四种不同接触表面蜡样芽孢杆菌菌膜形成的影响

为了解食源性致病菌蜡样芽孢杆菌在食品加工环境中菌膜形成能力,以玻璃、不锈钢、聚氯乙烯、聚丙烯为接触面,采用超声波平板菌落计数法测定不同环境因素(温度、pH、氯化钠、葡萄糖、苯甲酸钠及山梨酸钾)、不同材料表面蜡样芽孢杆菌(B.cereus)菌膜形成的变化趋势。结果表明:四种材质表面形成B.cereus菌膜能力的大小顺序为:玻璃 > 不锈钢 > 聚氯乙烯 > 聚丙烯。其中,30 ℃,pH7.0时菌膜形成量最大,添加低浓度葡萄糖(4.0%)或氯化钠(0.5%)对B.cereus菌膜形成有显著促进作用(p<0.05),添加0.15%苯甲酸钠、山梨酸钾的菌膜形成量显著高于添加0.10%的菌膜形成量(p<0.05)。本研究为蜡样芽孢杆菌风险评估提供基础数据,为食品工业蜡样芽孢杆菌菌膜的预防和控制奠定基础,为改进蜡样芽孢杆菌的清洗控制措施提供参考。     

生鲜食品中蜡样芽孢杆菌毒力基因及耐药性研究

目的:研究生鲜食品中蜡样芽孢杆菌的毒力基因及耐药性。方法:在成都市周边农贸市场和路边小摊采集各种生鲜食品共100份,采用MYP选择性培养基初步分离蜡样芽孢杆菌菌株,采用16S rRNA序列比对分析鉴定得到蜡样芽孢杆菌分离菌株,采用PCR检测分离鉴定菌株中蜡样芽孢杆菌13个毒力基因的携带情况,进一步采用纸片扩散法检测菌株对18种抗生素的耐药性。结果:从100份生鲜食品样本中,检出蜡样芽孢杆菌24株,检出率为24%,其中,路边小摊的检出率(70%)高于农贸市场(18.9%)。呕吐型基因ces和cer的检出率较低,仅在1株中发现;腹泻型基因nheC有24株检出;cytK有13株检出;bceT有12株检出;hblA有11株检出;nheB有10株检出;hblC、hblD、nheA各有9株检出;hly-Ⅱ有7株检出;hblB、entFM检出率为0;蜡样芽孢杆菌的4个看家基因rpoB、gyrB、groEL、vrrA在24株分离菌株中检出率为100%。同时发现,生鲜食品来源的蜡样芽孢杆菌分离菌株对杆菌肽B、磺胺甲亚唑、苯唑西林、青霉素表现出较高耐药性,耐药率大于96%;而对阿米卡星、氯霉素、庆大霉素、亚胺培南耐药率小于7%。结论:本研究通过分离鉴定生鲜食品源蜡样芽孢杆菌,研究其毒力基因携带及耐药性,为进一步评价生鲜食品中蜡样芽孢杆菌的安全风险提供参考数据。     

我国市售婴儿配方乳粉中蜡样芽胞杆菌污染及其毒力基因调查

目的了解国内市售婴儿配方乳粉中蜡样芽胞杆菌污染及毒力基因分布情况。方法采用MPN法定量检测婴儿配方乳粉中的蜡样芽胞杆菌,在对分离菌株正确鉴定的基础上,开展腹泻型和呕吐型毒素产生相关毒力基因的分布研究。结果 135份婴儿配方乳粉中有57份检出蜡样芽胞杆菌,检出率为42.22%。平均污染水平为7.14 MPN/g。国产产品蜡样芽胞杆菌污染较进口产品重,网售产品较超市销售产品重,差异有统计学意义(P0.05)。共发现24种蜡样芽胞杆菌毒力基因携带模式,其中nhe基因携带率最高,达92.98%(53/57),其次为ent FM基因(71.93%),70.18%(40/57)的菌株同时携带nhe和ent FM基因。亚型分型结果显示nhe A、nhe B和nhe C基因的携带率分别为88.72%、88.72%和49.12%。溶血素BL基因携带率分别为hbl A 24.56%、hbl C 22.81%和hbl D 17.54%,cyt K基因携带率为22.81%。有8株菌既携带nhe的3个基因又携带hbl的3个基因。结论我国市售婴儿配方乳粉蜡样芽胞杆菌污染较重,分离到的蜡样芽胞杆菌菌株普遍携带毒力基因,建议加强对婴儿配方乳粉中蜡样芽胞杆菌污染的监管,并开展膳食暴露该菌对婴儿健康影响的风险评估,为制定婴儿配方乳粉中蜡样芽胞杆菌的限量标准提供依据。     

