Fate of Listeria on various food contact and noncontact surfaces when treated with bacteriophage

Study objective was to determine efficacy of a bacteriophage suspension against Listeria spp. when applied to three common types of materials used in food manufacturing facilities. Materials included two food contact materials (stainless steel and polyurethane thermoplastic belting) and one noncontact material (epoxy flooring). Coupons of each material were inoculated with a cocktail containing L. monocytogenes and L. innocua (4 to 5-log₁₀ CFU/cm²). Two phage concentrations and a control, 0, 2 × 10⁷ and 1 × 10⁸ PFU/cm² were evaluated. Treated samples were held at 4 or 20°C for 1 and 3 hr to determine the effect of temperature and treatment time. Reductions in Listeria populations ranged from 1.27 to 3.33 log₁₀ CFU/cm² on stainless steel, from 1.17 to 2.76 log₁₀ CFU/cm² on polyurethane thermoplastic belting, and from 1.19 to 1.76 log₁₀ CFU/cm² on epoxy resin flooring. Higher phage concentration (1 × 10⁸ PFU/cm²), longer treatment time (3 hr), and processing area temperature of 20°C showed a greater (p ≤ .05) reduction of Listeria on the stainless-steel and polyurethane thermoplastic belting coupons. Overall, Listeria reduction by phage treatment occurred on all three materials tested, under all conditions.     

Occurrence of Listeria monocytogenes in food in Chile.

Out of 2145 food samples analysed 77 were found contaminated with Listeria monocytogenes in Santiago, Chile. Samples were: 603 ice-cream (3.5% contaminated), 256 soft cheese (0.8%), 155 hard cheese (0%), 229 baby milk bottles (0%), 634 processed meat products (3.6%) and 268 crustaceous shellfish (11.6%). Three different isolation media were used: for 318 samples, Modified McBride Agar (MMA), Lithium chloride Phenylethanol Moxalactam agar, and Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar; for 1827 samples MMA was replaced by Listeria Selective Agar Oxford Formulation. Isolates were classified as follow: serovar 1/2a (25 isolates), serovar 4b (20), serovar 1/2b (19), serovar 3b (7), serovar 1/2c (2), untypable (4). A high variety of phagovars was detected although 52% of strains was untypable.     

Validation of a traditional Italian-style salami manufacturing process for control of Salmonella and Listeria monocytogenes

Italian-style salami batter (formulated with pork shoulder) was inoculated with ca. 7.0 log CFU/g of either Salmonella or Listeria monocytogenes. Salami links (55-mm cellulose casings) were fermented at 30 degrees C for 24, 40, or 72 h and then dried to target moisture/protein ratios (MPRs) of 1.9:1 or 1.4:1. Links were sampled after fermentation (24, 40, and 72 h) and after combined fermentation-drying treatments (MPRs of 1.9:1 and 1.4:1 for all fermentation periods), and microbiological and proximate analyses were performed at each sampling. Pathogen populations were enumerated by direct plating on selective agar and by an injured-cell recovery method. When enumerated by the injured-cell recovery method, Salmonella populations were reduced by 1.2 to 2.1 log CFU/g after fermentation alone (24 to 72 h) and by 2.4 to 3.4 log CFU/g when fermentation was followed by drying. Drying to an MPR of 1.4:1 was no more effective than drying to an MPR of 1.9:1 (P > 0.05). When enumerated directly on selective media, Salmonella populations were reduced from 1.6 to 2.4 log CFU/g and from 3.6 to 4.5 log CFU/g for fermentation alone and fermentation followed by drying, respectively. L. monocytogenes populations were reduced by <1.0 log CFU/g following all fermentation and combined fermentation-drying treatments, regardless of the enumeration method. These results suggest that the Italian-style salami manufacturing process evaluated does not adequately reduce high pathogen loads. Processors may thus need to consider supplemental measures, such as raw material specifications and a final heating step, to enhance the lethality of the overall manufacturing process.     

