蜡样芽胞杆菌生长特性及大蒜精油对其抑菌活性的研究

主要研究了37℃条件下3种蜡样芽胞杆菌在营养肉汤中的生长状况,建立了37℃下蜡样芽胞杆菌在营养肉汤中的Boltzmann牛长模犁,3条牛长曲线相关系数鼯均大于0．97;检测了不同培养时间蜡样芽胞杆菌的产芽胞情况,结果表明1号菌株和14号菌株较早产芽胞,培养相同时间,产芽胞数：1号菌株,4号菌株〉标准菌株;采用牛沣杯法和平板计数法研究了大蒜精油对蜡样芽胞杆菌的抑制效果,结果表明浓度为lO。的人蒜精油对3种蜡样芽胞杆菌都有很好的抑制效果,3种菌株中l号菌株最难抑制。     

Optimisation of Bacillus cereus biofilm removal in the dairy industry using an in vitro model of cleaning‐in‐place incorporating serine protease

This article addresses a major hygienic issue in the dairy industry, namely biofilm removal. Response surface methodology was deployed to optimise Bacillus cereus biofilm removal conditions using serine protease. The currently practiced alkali cleaning‐in‐place (CIP) method was also optimised, and while the optimised protease CIP resulted in complete removal of biofilm cells, the reference CIP (with alkali) resulted in a reduction of 4.08 log biofilm cells/cm² and the optimised alkali CIP resulted in a reduction of ～4.92 log biofilm cells/cm². Furthermore, the amount of the biofilm matrix removed in the optimised protease CIP was significantly higher than that of alkali CIP. It was concluded that the optimised protease CIP has better applicability.     

Bacillus subtilis HJ18‐4 from Traditional Fermented Soybean Food Inhibits Bacillus cereus Growth and Toxin‐Related Genes

Bacillus subtilis HJ18‐4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad‐spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18‐4. Expression of B. cereus toxin–related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18‐4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18‐4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18‐4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.     

一株产抑菌物质的蜡样芽胞杆菌的鉴定及其抑菌物质性质

从酱渣中分离到一株产抑菌物质的新菌株,暂命名为菌株B。研究表明,该菌株经碱中和后的无细胞发酵液对革兰氏阳性菌、革兰氏阴性菌和真菌均有抑制作用;其发酵上清液经硫酸铵盐析和透析后,仍然有抑菌活性;双缩脲和茚三酮实验表明,抗菌活性物质为肽类物质。生理生化实验、16S rDNA序列比对及系统发育分析表明,菌株B为蜡样芽胞杆菌,其生长和抑菌最适培养基为NB培养基,在pH 7.0的NB培养液中培养40h其抑菌活性达到最大。     

蜡样芽孢杆菌胶原蛋白酶基因的异源表达与活性分析

研究采用降解骨胶原蛋白的蜡样芽孢杆菌MBL13-U作为原菌种,并对蜡样芽孢杆菌MBL13-U进行全基因组测序,克隆获得胶原蛋白酶(ColM13)基因。将目的基因与大肠杆菌的表达载体pET30a进行连接,转入大肠杆菌宿主菌株BL21,获得降解骨胶原蛋白的工程菌pET30a-ColM13/BL21。通过SDS-PAGE测定异源表达的重组蛋白酶ColM13的分子质量,并测定不同诱导时间和诱导浓度对ColM13酶活性的影响,最终确定重组蛋白酶ColM13的酶活最适条件:6‰异丙基硫代半乳糖苷(IPTG,100 mmol/L),37℃诱导6 h,优化后酶活力最高为64.99 U/mL,较优化前提高4.24倍。通过水解圈试验、紫外光谱和扫描电子显微镜等方式验证,表明工程菌构建成功,这为我国畜禽骨骼资源的深度开发和综合利用提供了新的研究思路。     

