酱卤肉中沙门氏菌和单核细胞增生李斯特菌生长特性及预测模型建立

依据流行病学的调查结果,酱卤肉中检出率较高的致病菌为沙门氏菌和单核细胞增生李斯特菌。本实验对该两种代表性致病菌以酱卤肉作为培养基成分在不同温度下的生长特性进行研究,建立了不同温度下沙门氏菌和单核细胞增生李斯特菌的生长一级模型和二级模型。在沙门氏菌和单核细胞增生李斯特菌培养温度范围内得出酱卤肉制品中沙门氏菌的动力学模型为：lgN＝A＋（10.2－A）×exp{－exp[e×（－3.173 1T2＋2.02T＋1.812）×（1.252T2－7.746T＋1.22－t）/（10.2－A）＋1]};酱卤肉中单核细胞增生李斯特菌的动力学模型为：lgN＝A＋（17.9－A）×exp{－exp[e×（－9.024T2＋5.774T－4.402）×（1.378T2－7.995T＋1.172－t）/（17.9－A）＋1]}。运用该预测模型可描述自然状态下酱卤肉制品中的两种代表性致病菌数量的动态变化,为该类产品的生产、运输、贮藏及销售过程中的食品安全保证提供良好的理论依据,用以减少由致病菌引发的食品安全风险,并为建立酱卤肉企业的食品安全管理体系提供科学基础。     

inlA和inlB基因缺失对单核细胞增生性李斯特菌侵袭HT29结肠癌细胞的影响

单核细胞增生性李斯特菌（Listeria monocytogenes,LM）是人畜共患的食源性致病菌,其可以穿透多个宿主屏障,而内化素蛋白家族被认为是LM穿透宿主屏障过程中起重要作用的毒力因子。本研究利用同源重组的方法构建了LM野生菌株EGDe的inlA和inlB基因双缺失菌株,利用实时荧光定量聚合酶链式反应检测其主要毒力基因表达的变化,并以HT29结肠癌细胞为对象,研究inlA和inlB基因缺失对LM侵袭宿主细胞能力的影响。结果表明基因的缺失对其生长能力没有影响,但多个毒力基因的表达发生了不同程度的变化,同时发现inlA和inlB基因的缺失使LM侵袭HT29结肠癌细胞的能力显著下降（P＜0.05）。本研究成功构建LM的inlA和inlB基因双缺失菌株,并初步研究了基因缺失对LM侵袭宿主细胞能力的影响,为深入研究内化素InlA和InlB在LM入侵宿主细胞过程中的具体作用提供了支持。     

Survival and Metabolic Activity of Listeria monocytogenes on Ready‐to‐Eat Roast Beef Stored at 4 °C

Three brands of commercial roast beef were purchased and artificially inoculated with a 5‐strain Listeria monocytogenes cocktail at 2 inoculation levels (approximately 3 and 6 Log CFU/g). Although all 3 brands contained sodium diacetate and sodium lactate, inoculated Listeria cocktail survived for 16 d in all 3 brands; significant increases in L. monocytogenes numbers were seen on inoculated Brand B roast beef on days 12 and 16. Numbers of L. monocytogenes increased to 4.14 Log CFU/g for the 3 Log CFU/g inoculation level and increased to 7.99 Log CFU/g for the 6 Log CFU/g inoculation level by day 16, with the pH values being 5.4 and 5.8 respectively. To measure the cell viability in potential biofilms formed, an Alamar blue assay was conducted. Brand B meat homogenate had the highest metabolic activities (P < 0.05). By comparing its metabolic activities to Brands A and C and the inoculated autoclaved meat homogenates, results indicated that the microflora present in Brand B may be the reason for high metabolic activities. Based on the denaturing gradient gel electrophoresis and the Shannon–Wiener diversity index analysis, the “Brand” factor significantly impacted the diversity index (P = 0.012) and Brand B had the highest microflora diversity (Shannon index 1.636 ± 0.011). Based on this study, results showed that antimicrobials cannot completely inhibit the growth of L. monocytogenes in ready‐to‐eat roast beef. Native microflora (both diversity and abundance), together with product formula, pH, antimicrobial concentrations, and storage conditions may all impact the survival and growth of L. monocytogenes.     

