鼠伤寒沙门菌TA97、TA98、TA100、TA102即用型标准菌株的研制

目的 研制鼠伤寒沙门菌TA97、TA98、TA100、TA102即用型标准菌株。方法 对高、低浓度鼠伤寒沙门菌TA97、TA98、TA100、TA102的冻干菌株进行计数，测定了增菌3、5、7、18 h后冻干菌的自发回变率，依据CNAS-GL003 2018《能力验证样品均匀性和稳定性评价指南》（CNAS-GL003）和GB 15193.4—2014《食品安全国家标准 细菌回复突变试验》（GB 15193.4—2014），测定即用型标准菌株的均匀性、储藏和运输稳定性、自发回变率和诱变剂突变率。比较20株即用型标准菌株和新鲜菌株的自发回变率和诱变剂突变率测试结果的标准偏差。结果 高浓度冻干样品浓度为10⁷~10⁸ CFU/样品，低浓度样品浓度为10⁵~10⁶ CFU/样品；增菌18 h后自发回变率的结果基本符合GB 15193.4—2014要求；4株即用型标准菌株的均匀性结果的F值分别为1.12、1.05、1.68、1.38，F<F_0.05（19，20），符合CNAS-GL003要求。4株冻干菌株在25 ℃ 5 d、4 ℃ 14 d、-20 ℃ 6个月的储存条件，检测结果符合储存和运输稳定性要求。20株即用型冻干标准菌株的自发回变率较新鲜菌株标准偏差小。结论 本研究研制出适用于Ames试验的即用型标准菌株，其均匀性、储存和运输稳定性良好，自发回变的稳定性优于新鲜菌株。     

食品检验用解脂耶氏酵母菌标准物质制备研究

目的 为满足实验室酵母菌日常检验质量控制需求及实验室间比对,制备解脂耶氏酵母菌检验用标准物质。方法 通过生化、基质辅助激光解吸电离飞行时间质谱及ITS位点测序对研究用菌株进行菌种鉴定确认后,刮取适宜菌苔于保护剂中混匀后冻干,制备10_⁴ CFU/样品浓度的标准物质。参照CNAS-GL003等标准要求进行均匀性检验后,采用单因素方差分析对结果进行评价。将样品放于25 ℃和37 ℃及-20 ℃、4 ℃条件下,分别进行运输稳定性检验及储存稳定性检验。依据国家标准GB 4789.15,将研制的本标准物质加入7类食品基质共20件样品中进行检验,验证真实食品样品中适用性。组织3家实验室,对本标准物质进行协作标定。结果CMCC98025经生化鉴定为解脂耶氏酵母菌,准确度为93%;MALDI-TOF MS鉴定结果为解脂耶氏酵母,分数为2.012;ITS位点测序结果与NCBI Genbank中已有序列比对,匹配最优结果为解脂耶氏酵母Yarrowia lipolytica(Accession number ：CP061015.1;Query Cover：95%;Ident：100%)。均匀性测试结果符合正态分布,通过单因子方差分析计算F_0.05(19,20)=2.137,F值为1.697,F＜F_0.05,符合均匀性要求。25 ℃及37 ℃培养箱中放置7天,标准物质中菌含量仍保持在10_⁴ CFU/样品,可保持稳定。标准物质于4 ℃储存28天及-20 ℃储存90天,菌含量为10_⁴ CFU/样品,复苏率均在91%以上,说明4 ℃放置较短时间及-20 ℃放置较长时间样品稳定。7类食品样品基质中加入本标准物质后进行检验,均可检出,回收率为81.3%。经三家实验室协作标定,本标准物质平均浓度为2.0～3.0×10_⁴ CFU/样品。结论 本研究制备的解脂耶氏酵母菌标准物质均匀性、稳定性、真实食品样品中应用验证及协作标定结果均符合要求,可应用于食品中解脂耶氏酵母菌定性检验,今后可作为实验室日常检验工作的阳性对照质控样品,也可作为实验室间比对样品发放,以保障日常检验工作结果可靠性,进一步提升检验机构人员的检验水平。     