成都市市售食品中蜡样芽胞杆菌污染状况与毒力基因分析

目的研究成都市市售食品中蜡样芽胞杆菌毒力基因携带及对抗生素的耐药情况。方法 2014—2016年,在成都市农贸市场和路边摊点共采集食品样品330份,按照GB 4789.14—2014《食品安全国家标准食品微生物学检验蜡样芽胞杆菌检验》分离疑似菌株,应用管家基因和16S rDNA测序鉴定蜡样芽胞杆菌,并针对分离菌株携带的毒力基因及对抗生素的耐药性进行检测。结果 2014—2016年,蜡样芽胞杆菌总检出率为17.6%(58/330),不同类型食品蜡样芽胞杆菌检出率差异有统计学意义(χ~2=29.683,P0.01),不同年份的食品样品蜡样芽胞杆菌的分离率差异无统计学意义(χ~2=5.835,P0.05)。米面制品、即食凉拌类和腌卤制品是蜡样芽胞杆菌的主要污染食品。腹泻型毒素基因(hbl,nhe,bceT,cytK,entFM)的检出率远高于呕吐型毒素基因(ces和cer)。分离菌株对四环素、红霉素、克林霉素的耐药率分别为29.3%(17/58)、24.1%(14/58)和22.4%(13/58)。结论成都市市售食品中蜡样芽胞杆菌分离菌株携带毒力基因类型多样,对四环素、红霉素、克林霉素的耐药率较高,对食品安全具有潜在威胁。     

青藏高原狭果茶藨子对蜡样芽孢杆菌的抑菌活性及作用机理

以青藏高原狭果茶藨子为原料,探究其对蜡样芽孢杆菌的抑菌作用及作用机制,利用纸片扩散法和二倍稀释法评价抑菌活性,通过测定膜蛋白荧光光谱、碱性磷酸酶、Na+K+-ATP酶、呼吸链脱氢酶活性等探究抑菌机制.结果表明,狭果茶藨子提取物对蜡样芽孢杆菌有明显的抑制作用,最低抑菌浓度和最低杀菌浓度分别为3.13和6.25 mg/mL...     

非致病性蜡样芽孢杆菌的益生功能与应用

蜡样芽孢杆菌广泛分布于环境当中。传统的致病性蜡样芽孢杆菌由于携带毒力基因,具有编码肠毒素和腹泻毒素的能力,被人类误食后会引起食物中毒甚至导致严重的疾病,因而引起人们的广泛关注。近年来,非致病性蜡样芽孢杆菌所具有的功效逐渐被人们所发现,并且应用于动物饲料的添加剂中;然而关于非致病性蜡样芽孢杆菌的益生功能却鲜有报道,该研究综述了非致病性蜡样芽孢杆菌的益生功能及其在食品领域的应用,主要包括抑菌功能、抗炎作用以及促生长作用等,并且对未来的研究发展方向做出了展望。     

酱腌菜中蜡样芽孢杆菌的检测与分析

对来自超市的50个酱腌菜样品进行了蜡样芽孢杆菌的检测与分析。研究结果表明,各样品中都有蜡样芽胞杆菌检出,数量为2×102～2×104 CFU/g,其中糖醋渍菜中蜡样芽孢杆菌的检出率较高。本调查分析对我国酱腌菜中蜡样芽孢杆菌污染的溯源奠定基础。建议完善酱腌菜中蜡样芽孢杆菌的限量标准。     

蜡样芽孢杆菌产磷脂酶D发酵条件优化

通过单因素和正交试验,对蜡样芽孢杆菌（Bacillus cereus）产磷脂酶D发酵条件进行了优化。结果表明,培养基最佳组成为卵黄10 g/L、蛋白胨15 g/L、牛肉粉2 g/L、MgSO4·7H2O 1 g/L、氯化钠3 g/L;最佳发酵条件为：培养基初始pH8.0,接种量1.0%,于36 ℃,200 r/min培养8 h。在最适发酵条件下磷脂酶D酶活达4.21 U/mL。     