婴幼儿配方乳粉及婴幼儿米粉中的蜡样芽胞杆菌流行情况、耐药性及分子特征研究

目的 了解流通环节市售婴幼儿配方乳粉和婴幼儿米粉中的蜡样芽胞杆菌流行情况、抗生素耐药性和毒力基因等分子特征。方法 采用GB 4789.14—2014《食品卫生微生物学检验 蜡样芽胞杆菌检验》对样品中的蜡样芽胞杆菌进行检测与生化鉴定。进一步通过抗生素敏感实验和全基因组测序对11株代表性分离株进行研究,获得耐药表型及耐药、毒力基因等分子特征信息。结果 婴幼儿配方乳粉和婴幼儿米粉中蜡样芽胞杆菌的检出率分别为11.97%（56/468）和20.74%（28/135）。11株代表性蜡样芽胞杆菌均为多重耐药菌株,全基因组测序结果发现11株分离株共携带8个耐药基因和14个毒力基因,其中主要携带磷霉素、β-内酰胺类抗生素抗性基因,以及肠毒素基因（hbl和nhe）,另发现1株携带ces基因簇蜡样芽胞杆菌菌株。11株分离株具有遗传多样性,11株分离株分属10个ST型。结论 婴幼儿配方乳粉和婴幼儿米粉中的蜡样芽胞杆菌检出率较低,但代表菌株的耐药谱和遗传特征具有多样性,应进一步加强监测及采取有效措施进行控制,以保障相关食品的安全性。     

蜡样芽孢杆菌LJ01中亚硝酸盐还原酶的基因克隆和生物信息学分析

为研究蜡样芽孢杆菌LJ01(Bacillus cereus LJ01)中亚硝酸盐还原酶(NiR)的酶学性质,获得高表达量的基因工程菌,本文对B.cereus LJ01的NiR序列进行了生物信息学分析,并将NiR基因克隆到表达载体pET-28a(+)和pET-32a(+)中,构建了基因工程菌pET-28a(+)-nir-BL21和pET-32a(+)-nir-BL21,随后探究了不同诱导条件对重组NiR表达量的影响。结果表明NiR编码蛋白的理论分子量约为60 kDa,理论pI为5.47,二级结构主要为α-螺旋和无规则卷曲,是不具有跨膜结构的亲水性蛋白。随着诱导温度的升高,重组NiR的表达量逐渐减少,诱导温度为16℃时重组NiR表达量最高。在同一诱导温度下,NiR在pET-28a(+)-nir-BL21中的表达量明显高于pET-32a(+)-nir-BL21,因此选用pET-28a(+)-nir-BL21作为基因工程菌。从B.cereus LJ01中克隆了NiR基因,构建了基因工程菌pET-28a(+)-nir-BL21,为该重组NiR的理化性质研究和食源芽孢杆菌中NiR的异源表达奠定了基础。     

溶菌酶复合涂膜贮藏鲜枣工艺研究

本文以溶菌酶（Lysozyme,LYZ）为原料,在pH2.0和90 ℃的条件下,对溶菌酶进行酸热诱导处理使其内部结构产生淀粉样纤维化转变。应用纳米粒度仪、原子力显微镜（AFM）、圆二色谱仪（CD）、荧光分光光度计表征了溶菌酶淀粉样纤维的结构特征。通过流延法制备了天然溶菌酶薄膜和溶菌酶淀粉样纤维薄膜,考察抗拉强度（TS）、断裂伸长率（E）、水蒸气透过率（WVP）、水溶性（WS）四个性能指标。此外,通过将香兰素（Vanillin）添加至溶菌酶溶液中并制备成薄膜,测定其对大肠杆菌（E. coli）和枯草芽孢杆菌（B. subtilis）的抑制效果。结果表明,淀粉样纤维化后溶菌酶的平均粒径明显增大,原子力显微镜结果证实溶菌酶从球状结构转变为纤维结构,圆二色谱结果表明蛋白质二级结构由α-螺旋转变为β-折叠结构。淀粉样纤维化后制成的薄膜表面结构更加均匀光滑,抗拉强度增强,水蒸气透过率和水溶性有显著降低（P<0.05）。此外,溶菌酶淀粉样纤维与香兰素复合薄膜对大肠杆菌和枯草芽孢杆菌的抑制性能有极大提升,抑制区域分别扩大到453.75 mm² 和293.78 mm²。该研究制备了一种可降解、环境友好的生物包装材料,为食品包装领域的进一步研究提供了新思路。     