冷链即食食品生产加工环境中李斯特氏菌属检测方法的建立与应用

目的 开发一种建立可用于冷链即食食品生产加工环境中李斯特氏菌属的分离与检测方法,应用于对生产企业中的环境进行监控。方法 本研究结合GB4789. 30-2016国标方法和美国美国食品药品监督管理局细菌学分析手册Food and Drug Administration Bacteriological Analytical Manual（FDA BAM ）FDA BAM 单增李斯特氏菌检测方法,通过制备冻干定量菌球模拟增菌实验,对增菌液中萘啶酮酸和吖啶黄素的添加时间、头孢曲松钠的添加浓度进行了研究,确定最佳增菌方式和头孢曲松钠浓度,并将其应用到实际冷链即食食品生产企业环境中李斯特菌属的分离检测中。,确认该方法的可行性。结果 3种李斯特氏菌属和背景菌的冻干菌球的浓度分别为102 CFU/球及103 CFU/球。通过模拟增菌实验,延后4小时 h使用添加剂,可使李斯特氏菌属及单增李斯特氏菌的检出率均提高10%,在此基础上头孢曲松钠添加量在6 μg/mL至~8 μg/mL时,李斯特氏菌属及单增李斯特氏菌的检出率均为100%。结论 以上该方法应用在生产企业实际环境监测中,可提高李斯特氏菌属的检出率。该方法可应用于为冷链即食食品生产企业中李斯特氏菌属的环境监控,为生产企业的环境监控提供了技术保障。     

食品中单增李斯特氏菌检测PCR-免疫胶体金 试纸条方法的建立

目的 建立食品中单核增生李斯特氏菌快速检测PCR-免疫胶体金试纸条法。方法 通过设计特异性引物建立单增李斯特氏菌检测PCR方法并使用免疫胶体金技术建立PCR产物快速检测试纸条; 用试验菌株检测PCR-免疫胶体金试纸条方法的检测特异度与敏感度。使用新建方法对市售肉制品和乳制品中单核增生李斯特氏菌进行检测, 验证该方法在食品检测中的可行性。结果 PCR-免疫胶体金法具有良好的特异度, 敏感度比标准琼脂糖凝胶电泳法高100倍。采集乳品样品131份, 阳性样品1份, 检出率1.53%; 肉制品224份, 阳性样品4份, 检测率1.79%。结论 建立的单增李斯特氏菌检测PCR-免疫胶体金试纸条法特异度好, 敏感度高, 适用于食品中单增李斯特氏菌的检测。     

Isolation and lytic activity of the Listeria bacteriophage endolysin LysZ5 against Listeria monocytogenes in soya milk

The endolysin gene (lysZ5) from the genome of the Listeria monocytogenes phage FWLLm3 was cloned in Escherichia coli and characterized. Comparative sequence analysis revealed that lysZ5 resembled the murein hydrolase ply511 encoded by L. monocytogenes phage A511. The encoded protein LysZ5 had a predicted molecular mass of 35.8 kDa and was expressed in E. coli as an N-terminal fusion protein of 41.5 kDa. Addition of purified fusion protein to lawns of indicator bacteria showed that LysZ5 could lyse L. monocytogenes, Listeria innocua and Listeria welshimeri, but not Staphylococcus aureus or Enterococcus faecalis. The purified protein was able to kill L. monocytogenes growing in soya milk, with the pathogen concentration reduced by more than 4 log₁₀ CFU ml⁻¹ after 3 h incubation at 4 °C. As far as we know, this is the first report of a Listeria phage endolysin to control pathogens in soya milk and to demonstrate endolysin activity in foods at refrigeration temperatures. Moreover, LysZ5 may also be useful for biocontrol in other ready-to-eat foods.     