蜡样芽孢杆菌高产新型中性蛋白酶发酵条件的优化

目的:优化蜡样芽孢杆菌发酵生产新型中性蛋白酶的培养条件。方法:采用Plackett-Burman试验设计,筛选影响蜡样芽孢杆菌高产新型中性蛋白酶酶活力的关键因子;通过Box-Behnken响应面试验设计获得新型中性蛋白酶发酵的最佳工艺条件。结果:筛选出影响蜡样芽孢杆菌高产新型中性蛋白酶的主要因子为葡萄糖、蛋白胨和pH值。获得的最优化发酵培养基组成和培养条件:葡萄糖35 g/L,蛋白40.38 g/L,氯化钠15 g/L,硫酸镁1.5 g/L,Tween-8010 g/L,装液量50 mL/250 mL,pH 7.15,菌龄12 h,发酵时间36 h。在上述条件下,蜡样芽孢杆菌发酵生产新型中性蛋白酶的酶活力高达696.51 U/mL,是未优化前的1.93倍。结论:研究结果为新型中性蛋白酶的产业化生产和应用提供了良好基础。     

Enterotoxigenic Profiling of Emetic Toxin‐ and Enterotoxin‐Producing Bacillus cereus,Isolated from Food,Environmental, and Clinical Samples by Multiplex PCR

Bacillus cereus comprises the largest group of endospore‐forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin‐ (8 patterns) and enterotoxin‐producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.     

Alkaline protease from Bacillus cereus VITSN04: Potential application as a dehairing agent

The objective of this work is to use protease enzyme as an ecofriendly alternative to chemicals in dehairing. An alkaline protease producing bacterium was isolated from protein-rich soil sample. The bacterium was identified as Bacillus cereus VITSN04 by 16S rRNA gene sequencing method. Growth characteristics and protease activity were studied in yeast, malt, beef, nutrient broth and soybean casein digest media and the enzyme secretion was found to correspond with growth. Maximum protease production was obtained in soybean casein digest medium at 16h with the activity of 200.1±0.68U/ml and a correlation coefficient of 0.965 between growth and enzyme production. The crude enzyme was found to have maximum activity at 30°C and pH 8.0. The protease was purified by ammonium sulphate precipitation, Sephadex G-50 and G-100 gel filtration chromatography. The purified protease was homogeneous on non-denaturing PAGE and its molecular weight was estimated to be 32kDa. The purified protease was of the serine type as it was inhibited by phenylmethylsulphonyl fluoride. The crude enzyme preparation was found to be effective in dehairing goat skins in leather processing.     

恒温隔绝式PCR快速检测蜡样芽孢杆菌方法的建立及应用

该研究以编码旋转酶B亚基的gyrB基因为靶基因,利用恒温热隔绝式PCR（Insulatedisothermal PCR,IiPCR）,建立一种快速检测蜡样芽孢杆菌的方法。同时,将建立的方法与聚合酶链式反应（Polymerase Chain Reaction,PCR）和实时荧光定量PCR（SYBER Green Ⅰ荧光染料法和TaqMan探针法）检测方法相比较,并应用于不同类型零售食品中的蜡样芽孢杆菌检测。结果表明建立的检测方法能在40 min内迅速判定出结果（+、-、？）;与大肠杆菌、金黄色葡萄球菌、单增李斯特菌、肠炎沙门氏菌等均无交叉反应,显示出良好的特异性;方法的最低检出限为1.5×102 CFU/mL,优于普通PCR,与实时荧光定量PCR检出限相当;对42份零售食品进行检测,共发现45.23%的样本被蜡样芽胞杆菌污染（19/42）。这一结果与常规PCR检测结果一致,但检测时间至少节省了4 h以上。该研究为蜡样芽孢杆菌提供了一种快速、便捷、特异性强、灵敏度高、适用于现场实时检测的检测方法。     

两株枯草芽孢杆菌蛋白酶的提取及产酱香研究

对两株产酱香的枯草芽孢杆菌E20和B1⑥分泌的胞外蛋白酶进行了提取和产酱香研究.其合适提取溶液为硫酸铵溶液,当提取浓度为20%,饱和浓度为70%时获得粗酶.Sephadex G100层析分别获得2个分子量不同的蛋白峰,并且都具有酶活性;提取的酶在固体大豆上发酵,均可产酱香.     