Detection of Virulence Genes and Growth Potential in Listeria monocytogenes Strains Isolated from Ricotta Salata Cheese

Ricotta Salata is a traditional ripened and salted whey cheese made in Sardinia (Italy) from sheep's milk. This product is catalogued as ready‐to‐eat food (RTE) since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens, such as Listeria monocytogenes, can represent a health risk for consumers. In September 2012, the FDA ordered the recall of several batches of Ricotta Salata imported from Italy linked to 22 cases of Listeriosis in the United States. This study was aimed at evaluating the presence and virulence properties of L. monocytogenes in 87 samples of Ricotta Salata produced in Sardinia. The ability of this product to support its growth under foreseen packing and storing conditions was also evaluated in 252 samples. Of the 87 samples 17.2% were positive for the presence of L. monocytogenes with an average concentration of 2.2 log₁₀ cfu/g. All virulence‐associated genes (prfA, rrn, hlyA, actA, inlA, inlB, iap, plcA, and plcB) were detected in only one isolated strain. The Ricotta Salata samples were artificially inoculated and growth potential (δ) was assessed over a period of 3 mo. The value of the growth potential was always >0.5 log₁₀ cfu/g under foreseen packing and storing conditions. This study indicates that Ricotta Salata supports the L. monocytogenes growth to levels that may present a serious risk to public health, even while stored at refrigeration temperatures.     

抗单增李斯特菌枯草芽孢杆菌C3增菌培养条件优化

枯草芽孢杆菌对单增李斯特菌有强烈抑制作用,该文对其增菌培养基和培养条件进行优化,为今后将其应用于食品和饲料奠定基础。在对培养基成分和培养条件进行单因素试验的基础上,设计5因素4水平正交试验对培养基成分进行优化。结果表明,培养基成分优化为葡萄糖1.0%,酵母浸粉1.0%,NaCl 0.5%,KH2PO4 0.2%,MgSO4·7H2O 0.2%,培养条件优化为pH5.4,装液量50 mL/250 mL,接种量3%,温度37 ℃,150 r/min培养14 h,在此条件下,活菌数可达到7.1×1010 CFU/mL,比优化前提高了7.89倍。     

双基因敲除减毒单增李斯特菌(ΔactA/ΔinlB)的构建及其生物学初步鉴定

减毒单增李斯特菌具有成为疫苗活载体的潜力,可同时引起MHCⅠ和MHCⅡ类抗原递呈系统,具有强烈激发CD8+和CD4+细胞免疫的能力。构建毒力基因缺失菌株进而评估其生物活性对于其作为疫苗活载体的开发尤为重要。本文采用同源重组技术构建单缺失菌株Lm-△act A和双缺失菌株Lm-△act A△/inl B,从生长状态、毒力基因表达和对Hep G2细胞侵袭能力方面探讨减毒菌株生物学特性。生长活性测定显示两种减毒菌株和野生型Lm(EGD-e)三者生长状况无差异;实时定量PCR结果显示act A基因缺失后,inl A基因表达水平上升两倍;act A和inl B基因双缺失后,plc B和hly基因的表达水平都有较大幅度的上升;侵袭Hep G2细胞结果显示act A基因缺失后侵袭能力增强、act A和inl B基因共同缺失后侵袭能力减弱。因此,减毒菌株的构建及其生物特性研究不仅对食源性致病菌Lm致病机理具有重要意义,也为构建预防人类和动物疫病的疫苗载体奠定了基础。     

In vitro Antilisterial Effects of Citrus Oil Fractions in Combination with Organic Acids