AnDiaTec Salmonella sp. PCR-ELISA for analysis of food samples

A commercially available PCR kit (AnDiaTec Salmonella sp. PCR-ELISA) was developed and evaluated for the detection of Salmonella sp. in food samples. The test is based on PCR amplification and hybridization of the amplified DNA to a microtiter plate followed by the detection of PCR product in the manner of an enzyme-linked immunosorbent assay test. The sensitivity and specificity were evaluated first with Salmonella pure cultures and artificially contaminated food samples, including food types for which an inhibition of the PCR reaction was expected. Both experiments proved a very good sensitivity, specificity, and reliability of the test with a very broad range of food types. In a second evaluation study, more than 1,100 food samples of different types were tested in parallel with the PCR method and with the International Standardization Organization 6579 bacteriological reference method. The results of this evaluation study and the results from other experiments on dilutions of artificially contaminated food samples led to the establishment of a positive-negative cutoff value (optical density at 450 nm of more than 0.9) with respect to the conventional bacteriological method. Using this positive-negative cutoff, 98% agreement to the bacteriological method was obtained.     

检测食品中志贺氏菌的双抗夹心ELISA方法的研究

研究获得纯化抗志贺氏菌IgY,经检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320;以志贺氏菌免疫新西兰大耳白兔,获得抗志贺氏菌的兔抗体,效价可达1∶12800。以此两种抗体建立双抗夹心ELISA,正交试验分析表明,兔抗体包被条件为4℃过夜,采用不封闭,抗原与包被抗体结合条件为37℃、1h;加入检测抗体(IgY)的浓度为0.25mg/mL,其结合条件为37℃、1h。该方法对纯培养菌液检出限为105cfu/mL,具有良好的敏感性;10株其他菌株的检测结果表明,该方法只与志贺氏菌发生特异性反应,不与其他菌株的抗原发生交叉反应。染菌的食品样品经选择性增菌后进行双抗夹心ELISA检测,含0.1～1cfu/mL志贺氏菌的样品在增菌13h后可检出阳性反应,含1～10cfu/mL志贺氏菌的样品在增菌11h后可检出阳性反应。     

食品中单核细胞增多李斯特菌的快速检测

单核细胞增多李斯特菌是一种能引起人畜共患病和食源性疾病的致病菌。本文简述了单增李斯特菌的特性和毒力因子,介绍了从分子生物学和免疫学发展起来的核酸探针杂交、PCR、ELISA、免疫传感器等快速检测方法。这些快速检测方法与传统检测方法相比不仅缩短了检测时间,提高了检测效率,还提高了检测方法的灵敏度和特异性,对于单增李斯特菌的检测具有良好的应用前景。      

食品检测用单核细胞增生李斯特氏菌标准物质的研制

目的建立食品检测用单核细胞增生李斯特氏菌标准物质的制备方法,研制均匀稳定的标准物质。方法利用冷冻干燥法制备103 CFU/样品的标准物质,参照CNAS-GL 29《标准物质/标准样品定值的一般原则和统计方法》,抽取20件样品,利用平板计数法测定标准物质的均匀性,并对结果进行统计分析;将样品分别置于-20、25、37℃条件下保藏,对其储藏稳定性和运输稳定性进行评价。组织5家实验室进行协同标定,使用30种即食熟肉制品作为基质,并按照国标法检验标物物质的适用性。结果采用单因素方差分析进行均匀性检验, F=0.922,符合标准物质的要求。标准物质在-20℃保藏28 d, 25、37℃保藏7 d,样品仍然稳定。经5家实验室协同标定,样品含量均在103 CFU/样品的水平;标准物质加入到30种即食熟肉制品中,均可以检出单核细胞增生李斯特氏菌。结论本研究所制备的单核细胞增生李斯特氏菌标准物质的均匀性、储藏稳定性和运输稳定性均符合要求,适用性良好,可用于食品检测实验室的质量控制和食品中单核细胞增生李斯特氏菌检测结果的评价。     