基于全基因组测序的蜡样芽孢杆菌食品分离株分子特征及耐药性研究

目的 研究上海市食品中分离的蜡样芽孢杆菌(Bacillus cereus)的基因组特征及其耐药性。方法 本研究对上海市食品中分离的蜡样芽孢杆菌进行鉴定、药物敏感性实验和全基因组测序,利用BioNumerics软件对测序数据进行拼接组装,利用拼接数据进行多位点序列分型(MLST)、毒力基因和耐药基因分析,并与PubMLST数据库中的ST型进行比较;对耐药基因和耐药表型进行比较分析。结果 本研究中37株蜡样芽孢杆菌均可分型,其中7株为ST新型。37株蜡样芽孢杆菌分为34个ST型,其中3株菌为ST26型,其余ST型各占1株,呈高度多样性。37株蜡样芽孢杆菌均携带nheA、nheB、nheC基因,hblACD基因簇的携带率为37.8%,而完整ces呕吐基因簇的携带率只有16.2%,其中2株为分离自即食食品的ST26型。药敏实验显示37株蜡样芽孢杆菌对氨苄西林、青霉素和苯唑西林耐药性较高,与其携带耐药基因种类并不完全一致。结论 上海市食品中分离的蜡样芽孢杆菌菌株分子分型呈现高度多样性,应加强食品中蜡样芽孢杆菌分子生物学监测,为预防控制由其引起的食源性疾病提供科学依据。     

婴幼儿配方羊乳粉生产环节蜡样芽孢杆菌分离株多位点序列分型分析

为探讨我国婴幼儿配方羊乳粉生产环节阳性分离株的遗传多样性,并将其基因序列与已报道的乳源分离株的基因序列进行比对,确立其特征序列,为婴幼儿配方食品生产体系溯源提供依据,为探究其致病机理和有效防控提供参考依据。实验选取glpF、gmk、ilvD、pta、pur、pycA、tpi 7 个管家基因构建多位点序列分析分型方案,鉴定经随机扩增多态性DNA分型得到的42 株Bacillus cereus的序列类型。结果表明：42 株B. cereus分为7 个序列类型（sequence type,ST）,分别为ST-770（4.8%,2/42）、ST-1000（71.4%,30/42）、ST-1084（9.5%,4/42）、ST-1348（2.4%,1/42）、ST-1349（2.4%,1/42）、ST-1350（2.4%,1/42）和ST-1351（7.1%,3/42）;所有分离株被识别为205、142、23三个不同克隆谱系,2 个单态群（ST-770和ST-1351）和2 个独株（ST-1348和ST-1349）;发现4 个新的ST型和4 个新的等位基因,已上传至国际数据库得到新的序列号和等位基因号,分别为ST-1348、ST-1349、ST-1350、ST-1351和glp-253、glp-254、ilv-277、pyc-199。     

Typing and evaluating heat resistance of Bacillus cereus sensu stricto isolated from the processing environment of powdered infant formula

Bacillus cereus sensu lato is one of the most harmful bacterial groups affecting the quality and safety of powdered infant formula (PIF). In this study, samples were collected from the raw materials and processing environments of PIF. A total of 84 isolates were identified as Bacillus cereus sensu stricto (B. cereus s. s.) by 16S rRNA analysis, molecular typing technology, and physiological and biochemical tests. The 84 B. cereus s. s. strains were assigned to panC group II, group III, and group IV. Then, the 7 housekeeping genes glpF, gmk, ilvD, pta, pur, pycA, and tpi were selected for multilocus sequence typing. Results showed that the 84 isolates were clustered into 24 sequence types (ST), and 14 novel ST were detected. Among the 24 ST, ST999 (19/84, 22.62%) and ST1343 (13/84, 15.48%) predominated. The correlation between processing areas and ST showed that the processing environments of the production and packing areas were the most susceptible to contamination by B. cereus s. s. Spores of these ST showed different heat resistance phenotypes evaluated by the analysis of D_T (time in minutes of spore decimal reduction at each temperature) and Z values (temperature increase required to reduce the D_T value to one-tenth of the original). Spores from group III according to panC gene analysis were the most heat resistant. These findings will help us to better understand B. cereus s. s. contamination and control in PIF processing environments.     

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible.     

