不同温度培养诱导金黄色葡萄球菌对超高压失活的抗性及其建模

探讨不同培养温度处理诱导金黄色葡萄球菌(ATCC 6538)对超高压的抗性,并建立不同培养温度下金黄色葡萄球菌的超高压抗性模型。金黄色葡萄球菌经不同温度培养,在100~500 MPa的超高压条件下,选用线性、Weibull和Gompertz三种模型来拟合超高压抗性曲线,以决定系数(R2),均方误差(RMSE),精确因子(Af)和偏差因子(Bf)作为模型拟合度优劣的评判指标。实验结果表明,在100~500 MPa压力的作用下,线性模型的拟合效果不佳,R2最小值达到0.8870,Weibull和Gompertz模型对超高压的抗性具有较好的拟合性(R2≥0.9467),且Weibull模型的拟合效果最好,R2最大值达到0.9956,RMSE最小值为0.0312。因此,Weibull模型可以很好地拟合金黄色葡萄球菌以不同的培养温度胁迫后在超高压作用下的抗性曲线,随着培养温度的升高,金黄色葡萄球菌的超高压抗性呈增加趋势。      

应用基因芯片技术鉴定食源性金黄色葡萄球菌的研究

目的:建立快速鉴定食源性金黄色葡萄球菌的基因芯片技术。方法:采用PCR方法扩增金黄色葡萄球菌16SrRNA基因的DNA片段,序列进行BLAST比较并通过软件设计其特异性探针,采用基因芯片杂交技术鉴定食源性金黄色葡萄球菌样品。结果:对所有样品进行基因芯片杂交技术处理并扫描观察,金黄色葡萄球菌的杂交结果呈阳性,其检测结果与传统方法鉴定结果一致。结论:应用基因芯片鉴定技术可快速、准确地鉴定食源性金黄色葡萄球菌,值得推广应用。     

碳纳米生物传感检测金黄色葡萄球菌

目的建立一种基于碳纳米材料的适配体传感器快速检测金黄色葡萄球菌的方法。方法利用核酸适配体与其靶标之间的特异性结合能力以及碳纳米管的荧光猝灭特性,构建一种新型快速的金黄色葡萄球菌检测方法。结果该方法特异性良好,与非目标菌株无交叉反应。同时,该方法对金黄色葡萄球菌的检出限为10~5 CFU/mL,线性范围为10~5～10~9 CFU/mL,而且在此浓度范围之间呈现良好的线性关系,线性方程为Y=53.22X-39.99,线性相关系数r~2=0.992。结论该方法具有检测速度快、操作简便、检测成本低等特点,为金黄色葡萄球菌的快速检测提供了一种有效手段。     

Fate of Staphylococcus aureus in cheese treated by ultrahigh pressure homogenization and high hydrostatic pressure

We evaluated the influence of ultrahigh pressure homogenization (UHPH) treatment applied to milk containing Staphylococcus aureus CECT 976 before cheese making, and the benefit of applying a further high hydrostatic pressure (HHP) treatment to cheese. The evolution of Staph. aureus counts during 30 d of storage at 8°C and the formation of staphylococcal enterotoxins were also assessed. Milk containing approximately 7.3 log₁₀ cfu/mL of Staph. aureus was pressurized using a 2-valve UHPH machine, applying 330 and 30 MPa at the primary and the secondary homogenizing valves, respectively. Milk inlet temperatures (T_in) of 6 and 20°C were assayed. Milk was used to elaborate soft-curd cheeses (UHPH cheese), some of which were additionally submitted to 10-min HHP treatments of 400 MPa at 20°C (UHPH+HHP cheese). Counts of Staph. aureus were measured on d 1 (24 h after manufacture or immediately after HHP treatment) and after 2, 15, and 30 d of ripening at 8°C. Counts of control cheeses not pressure-treated were approximately 8.5 log₁₀ cfu/g showing no significant decreases during storage. In cheeses made from UHPH treated milk at T_in of 6°C, counts of Staph. aureus were 5.0 ± 0.3 log₁₀ cfu/g at d 1; they decreased significantly to 2.8 ± 0.2 log₁₀ cfu/g on d 15, and were below the detection limit (1 log₁₀ cfu/g) after 30 d of storage. The use of an additional HHP treatment had a synergistic effect, increasing reductions up to 7.0 ± 0.3 log₁₀ cfu/g from d 1. However, for both UHPH and UHPH+HHP cheeses in the 6°C T_in samples, viable Staph. aureus cells were still recovered. For samples of the 20°C T_in group, complete inactivation of Staph. aureus was reached after 15 d of storage for both UHPH and UHPH+HHP cheese. Staphylococcal enterotoxins were found in controls but not in UHPH or UHPH+HHP treated samples. This study shows a new approach for significantly improving cheese safety by means of using UHPH or its combination with HHP.     

