生菌候选菌株具有益生特性,将筛选的鲤鱼源粪肠球菌YL-1进行体外抑菌试验。结果发现,YL-1对病原菌嗜水气单胞菌有较强的抑菌作用;通过对YL-1上清液进行热处理,蛋白酶处理,中和酸等方式分析发现,其抑菌物质主要是对有机酸和胰蛋白酶敏感的一类物质。     

Enterococcus faecalis of human and poultry origin share virulence genes supporting the zoonotic potential of E. faecalis

Enterococcus faecalis is a major cause of nosocomial infections in humans and has been linked to severe extra‐intestinal infections in poultry. A zoonotic potential has been suggested and the aim of the present study was to investigate similarities in virulence gene profiles of E. faecalis originating from infections in humans and poultry respectively. A total of 106 isolates of E. faecalis [26 human clinical isolates, 60 poultry clinical isolates (including two small‐colony variants (SCVs) and 20 poultry cloacal isolates] were investigated for presence of seven virulence‐associated genes: ace, asa1, cylA, efaA, EF0591, esp and gelE. For each gene, the PCR‐amplification product was sequenced from one isolate in each group to explore intragenic variations between genes of human and poultry origin. Haemolytic and protease activities were assessed and isolates were assigned a sequence type (ST). Three of the seven genes investigated (ace, efaA and gelE) were present in all isolates. The asa1 was detected in 63/80 and 13/26 isolates of poultry and human origin respectively. For cylA, the numbers were 46/80 and 14/26 respectively. Among poultry isolates, esp and EF0591 were the least frequently observed genes (1/80 and 20/80 respectively); the prevalences among human isolates were 1/26 and 18/26 respectively. A high degree of similarity between genes in human and poultry isolates were confirmed by sequencing of amplification products. None of the cylA‐positive isolates demonstrated haemolytic activity, while the phenotypic expression of gelatinase varied. The ST16 was the only ST shared by human and poultry isolates. The SCV isolates did not show a unique virulence profile or phylogeny. In conclusion, regardless of the distinct phylogenetic background of most E. faecalis isolates of human and poultry origin, we found major similarities in virulence gene profile and gene sequences in isolates from the two sources, supporting the zoonotic risk associated with this organism.     

微量环丙沙星对人源大肠杆菌和粪肠球菌体外敏感性的影响

建立人离体肠道模拟模型，研究微量环丙沙星对人源肠道大肠杆菌和粪肠球菌敏感性的影响，进而用聚合酶链反应法扩增耐药菌的gyrA基因的耐药决定区，并分析其耐药机制。结果显示，大肠杆菌连续培养后存活菌株对微量环丙沙星耐药，此耐药菌对其他抗菌药敏感；粪肠球菌绎连续培养，对环丙沙星和其他抗菌药物仍敏感；耐药大肠杆菌的gyrA基因发生突变，248位碱基由C变为T，259位由G变为T，相应地，该基因编码的蛋白质在83位的丝氨酸和87位的天冬氨酸分别改变为亮氨酸和酪氨酸。研究表明，微量环丙沙星对人肠道菌群具有不同的选择作用，能诱导大肠杆菌产生耐药性。这为动物源食品中环丙沙星残留的安全性评价提供了试验依据。     

Reduction of Salmonella in gnotobiotic Japanese quails caused by the enterocin A-producing EK13 strain of Enterococcus faecium