RapidChek检测食品中单增李斯特菌的方法评价

目的评价RapidChek法检测食品中单增李斯特菌的检测效果并验证。方法采用t检验,比较RapidChek方法与GB 4789.30-2016方法的培养基增菌效果。依据ISO 16140:2003《食品和动物饲料微生物学-可替代方法的验证方案》,比较RapidChek检测方法与GB 4789.30-2016检测方法的检测效果,涉及的性能指标有相对准确性、相对特异性和相对灵敏度、包含性和排他性。结果 RapidChek检测方法增菌培养基效果优于GB 4789.30-2016方法增菌培养基LB_1。根据RapidChek检测方法、GB 4789.30-2016培养基+RapidChek试纸条方法、RapidChek培养基+GB 4789.30分离鉴定方法的统计检验得出,3种方法与参照方法在相对准确性、相对特异性和相对灵敏度方面无显著性差异。在包含性和排他性方面,RapidChek检测方法与GB 4789.30-2016检测方法的检测结果均一致。结论在所验证的指标上,RapidChek检测方法(包括与GB4789.30-2016组合的方法)与GB 4789.30-2016方法无差异。     

Rapid polymerase chain reaction/DNA probe membrane-based assay for the detection of Listeria and Listeria monocytogenes in food

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.     

Optimum conditions for combined application of Leuconostoc sp. and Saccharomyces sp. to sourdough

Investigation of the optimum conditions for the combined application of Saccharomyces sp. (yeast) and Leuconostoc sp. (lactic acid bacteria, LAB) isolated in a previous study to the development of novel sourdough was carried out by response surface analysis. First, the cell growth conditions were analyzed. LAB showed good proliferation under conditions of 30–35°C, low pH, and high acidity, whereas the growth of yeast was inhibited. The growth of yeast was optimum at 25°C for 24 h. Based on these results, analysis of sourdough was carried out by varying the LAB population, temperature, and time after fixing the number of yeast. It was determined by response surface analysis that the optimal conditions for fermentation are LAB population of 10⁵ CFU/mL, temperature of 25°C, and reaction time of 24 h. From these results, the growth of LAB should be constantly maintained, and an appropriate pH that does not inhibit the growth of yeast due to the presence of generated organic acid is required to allow for the unique properties of sourdough. This study could give useful information for the development of novel sourdough.     

Comparison of a cold enrichment and the FDA method for isolating Listeria monocytogenes and other Listeria spp. from ready-to-eat food on retail sale in the U.K

A cold enrichment and the modified FDA selective enrichment method were compared for their ability to detect Listeria monocytogenes and other Listeria species from various ready-to-eat foods on sale in the UK. Of 57 food samples examined using cold enrichment, five yielded L. monocytogenes, and two L. innocua. The FDA enrichment method yielded three samples positive for L. monocytogenes only. Foods examined included soft cheeses, fermented meat sausages, pates and salads.     

Efficacy of bacteriocin-containing cell-free culture supernatants from lactic acid bacteria to control Listeria monocytogenes in food

Consumer demands have led to an increased interest in the use of natural antimicrobials for food protection. With the objective of developing novel products for enhancing the microbial safety of food, we have tested cell-free culture supernatants (CFS's) of eight antagonistic bacterial strains for their efficacy to inhibit Listeria monocytogenes in different food matrices. The antagonistic strains represented different members of the order Lactobacillales as well as one isolate of Staphylococcus sciuri and all showed strong inhibition of L. monocytogenes on agar plates. Cell-free supernatants were obtained after growing the bacteria in a yeast extract-glucose broth. In six of the CFS's, different class IIa bacteriocins, namely leucocin A, leucocin B, mundticin L, pediocin PA-1, sakacin A, and sakacin X, were identified as the major anti-listerial compounds. For the other two strains, the active substances could not be ascertained conclusively. The minimal effective concentration (MEC) of the individual CFS's to achieve a 2.3 log₁₀ reduction of L. monocytogenes was determined in culture broth, whole milk, and ground beef at 4 °C. While all bacteriocin-containing CFS's were effective in broth at concentrations from 52 to 205 AU/ml, significant higher concentrations were needed when applied in food. Best results were obtained using CFS's containing pediocin PA-1, that displayed only three- and ten-times higher MEC's in milk (307 AU/ml) and ground meat (1024 AU/g) compared to broth, respectively. A twenty-fold increase in the MEC (2048 AU/ml) was observed for a mundticin L-containing fermentate, and a CFS containing leucocin A and B was inactivated more than fifty-fold (> 1280 AU/ml) in both food matrices. Remarkably, the sakacin A and sakacin X containing CFS's displayed very selective inactivation rates, in which sakacin A was only effective in meat (512 AU/g), while sakacin X was only effective in milk (2048 AU/ml). In all cases, inhibition of L. monocytogenes was only transient and surviving or resistant bacteria started growing after prolonged storage. These results highlight the importance of careful testing the effectiveness of bacteriocins in the food systems for which they are intended to be applied against the selected target and non-target bacteria. Furthermore, the outgrowth of surviving or resistant bacterial populations points out that the tested bacteriocins are not suited to assure full inhibition of L. monocytogenes in a food product, if not applied in combination with additional preservative measures.     