A proteomic approach to cold acclimation of Staphylococcus aureus CECT 976 grown at room and human body temperatures

Staphylococcus aureus is an important pathogenic microorganism that has been associated with serious infection problems in different fields, from food to clinic. In the present study, we have taken into account that the main reservoirs of this microorganism are the human body and some parts of food processing plants, which have normal temperatures of around 37 and 25 °C, respectively. It can be expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food matrices. Since temperature abuse for foods occurs at approximately 12 °C, it is expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food. For this reason, we have performed a proteomic comparison between exponential- and stationary-phase cultures of S. aureus CECT 976 acclimated to 12 °C after growing at 25 °C or 37 °C. The analysis led to the identification of two different protein patterns associated with cold acclimation, denominated pattern A and pattern B. The first was characteristic of cultures at stationary phase of growth, grown at 25 °C and acclimated to 12 °C. The second appeared in the rest of experimental cases. Pattern A was distinguished by the presence of glycolytic proteins, whereas pattern B was differentiated by the presence of general stress and regulatory proteins. Pattern A was related through physiological experiments with a cross-resistance to acid pH, whereas pattern B conferred resistance to nisin. This prompted us to conclude that both molecular strategies could be valid, in vivo, for the process of acclimation of S. aureus to cold temperatures.     

金黄色葡萄球菌菌膜形成过程中过氧化氢酶变化的研究

选择食品工业中出现的典型细菌菌膜(金黄色葡萄球菌菌膜),探讨单一细菌菌膜形成中细菌自身产生的具有氧化还原活性的生物酶在菌膜形成与生长过程中的变化,并将其与菌膜的实时变化相对照,探索菌膜形成过程中的氧化还原活动的变化,以寻求食品工业中可有效地控制菌膜污染的方法。对金黄色葡萄球菌菌膜中过氧化氢酶量的变化进行实时监测,并对不同培养时间段的菌膜所得到的检测峰电流进行作图分析,结果显示:细菌中过氧化氢酶的量随着菌膜形成量的上升而上升,随着菌膜的自溶分解现象而减小。可为金黄色葡萄球菌菌膜中的氧化代谢活动研究提供理论依据与研究基础。     

酸性氧化电解水对金黄色葡萄球菌生物被膜清除效果的研究

研究酸性氧化电解水对金黄色葡萄球菌生物被膜的清除效果。通过观察放大5 000倍的金黄色葡萄球菌生物被膜的扫描电镜照片发现,经酸性氧化电解水处理后,细菌外部基质基本被破坏,细胞破裂严重,生物被膜态细菌量下降,清除效果明显。增加保存酸性氧化电解水的时间、提高保存温度以及存在有机干扰物时均会显著降低清除效果;闭口储存的电解水清除生物被膜的能力高于敞口保存。有效氯、pH值和氧化还原电位对清除效果具有协同作用:有效氯含量较低时,电解水清除能力影响明显,有效氯含量较高时,清除效果无明显影响;低pH值条件下,清除效果较好;氧化还原电位与清除效果有明显正相关关系。     

Production of enterotoxin A and thermonuclease by Staphylococcus aureus in legumes

Growth, enterotoxin A (SEA) and thermonuclease (TNase) production of S. aureus (Strains CP 7 and FRI 722) was determined in media produced from the following heat or irradiation sterilized legumes: peas, black beans, mung beans, adzuki beans and soybeans. Media containing the five legumes alone or in combination with Brain Heart Infusion Broth (BHI) were tested. With the exception of heat-sterilized black beans and adzuki beans, S. aureus growth was excellent in all media with cell counts after 48 h (25 °C) exceeding 10⁸ cfu/ml. In black beans and adzuki beans cell counts were 1–2 log-cycles lower. Enterotoxin A was produced in amounts of 33 to 72 ng/ml in BHI after 48 h. Almost no toxin was produced in the four different beans following heat or irradiation treatment; in peas the toxin concentration reached 14 to 15 ng/ml. In the medium prepared from irradiated soybeans and BHI the final toxin concentration was about the same as in BHI alone. In all the other media consisting of a combination of legumes with BHI toxin concentrations were there to four times higher than in BHI alone. Production of thermonuclease showed variation and did not always correlate with enterotoxin production.     