多黏菌素类抗生素耐药性编码基因检测用质粒标准样品研制

目的 研制适合多黏菌素类抗生素耐药性编码基因检测用质粒DNA标准样品。方法 由National Center for Biotechnology Information（NCBI）数据库检索得到mcr-1、mcr-3、mcr-5基因参考序列,构建重组质粒和重组菌株;将重组菌传代至15代检测目标基因的遗传稳定性。提取重组质粒,真空干燥,制得标准样品。将标准样品水化、连续10倍梯度稀释,测定聚合酶链式反应（PCR）和实时荧光PCR（RT-qPCR）扩增目标基因的检出限。随机抽取质粒DNA标准样品,采用PCR法和紫外分光光度计法检验样品的均匀性及其在4 ℃、37 ℃、-20 ℃的贮存稳定性。结果 成功获得多黏菌素类抗生素耐药机制编码基因mcr-1、mcr-3、mcr-5基因片段,重组菌15代传代中目的基因遗传稳定。mcr-1、mcr-3、mcr-5标准样品PCR和RT-qPCR最低检出限分别为1.67×10⁴ 拷贝数/μL和1.67 拷贝数/μL、1.31×10⁶ 拷贝数/μL和13.1 拷贝数/μL、1.55×10⁵ 拷贝数/μL和1.55 拷贝数/μL。T检验表明,各基因随机抽取的12管标准样品质量间的F值均小于F_临界值,且PCR均可检出,均匀性符合要求。标准样品在4 ℃贮存90 d、37 ℃贮存14 d、-20 ℃贮存360 d,定性、定量检验结果稳定、无极显著性差异。结论 本实验制备的mcr-1、mcr-3、mcr-5质粒DNA标准样品目的基因遗传稳定,均匀性和贮存稳定性良好。本研究结果可用于多黏菌素类抗生素耐药机制的快速预测和相关基因检测的质控样品。     

食品检测用肠道侵袭性大肠埃希氏菌标准物质的研制

目的 为满足肠道侵袭性大肠埃希氏菌(enteroinvasive Escherichia coli, EIEC)准确检测的实验室质量控制和能力验证需求,研制具有我国自主知识产权、稳定性良好且具有清晰基因组信息背景的即用型EIEC标准物质。方法 利用二代高通量测序技术对EIEC(CMCC 44840)进行全基因组测序,明确CMCC 44840的种属、血清型、多位点序列分型(MLST)和毒力基因;采用冷冻干燥技术制备活菌含量为10³ CFU的EIEC冻干样品;参照CNAS-GL017-2018进行均匀性检验,并采用单因素方差分析对结果进行统计分析;将样品分别于-20 ℃、4 ℃、25 ℃和37 ℃条件下保藏,对其储藏稳定性和运输性进行评价;利用5种食品基质样本进行标准物质的使用效果验证,同时组织3家实验室进行协同标定。结果 CMCC 44840基因组大小为4.96 Mb,GC含量为50.7%,编码基因5 424个,种属鉴定结果为大肠埃希氏菌,(Escherichia coli)血清预测结果为O28ac:H7,MLST为ST311型,携带ipaH、virB、virF等毒力基因;制备的EIEC冻干样品均匀性检验结果F=1.79,符合标准物质均匀性要求;冻干样品在25 ℃和4 ℃下保藏7 d,-20 ℃和4 ℃下保藏60 d,菌含量均保持在10³CFU水平,在37 ℃下第3 d菌含量出现下降,低于10³CFU水平;添加EIEC冻干样品的20件不同食品基质样品经增菌后均检出EIEC;3家协同标定实验室测定EIEC冻干样品菌含量均为10³ CFU,且实验室间无显著性差异(F=0.59)。结论 本研究所制备的EIEC标准物质所用菌株具有清晰的基因组序列信息,均匀性和稳定性均符合要求,适用性良好,能够满足食品检测实验室的质量控制和能力验证的需求。     