抗菌肽cp1对蜡样芽孢杆菌的抑菌作用及在巴氏杀菌乳中的应用

目的 探究抗菌肽cp1对蜡样芽孢杆菌的抑菌作用,并进一步分析抗菌肽cp1在巴氏杀菌乳中的应用。方法 以蜡样芽胞杆菌为研究对象,通过测定抑菌圈直径(diameter of inhibition zone, DIZ)、最小抑菌浓度(minimal inhibitory concentration, MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)评价抗菌肽cp1对蜡样芽胞杆菌的抑菌效果。通过分析生长曲线、细胞内三磷酸腺苷(adenosine triphosphate, ATP)、膜电位、内容物渗出和细胞形态的变化,来揭示可能的作用机制。在此基础上,并探究抗菌肽cp1在巴氏杀菌乳贮藏中的应用。结果 当抗菌肽cp1质量浓度为10μg/mL时,其对蜡样芽胞杆菌的DIZ为(16.19±1.29) mm;抗菌肽cp1对蜡样芽孢杆菌的MIC、MBC分别为4μg/mL和8μg/mL;当抗菌肽cp1质量浓度为2 MIC时,蜡样芽胞杆菌几乎不生长;抗菌肽cp1导致细胞内ATP含量降低,细胞膜超极化,细胞液(包括核酸和蛋白质)漏出,细胞形态破坏;在巴氏杀...     

Model for Combined Effects of Temperature, pH and Water Activity on Thermal Inactivation of Bacillus cereus Spores

Initially, the effect of water activity (a_w) on heat resistance of Bacillus cereus spores (decimal reduction time) was investigated. A linear relationship was found between log D and 1-a_w. The combined effects of temperature (85-105°C), pH (4.5-6.5) and water activity (0.80-1) were then studied. A four parameter model was fitted to the data. This model appeared to be parsimonious with each parameter having a biological significance. Interactions between factors were observed but they accounted for <2.4% of the total variation and they were not taken into account by the model.     

大蒜提取物对蜡样芽孢杆菌抑制效果的研究

该文采用抑菌圈法研究了大蒜的水提取物、乙醇提取物和酸性水溶液的提取物对蜡样芽孢杆菌的抑制作用.结果表明大蒜的乙醇提取物的抑菌效果好过水提取物,酸性的水溶液提取物效果最好,可以较好地控制蜡样芽孢杆菌的生长,有较好的应用前景.     

一株降解呕吐毒素蜡样芽孢杆菌的筛选与鉴定

目的:分离筛选能够降解呕吐毒素(deoxynivalenol,DON)的芽孢杆菌,以用于该毒素的生物降解。方法:采集霉变秸秆、土壤和粪便样品,先将样品加热80℃后,取上清液接种到以DON为唯一碳源的分离培养基富集DON降解菌。以LB培养基分离纯化富集菌,然后对分离到的菌株进行DON毒素降解能力检测。对降解能力最强的菌株进行形态学观察、生理生化实验和16S r DNA序列鉴定。结果:从16株分离菌中筛选得到一株对DON降解能力最强的菌株B.JG05,降解率最高可达80.61%,且对含DON饲料的降解率为82.68%。该菌株呈短杆状,能形成芽孢;生理生化特性符合蜡样芽孢杆菌的基本特征;16S r DNA序列进化树分析表明该菌株与蜡样芽孢杆菌的亲缘关系最近。结论:筛选获得了一株高效降解DON的蜡样芽孢杆菌B.JG05,为饲料和食品中DON毒素的生物降解提供了可能。     

枯草芽孢杆菌HS18碱性蛋白酶分离纯化及其性质研究

从合浦珠母贝肠道中分离鉴定到酶菌株枯草芽孢杆菌（Bacillus subtilis）HS18,将枯草芽孢杆菌HS18的发酵液,经过硫酸铵盐析、DEAE-Sephadex A-50柱层析、Sephadex G-100柱层析3步纯化后得到了单一的酶蛋白,回收率为11.4%;经SDS-聚丙烯酰胺凝胶电泳测得酶蛋白分子量约为31ku;该蛋白酶最适反应温度为50℃,最适pH10.0;K+及Na+对酶有明显激活作用;丝氨酸蛋白酶特异性抑制剂（PMSF）能强烈抑制酶活性。表明所纯化到的蛋白酶为丝氨酸碱性蛋白酶。