酶底物法快速测定食品中金黄色葡萄球菌

目的 建立酶底物法快速测定食品中金黄色葡萄球菌含量的分析方法。方法 参照GB 478910-2016将食品进行前处理后, 放入加有新型酶底物的金黄色葡萄球菌培养基中, 将培养基置入恒温箱或食源性致病菌快速检测仪中, 进行8 h培养。当食品中含有金黄色葡萄球菌, 会与新型酶底物反应过程中释放出绿色物质或者荧光基团。结果 在市场上购买50份样品使用新型培养基进行培养检测, 并与GB 478910-2016检测结果对比。所使用的新型培养基与国标方法得出的检测结果符合率为99.4%, 结果基本一致。结论 新型酶底物培养基与食源性致病菌同步快速检测仪配合使用结果准确, 并且检测时间相比传统方法也大幅度的缩短, 为食品卫生质量监管提供技术手段。     

酸奶制作中金黄色葡萄球菌污染的模拟实验研究

金黄色葡萄球菌是乳制品中检出率较高的食源性致病菌.该论文采用模拟实验的方式,探讨了在金黄色葡萄球菌污染的情况下,酸奶制作过程中乳酸菌和金黄色葡萄球菌消长的动态规律,并初步估计了被污染酸奶中金黄色葡萄球菌耐热肠毒素的危害水平.研究结果显示,当发酵温度偏低、奶粉添加量偏高和金葡菌污染程度较高时,金黄色葡萄球菌生长速率和最大菌数明显提高.参考有关文献进行估算,对于一个体重为61kg的成人来说,如果酸奶食用量少于1300mL,一般不会有酸奶金黄色葡萄球菌耐热肠毒素中毒的危险;但对于儿童来说则有一定风险.本研究对于酸奶制作工艺控制及酸奶的安全消费具有参考意义.     

Predicting heat inactivation of Staphylococcus aureus under nonisothermal treatments at different pH

The aim was to assess whether heat resistance data obtained from isothermal treatments allow the estimation of survivors of Staphylococcus aureus under nonisothermal conditions and to find a model that accurately predicts its heat inactivation at constantly rising heating rates (0.5-9 degrees C/min) in media of different pH (4.0-7.4). S. aureus showed a higher heat resistance under isothermal treatments at pH 4.0 than at pH 5.5-7.4. However, under nonisothermal treatments S. aureus increased its heat resistance at pH 5.5-7.4 and became more thermotolerant than at pH 4.0. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments did not adequately fit experimental values. Whereas the number of survivors was much higher than estimated at pH 5.5-7.4, that obtained at the slower heating rates at pH 4.0 was lower. An equation based on the Weibullian-like distribution (log10 S(t) = (t/delta)p) accurately described survival curves obtained under nonisothermal conditions. A nonlinear relationship was observed among the scale parameter (delta) and the heating rate which allowed the development of two equations capable of predicting the inactivation rate of S. aureus under nonisothermal treatments. This study might contribute to prevent public health risks in foods requiring long heating lag phases during their processing.     

应用基因芯片技术检测食品中金黄色葡萄球菌

采用基因芯片技术对食品中金黄色葡萄球菌进行检测,在检测靶基因扩增后用制备的基因芯片进行杂交反应。应用基因芯片对从食品中分离的金黄色葡萄球菌进行检测,得到金黄色葡萄球菌特异性的杂交图谱,从而达到对食品中金黄色葡萄球菌进行检测的目的。实验证明,制备的基因芯片能够准确、快速检测食品中金黄色葡萄球菌。     

应用干制培养基检测食品中金黄色葡萄球菌

目的建立金黄色葡萄球菌X干制培养基(Staphylococcus aureus X medium, XSA)检测食品中金黄色葡萄球菌的分析方法。方法依据GB 4789.10—2016《食品安全国家标准食品微生物学检验金黄色葡萄球菌检验》中前处理的方法制备样品,将样品添加到7.5%NaCl肉汤增菌后接种至干制培养基XSA上进行培养,通过鉴定实验进行定性检测和菌落平板计数进行定量检测,并用国标法同时进行定性及定量检测。结果在定性检测方面,该方法和国标方法假阴性率和假阳性率均为0%,但是检测时间由92 h缩短为48 h,在定量检测方面,该方法与国标法的对数偏差值的绝对值为0.3979<0.45,并且在6次检测同一个样品时的结果相对标准偏差较小,从而说明该方法和国标法的准确度相当,检测时长由78h缩短至24h,大大提高了检验效率。结论该方法操作简便快捷,结果稳定,适用于食品中金黄色葡萄球菌的检测。     

Taqman探针实时PCR检测金黄色葡萄球菌的研究

建立了Taqman探针实时PCR方法,针对乳中携带sea基因的金黄色葡萄球菌进行检测。研究所设计的引物具有良好的特异性,而且Taqman探针实时PCR方法检测sea基因的灵敏度高,最低检出限为69 fg,并且该方法可在8 h内完成人工污染乳中金黄色葡萄球菌的检测,最低检出限为83 cfu/mL。研究所建立的方法具有特异性强、灵敏性高及操作简便等优点,适宜于牛乳中金黄色葡萄球菌污染的调查及监测。     