ABSTRACT:  The objectives of this study were to screen activity of citrus essential oil fractions (EOs) alone and in combination with organic acids against 2 species of Listeria . Five citrus EOs were initially screened by disc diffusion assay for antibacterial activity. Cold pressed terpeneless Valencia orange oil (CP terpeneless oil) had the strongest bacteriostatic (MIC) and bactericidal (MBC) properties at 0.55% and 1.67%, respectively. Four organic acids were tested for effectiveness against Listeria . Citric and malic acids proved to be the most effective with MBC of 1.1% alone. Assays were conducted to determine synergistic effects of EOs and citric or malic acids. There was a significant decrease in MIC and MBC to 0.04% EO plus 0.12% malic or citric acid. EOs from citrus paired with organic acids offer the potential as an all-natural antimicrobial for improving the safety of all-natural foods.     

Role of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) in bacterial heat resistance as a function of treatment medium pH

This investigation aimed to determine the role of general stress-response alternative sigma factors σ^S (RpoS) and σ^B (SigB) in heat resistance and the occurrence of sublethal injuries in cell envelopes of stationary-phase Escherichia coli BJ4 and Listeria monocytogenes EGD-e cells, respectively, as a function of treatment medium pH. Given that microbial death followed first-order inactivation kinetics (R² > 0.95) the traditional D_T and z values were used to describe the heat inactivation kinetics.Influence of rpoS deletion was constant at every treatment temperature and pH, making a ΔrpoS deletion mutant strain approximately 5.5 times more heat sensitive than its parental strain for every studied condition. Furthermore, the influence of the pH of the treatment medium on the reduction of the heat resistance of E. coli was also constant and independent of the treatment temperature (average z value = 4.9 °C) in both parental and mutant strains.L. monocytogenes EGD-e z values obtained at pH 7.0 and 5.5 were not significantly different (p > 0.05) in either parental or the ?sigB deletion mutant strains (average z value = 4.8 °C). Nevertheless, at pH 4.0 the z value was higher (z = 8.4 °C), indicating that heat resistance of both L. monocytogenes strains was less dependent on temperature at pH 4.0. At both pH 5.5 and 7.0 the influence of sigB deletion was constant and independent of the treatment temperature, decreasing L. monocytogenes heat resistance approximately 2.5 times. In contrast, the absence of sigB did not decrease the heat resistance of L. monocytogenes at pH 4.0.The role of RpoS in protecting cell envelopes was more important in E. coli (4 times) than SigB in L. monocytogenes (1.5 times). Moreover, the role of σ^S in increasing heat resistance seems more relevant in enhancing the intrinsic resilience of the cytoplasmic membrane, and to a lesser extent, outer membrane resilience.Knowledge of environmental conditions related to the activation of alternative sigma factors σ^S and σ^B and their effects on heat resistance would help us to avoid and/or identify situations that increase bacterial stress resistance. Therefore, more efficient food preservation processes might be designed.     

Anti-Listeria monocytogenes activity of enterocins microencapsulated by ionic gelation

The encapsulation of enterocins synthesized by Enterococcus faecium CRL1385 through ionic gelation with calcium ions was analyzed. Different enterocins samples were lyophilised and encapsulated using low-methoxyl pectin as the coating material. Lipids present in milk butter were also added to control the release of antimicrobial peptides from the capsules. The morphology of fresh and freeze-dried capsules was examined using light microscopy and scanning electron microscopy, respectively. Antimicrobial activity of encapsulated bacteriocins was assessed against Listeria monocytogenes 01/155 using the agar diffusion technique and direct contact in microplates. The capsules with higher lipid content showed a more spherical and uniform shape. Pathogen inhibition was observed for capsules prepared with different bacteriocin solutions both on solid (halo diameter = 8.5–13.5 mm) and in an aqueous medium (ca. 2 log orders decline in L. monocytogenes viability). The outcomes suggest that bacteriocin encapsulation through ionic gelation can be a potential alternative for the application of these antimicrobial peptides as biopreservatives in food.