The effect of the enterocin A-producing EK strain of Enterococcus faecium on Salmonella dusseldorf SA31 was tested in gnotobiotic Japanese quails. Sixteen 3-day-old gnotobiotic Japanese quails, hatched from disinfected eggs placed in sterile boxes, were divided into two groups of 8 birds: a control group, which was inoculated orally with the SA31 strain (1×10⁷ CFU/ml), and the experimental group, which was inoculated orally with 200 l of E. faecium (1×10⁹ CFU/ml), 16 h before infection with the S. dusseldorf. The latter group then received the same average dose of E. faecium daily in drinking water. Faecal samples were taken 8, 24, 48 and 168 h after the inoculation of S. dusseldorf and examined for S. dusseldorf and E. faecium (EK13). The quails were then killed and the number of the EK13 strain of E. faecium and of S. dusseldorf in the caecum and ileum were estimated. A reducing effect of the EK13 strain against the SA31 strain in faeces was detected in the samples taken at 24 and 48 h from the group with the EK13 strain. Significant reductions were also found in the numbers of S. dusseldorf SA31 strain in the caecum but not in the ileum.     

乳酸菌制剂在黑毛和牛育肥中的使用效果

[目的]旨在通过黑毛和牛（Japanese Blackcattle）育肥后期投服乳酸菌制剂Entero—coccus faecalis FK-23粉末,探讨付与牛肉的附加价值和该制剂的实用性。[方法]供试牛10头,随机分为试验组和对照组,试验组5头,每头每日投服乳酸菌制剂8．0g。测量体重（BW）,静脉采血检测白细胞数（WBC）,维生素A（VA）,维生素E（VE）．总胆固醇（TC）,谷草转氨酶（GOT）,血尿素氮（BUN）及血糖（GLC）浓度;HPLC测验中性脂肪的脂肪酸成分。[结果]试验组与对照组比较。食欲亢进,日增重（DG）明显增加（P〈0．05）,血清VE和TC明显升高（P〈0．05）,VA维持预计水平走向不变。中性脂肪的不饱和脂肪酸比例出现增高趋势,出栏体重增长4．3％,胴体重增加9．3kg。[结论]结果提示,黑毛和牛育肥后期投服乳酸菌E．faecalisFK-23制剂可以获得VE含量较高的高附加价值牛肉并能提高其生产性能。     

致羔羊脑炎粪肠球菌生物被膜形成能力及相关基因检测研究

为观察致羔羊脑炎粪肠球菌XJ05株生物被膜（BF）的形成能力并检测致羔羊脑炎粪肠球菌BF形成相关基因的携带情况,本研究以XJ05为研究对象,运用96孔板建立不同时间BF模型,染色后酶标仪测定D_{595 nm}值,并用倒置显微镜观察BF形态。同时检测11株菌中与BF形成相关基因（gelE、esp、ace、asa1、asa373、efaA、ef0591、ef3314、ahrc、eep）的携带情况,并对相关基因片段做同源性分析。结果显示,在不同时间段XJ05株BF的生成量与空白对照组相比差异显著（P<0.05）,24 h时D_{595 nm}值最高且与其他各个时间段相比均差异显著（P<0.05）。显微镜下观察6 h时BF初具规模,可见散落的网状结构,12~24 h矩阵网格结构明显,24 h时形成高度有序的网格结构,24 h后网状结构逐渐脱落,BF开始降解。11株菌中10种相关基因的携带情况不同,有6株携带8种基因,1株携带7种基因,1株携带6种基因,1株携带2种基因,有2株未检测到10种基因的任何一种。检出的基因片段同源性均在93.3%~100.0%之间。结果表明,XJ05株能形成完整的BF,11株分离的致羔羊脑炎粪肠球菌中有9株携带部分与BF形成相关的基因。     

Detection on Biofilm and Biofilm-associated Genes of Enterococcus faecalis Inducing Lamb Encephalitis