原料乳中蜡样芽孢杆菌部分致病基因的鉴定及毒力评价

研究原料乳中蜡样芽孢杆菌的潜在威胁,对我国10 个省市原料乳中蜡样芽孢杆菌的污染状况进行调查,并利用PCR 扩增技术及血平板检测的方法分析乳源性蜡样芽孢杆菌中的毒性分布。结果表明,原料乳中蜡样芽孢杆菌检出率为33%,检出水平为1 × 103～1 × 106 CFU/ml,检出的蜡样芽孢杆菌中25 株(75.8%)能产生溶血素BL,24 株(72.7%)含有bceT 基因,至少产生一种毒素的菌株有31 株占检出菌总数的93.9%。研究结果证实我国原料乳中的蜡样芽孢杆菌污染状况不容乐观,严格监控原料乳中的蜡样芽孢杆菌污染,对生产出优质乳制品具有重要意义。     

Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk

The growth and death or survival of Bacillus cereus in sterile skimmed milk fermented with 18 different lactic acid bacteria (LAB) were investigated. B. cereus alone in milk reached about 10(7)-10(8) colony-forming units (cfu)/ml. When B. cereus was cultivated together with different Lactobacillus or Lactococcus cultures at 30 or 37 degrees C, the B. cereus counts after 72 h of fermentation ranged between < 10 cfu/ml and about 10(6) cfu/ml. The inhibition patterns for the different Lactobacillus and Lactococcus cultures varied. All the Lactococcus cultures (with one exception) reduced pH to 5.3 or lower in 7 h. After 24 h, B. cereus was not detected in any of the fast Lactococcus-fermented milk samples. After 48 h, B. cereus was not detected for 4 of the 12 Lactobacillus cultures. These cultures reduced pH to below 5.0 in 24 h. The other Lactobacillus cultures also inhibited B. cereus, but the counts of B. cereus were still 10(4)-10(6) cfu/ml after 72 h. They also reduced pH at a slower rate. Survival of B. cereus was to a variable extent linked with formation of endospores. Proteinase K did not affect the antimicrobial activity observed. Acid production with decreasing pH, particularly the initial rate of pH decrease, appears to be most important for control of B. cereus with LAB.     

The members of the Bacillus cereus group are commonly present contaminants of fresh and heat-treated milk

To determine the level of milk contamination by Bacillus cereus sensu lato, 44 samples were collected from a dairy farm and two independent dairies in northeastern Poland. A total of 680 B. cereus sensu lato isolates were recovered. Based on spore counts, their highest level in milk was found during the spring and summer months. Although significant variations in chromosomal DNA polymorphisms among B. cereus sensu lato isolates were noted based on repetitive element sequence polymorphism (rep-PCR) and pulse-field gel electrophoresis (PFGE), indistinguishable B. cereus isolates were observed in all sampling points and locations. Both B. cereus sensu stricto/Bacillus weihenstephanensis and Bacillus thuringiensis cultured from milk harbored nheA, hblA, and cytK in, respectively, 80%, 55%, and 60% of the isolates. With respect to Bacillus mycoides/Bacillus pseudomycoides, 30% and 70% of theses isolates harbored, respectively, nheA and hblA. The presence of cytK was not detected in any isolate. Our data show the occurrence of potentially toxic B. cereus s.l. in both raw and heat-treated milk, thus emphasizing the requirement for precautions that prevent spore germination and vegetative proliferation by keeping the milk at low temperatures during all steps of production and dispensation to the consumers.     

蜡样芽胞杆菌生长/非生长界面模型的建立和评价

蜡样芽胞杆菌是软烤虾仁产品的主要变质菌,它是一种条件致病菌,通过产生腹泻毒素和呕吐毒素导致食物中毒。该研究旨在建立一种概率模型来预测出蜡样芽胞杆菌的生长/非生长情况或者生长概率。用lo-gistic回归模型建立不同温度、水分活度和pH环境因子作用下蜡样芽胞杆菌的生长/非生长界面模型。实验结果表明蜡样芽胞杆菌在脑心浸液肉汤培养基中生长的最低温度为9.99℃,最低水分活度为0.931,最小pH值为4.5。在此基础上建立的蜡样芽胞杆菌生长/非生长界面模型的χ2=49.73,P<0.000 1。用logistic回归模型建立的生长/非生长模型拟合效果达到极显著水平。模型的预测值同时很好地量化了环境因子对蜡样芽胞杆菌的协同作用,为软烤虾仁产品中蜡样芽胞杆菌的生长/非生长界面模型的建立提供了参考。