扇贝和利玛原甲藻混合基体中大田软海绵酸和鳍藻毒素-1标准样品的研制

目的 研制基于扇贝和利玛原甲藻混合基质的大田软海绵酸（OA）和鳍藻毒素-1（DTX1）定量分析标准样品。方法 以扇贝组织和含有OA和DTX1的利玛原甲藻的藻泥为原料，经充分混匀、冷冻干燥制备基体OA和DTX1标准样品，开展定性确证以及均匀性、稳定性检验和协同定值分析。结果 经确证该标准样品中含有OA和DTX1；经方差分析法（F检验）检验评价均匀性良好；考察长期稳定性和模拟运输条件下短期稳定性，样品性能稳定；该基体标准样品中OA和DTX1特性标准值及不确定度分别为（7.039±0.272） mg/kg （k=2）、（2.012±0.021） mg/kg（k=2）。结论 该混合基体标准样品填补了国内目前没有该类标准样品的空白，可以用于方法验证、检测过程质量控制、能力验证等实验室质量控制活动。     

食品检测用副溶血性弧菌标准物质的制备和评价

该研究制备并检测了不含基质的副溶血性弧菌标准物质。用质谱、生化、16S RNA基因序列测定3种方法对菌株CMCC20030分别确认种属。先采用冷冻干燥技术制备800个样品[103 CFU/样品(20 μL)]。然后随机抽取20个样品进行均匀性检验;再将样品存放25、37 ℃分别在1、3、5、7 d取出进行运输稳定性检验,将样品存放4 ℃分别于7、14、28 d进行短期保藏性检验,将样品存放-20 ℃分别于14、28、60 d的保藏稳定性检验。组织5家实验室对样品进行协同标定。最后用食品基质检测使用效果。菌株CMCC20030经3种方法均鉴定为副溶血性弧菌。均匀性检验中,F样品=1.930,小于FINV（0.05,19,20）。稳定性检验中,于-20 ℃保藏60 d、4 ℃保存28 d、25 ℃保藏7 d和37 ℃保藏3 d后,活菌含量103 CFU/样品。协同标定实验中,5家实验室结果均为103 CFU/样品。使用效果实验中,20种食品基质加入样品后均可以检出,本底对照均未检出。制备的不含基质的副溶血性弧菌样品满足标准物质要求,可依据不同目的灵活使用。     

湖州市市售凉拌菜中主要致病菌污染状况及快速定量风险评估

目的 了解湖州市市售凉拌菜中主要致病菌的污染状况,并初步评估其健康风险,为食源性疾病防制提供参考。方法 2017年和2020年随机采集市售凉拌菜461份,对其开展大肠埃希菌（Escherichia coli）、沙门氏菌（Salmonella）、单核细胞增生李斯特菌（Listeria monocytogenes）、副溶血性弧菌（Vibrio parahaemolyticus）、金黄色葡萄球菌（Staphylococcus aureus）以及蜡样芽孢杆菌（Bacillus cereus）检测,依据相关标准对其进行微生物污染状况评价,并运用食品微生物快速定量风险评估（sQMRA）模型初步评估其健康风险。结果 凉拌菜中大肠埃希菌不合格率最高（48.94%,46/94）,其平均污染水平为104.34 CFU/g,主要在农贸市场和网店等流通环节污染严重（P=0.004）。食源性致病菌中,单增李斯特菌、金黄色葡萄球菌、沙门氏菌和蜡样芽孢杆菌检出率分别为9.54%（44/461）、5.21%（24/461）、1.30%（6/461）和1.06%（1/94）,金黄色葡萄球菌≥10⁴ CFU/g和蜡样芽孢杆菌≥10⁵CFU/g的比例均为零。经评估,4种食源性致病菌的年估计总发病数为2 208例,总发病概率为7.21×10^-4,风险等级均为中风险。结论 湖州市市售凉拌菜整体卫生状况不佳,尤其是大肠埃希菌污染严重,4种主要食源性致病菌污染也存在一定风险,建议加强监督、评估与优先管理。     