The purpose of this study was to observe and analyze the ability of biofilm formation of the Enterococcus faecalis (E.faecalis) XJ05 and to detect if the E.faecalis which could induce lamb encephalitis carried the biofilm-associated genes. The E.faecalis XJ05 was used as the sample. The biofilm model was established by the 96-well microtiter plate and read by microplate reader in the D_{595 nm} at the same time observed by the inverted microscope. The primers were designed to examine if the 11 strains of E.faecalis carried the biofilm related genes and then the amplified products were sequenced and analyzed for homology. The results showed that the amount of BF produce in XJ05 strain was significantly different from blank control group at different time (P<0.05), and the highest D_{595 nm} value was at 24 h and significant difference compared with other time (P<0.05). The biofilm of 6 h was scattered when observed under the microscope and the time between 12 to 24 h biofilm to be more denser, after 24 h the biofilm became to degradate and loose gradually. It turned out that the 11 strains of bacteria carried different biofilm-associated genes, 8 biofilm-associated genes were found in 6 strains of bacteria, 7 biofilm-associated genes were find in 1 strain of bacterium, 6 biofilm-associated genes were found in 1 strain of bacterium, 2 biofilm-associated genes were found in 1 strain of bacterium, 2 strains of bacteria could not text out anyone of the detected genes.The homology of all detected genes were all between 93.3% to 100.0%. It concluded that the E.faecalis XJ05 could form the completed biofilm and there were 9 kinds carried the biofilm-associated genes in 11 strains of E.faecalis.     

粪肠球菌生长曲线的测定及其对小鼠脑组织的影响

为测定致羔羊脑炎粪肠球菌（Enterococcus faecalis）的生长曲线,寻求一种快速而准确的方法测定不同生长时期粪肠球菌数量,并客观评价其毒性强弱及其对小鼠脑组织的影响,试验采用平板菌落计数法和OD-Monitor振荡比浊法（D_λ值法）测定粪肠球菌的生长曲线,探究该菌在合适时间段内的吸光值（D_{600 nm}）与平板菌落计数法测定的活菌数（CFU）的关系。用粪肠球菌感染小鼠,观察记录小鼠的死亡情况,最后采用Karber法计算粪肠球菌感染小鼠的半数致死量（LD₅₀）。用LD₅₀的剂量感染小鼠,及时采集死亡小鼠脑组织,未死亡的小鼠72 h后全部剖杀取脑组织,一部分做涂片染色,制作病理切片,观察病理变化;一部分进行培养,用于PCR方法进行细菌的回收鉴定。结果显示,用两种方法测定此株粪肠球菌的生长曲线基本一致,在2~8 h生长迅速,为对数生长期,8~14 h生长缓慢,为稳定期,14 h之后死亡数增加,进入衰亡期;对12 h粪肠球菌D_{600 nm}与CFU的关系进行探讨,成功建立回归方程：y=20.769x-1.3422,R²=0.997;其感染小鼠的LD₅₀为7.77×10¹¹个活菌。以此剂量感染小鼠,脑组织涂片染色和培养染色,均能看到革兰氏阳性球菌;PCR结果显示,均出现了大小为112 bp的条带。对脑组织进行病理学观察发现该菌可导致脑组织充血、出血、形成微血栓,脑膜充血。通过生长曲线和其D_{600 nm}与CFU关系的建立,可实时监测粪肠球菌数量,为后期更深入研究粪肠球菌穿越血脑屏障的机制奠定重要的理论基础。     

屎肠球菌素E9的产生和特性研究

用琼脂扩散法分别检测了不同培养时间的屎肠球菌E9无细胞上清液和连接在菌体上的Enterocin E9活性,同时分析了Enterocin E9的生物学特性。结果显示：含Enterocin E9的上清液和菌体都具有抑菌活性,活性大小存在浓度依赖性。在一定时间范围内,其上清液和菌体的活性都随着培养时间的增加而逐渐升高。取上清液进行稳定性分析,结果发现Enterocin E9有很好耐热和耐酸碱性,经121℃处理20 min后其活性仍大部分保留,在pH2~12的范围内37℃下处理4 h仍保持较高活性;不同浓度的吐温-80对其活性无明显影响;室温放置7周后,活性无明显变化。上述实验说明屎肠球菌E9产生的细菌素不仅分泌到上清液中,同时还有较大部分与菌体结合。Enterocin E9稳定性良好,储藏条件简单,具有在畜牧业中应用的潜能。