脉冲强光对馒头表面金黄色葡萄球菌的杀菌效果

目的研究脉冲强光技术在馒头表面金黄色葡萄球菌杀菌中的效果及其机制。方法控制闪照距离为9~21 cm,单脉冲能量为100~500 J,闪照次数为0~20次,分析不同条件下金黄色葡萄球菌的失活规律,并将该优化条件应用于馒头表面金黄色葡萄球菌的杀菌试验,最后采用透射电子显微镜技术分析脉冲强光技术对金黄色葡萄球菌形态结构的影响,从而分析其杀菌机制。结果脉冲强光在闪照距离为9 cm,单脉冲能量为500 J的情况下,闪照16次能对金黄色葡萄球菌产生良好的杀菌效果。该脉冲强光处理技术能降低馒头表面的金黄色葡萄球菌1.72 log CFU/g。透射电镜观察结果说明了脉冲强光可破坏金黄色葡萄球菌的细胞膜和菌体胞质内容物,从而造成细胞的失活。结论脉冲强光可对金黄色葡萄球菌产生较强的杀菌效果,在馒头加工业中具有一定的应用前景。     

Inactivation of Staphylococcus aureus in milk using flow-through pulsed UV-light treatment system

ABSTRACT:  This study investigated the efficacy of pulsed UV-light for continuous-flow milk treatment for the inactivation of Staphylococcus aureus , a pathogenic microorganism frequently associated with milk safety concerns. Pulsed UV light is an emerging technology, which can be used for the inactivation of this pathogen in milk in a relatively short time. Pulsed UV light damages the DNA of the bacteria by forming thymine dimers that lead to bacterial death. The effect of sample distance from the quartz window of the UV-light source, number of passes, and flow rate was investigated. A response surface methodology was used for the design and analysis of experiments. Milk was treated at 5-, 8-, or 11-cm distance from a UV-light strobe at 20, 30, or 40 mL/min flow rate and treated up to 3 times by recirculation of milk to assess the effect of the number of passes on inactivation efficiency. Log₁₀ reductions varied from 0.55- to 7.26-log₁₀ CFU/mL. Complete inactivation was obtained in 2 cases and no growth was observed following an enrichment protocol. Predicted results were in agreement with the experimental data. Overall, this work demonstrates that pulsed UV-light has a potential for inactivation of milk pathogens.     

双重PCR-DHPLC技术快速检测水产品中金黄色葡萄球菌

根据编码金黄色葡萄球菌肠毒素A的sea基因、编码耐热核酸酶的nuc基因为目的基因,设计两对特异性引物,利用聚合酶链式反应结合变性高效液相色谱技术,建立水产品中金黄色葡萄球菌的双重PCR-DHPLC检测方法。以30株参考菌株进行特异性实验,除所试8株金葡菌出现目的片段和阳性吸收峰外,其余菌株均未检测到目的片段与阳性吸收峰,表明该方法具有良好的特异性。灵敏性实验结果表明,检测灵敏度达到菌液浓度50CFU/mL,比普通的凝胶电泳高一个数量级。利用建立的双重PCR-DHPLC检测方法对240份水产品进行检测,共检出22株金黄色葡萄球菌,检出率为9.2%,其中含有肠毒素基因有8株,占总样本的3.3%,与国标法检出阳性率比较无显著差异,证明该方法具有良好的实用性。实验证明,本研究建立的双重PCR-DHPLC方法不仅可以特异、灵敏、简便快速且高通量的实现对水产品中金葡菌的检测,而且也可通过对肠毒素基因的分析,方便地判断出该菌株致病性的强弱。     

云南省食源性金黄色葡萄球菌脉冲场凝胶电泳分型分析

目的采用脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)方法分析云南省2013～2015年食源性金黄色葡萄球菌分子分型带型,初步建立金黄色葡萄球菌PFGE分型的数据库。方法用限制性内切酶Smal I酶切113株金黄色葡萄球菌染色体DNA以进行PFGE分析,并利用BioNumerics软件对分离株的指纹图谱进行聚类分析。结果 113株食源性金黄色葡萄球菌的PFGE图谱的相似性系数在56.67%～100%之间,经聚类分析得到89个PFGE型别。结论建立云南省食源性金黄色葡萄球菌PFGE分型数据库,PFGE带型呈现多样性,对今后云南省金黄色葡萄球菌引起食物中毒的诊断和溯源工作有重要意义。     

2009-2018年广州市即食食品中金黄色葡萄球菌污染情况和菌型特征

目的 分析2009-2018年广州市零售即食食品中金黄色葡萄球菌污染情况,以及菌株肠毒素基因、耐药表型特征.方法 从广州市辖11个区的农贸市场、超市随机购买零售即食食品,增菌后分离金黄色葡萄球菌.对所有菌株进行多位点序列分型(MLST)、抗生素敏感性试验,并检测24种肠毒素基因.对耐甲氧西林金黄色葡萄球菌(MRSA)进...