宠物食品中沙门氏菌辐照模型的建立

为了建立宠物食品中沙门氏菌的辐照模型,研究了用60Coγ射线辐照不同理化性质的宠物食品,考核理化性质对沙门氏菌灭菌效果的影响.考察了初始菌数量、pH值、含水量、蛋白质含量、辐照后储存时间等因素对沙门氏菌杀灭效果的影响,得到了宠物食品中沙门氏菌的辐照模型.模型的建立为宠物食品中沙门氏菌的灭菌提供了新的方法,为其他类型菌株的辐照灭菌提供参考,在生产上具有一定的指导意义.     

The distribution of lactococcal bacteriophage in the environment of a cheese manufacturing plant

Survival of starter bacteria and their bacteriophages following spray irrigation of whey in the vicinity of a cheese plant was investigated. Phage in environmental samples that attacked starter strains used in the plant were detected infrequently in water, but persisted for up to six weeks in the soil. Enrichment of cow dung, grass and trough water samples was required to detect phage, indicating their presence at low levels. No starter bacteria could be re-isolated from grass samples from whey irrigated pasture. Inside the plant, whey contained the largest numbers of phage and when released into the environment (via irrigation practices) contributed to the recycling of phages through the plant. It did not, however, appear to be the initial source for starter infection by ‘new’ phage. The detection of phages in the bulk raw milk that were not active on starter strains that were being used in the plant, reemphasized raw milk as a potential source of new phage.     

食品中单核细胞增生单核细胞增生李斯特菌的重组酶聚合酶扩增检测方法的建立

目的 建立食品中单核细胞增生李斯特菌的重组酶聚合酶扩增(RPA)检测体系。方法 针对单核细胞增生李斯特菌的特异性基因hlyA,筛选出一组特异性强且高效的引物,建立单核细胞增生李斯特菌的RPA检测体系,并进行特异性、灵敏度检测。结果 建立的RPA方法在37 ℃恒温反应仅需30 min,能够特异地检测单核细胞增生李斯特菌,最低模板浓度可低至0.5 ng/μL。人工染菌试验显示,该RPA体系可以有效扩增出每2.5 g样品中人工染菌10⁴ CFU样品中的单核细胞增生李斯特菌。结论 RPA等温扩增方法特异较强、灵敏度较高,具有操作简便、不需要昂贵仪器、常温进行扩增反应等优点,适用于现场检测以及在基层实验室推广应用。     

即食食品中单增李斯特菌的半定量风险评估

开展某市即食食品中单增李斯特菌的半定量风险评估,参照微生物风险评估程序,对单增李斯特菌开展了危害识别、危害特征描述、暴露评估和风险特征描述。通过剂量反应关系推测易感人群和非易感人群由于摄入即食食品导致单增李斯特菌病的每年发病概率分别为3.71×10-7和3.39×10-9。基于2008~2011年监测各类生食蔬菜、生食水产品、菜肴(沙拉)等即食食品942组数据,构建了即食食品中单增李斯特菌的风险矩阵,由风险可能性和风险损失度计算得到易感人群通过摄入即食食品感染单增李斯特菌的风险等级属于五级风险等级中较小的一级,表明当地居民通过摄入即食食品感染单增李斯特菌病的风险较小。本文可为构建完整单增李斯特菌风险评估体系提供理论参考。