我国市售茶叶包装接触面积/体积比调查研究

目的 了解我国茶叶包装材料的使用情况,构建不同类别茶叶不同食品接触材料的接触面积/体积比（S/V）基础数据库。方法 采集市场销售的不同包装和类型的7大类茶叶,通过直接测量法及应用3D表面积测量仪获得413份茶叶样品的接触面积,结合不同类别茶叶产品的质量、规格等数据信息,计算其S/V。结果 茶叶的接触材料均为单一材质,不同类别茶叶接触材料的S/V的平均值为93.0 dm²/kg,范围为3.5~595.6 dm²/kg。 99.8%（412/413）的茶叶接触材料S/V≥6.0 dm²/kg,87.2%（360/413）的S/V介于6.0~200.0 dm²/kg之间。结论 我国大部分市售茶叶的S/V大于欧盟评估时采用的6.0 dm²/kg,利用本次调查研究获得的参数将会降低食品接触材料风险评估中的不确定性。     

食品检测用鼠伤寒沙门氏菌标准物质的研制

目的 研制均匀稳定的鼠伤寒沙门氏菌标准物质。方法 采用冷冻干燥技术制备含量为1.5-2.0×103 CFU /样品的菌球, 参照CNAS—GL29: 2010《标准物质/标准样品定值的一般原则和统计方法》, 随机抽取22件样品进行均匀性检验, 采用单因素方差分析对结果进行统计分析, 将样品分别于-20、4、25、37 ℃条件下保藏, 对其储藏稳定性和运输稳定性进行评价, 并组织3家实验室进行协同标定, 再使用45件食品作为基质, 按照国标法检验鼠伤寒沙门氏菌标准物质的适用性。结果 对22件标准物质的均匀性检测结果进行单因素方差分析, F=1.986, 符合标准物质的要求。标准物质在?20 ℃保藏28 d, 复苏率为103.1%; 在4 ℃保藏28 d, 复苏率为102.0%; 在25 ℃保藏14 d或者37℃保藏7 d, 样品中菌含量仍保持在103 CFU/样品的水平, 说明样品的短期储藏稳定性、长期储藏稳定性和运输稳定性都符合要求。经3家实验室协同标定, 样品活菌含量均在103 CFU/样品水平, 生化鉴定结果均符合沙门氏菌的特征; 标准物质加入到45种食品基质中, 均可以检出沙门氏菌。结论 本研究所制备的鼠伤寒沙门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沙门氏菌检测结果的评价。     

2019—2020年度食品中副溶血弧菌能力验证样品的研制及其应用

目的 制备食品中副溶血弧菌检验能力验证样品，并应用于2019—2020年度实验室能力验证考核。方法 将5种背景菌株和副溶血弧菌通过生化、基质辅助激光解吸电离飞行时间质谱鉴定确认菌株种属。采用冷冻干燥技术制备阳性样品和阴性样品，阴性样品仅含有背景菌株，阳性样品在背景菌的基础上添加有副溶血弧菌。随机抽取20份样品进行均匀性检验，将样品存放于-20 ℃、4 ℃进行保藏稳定性检验，将样品存放于25 ℃、37 ℃进行运输稳定性检验。向参加考核的实验室发放样品并提供作业指导书，回收各实验室结果进行统计。结果 菌株种属确认与预期结果一致，阴性样品和阳性样品的均匀性和稳定性均满足要求。2019年和2020年考核结果满意率分别为87.5%和90%。结论 本研究制备的样品满足食品中副溶血弧菌能力验证项目的要求，通过组织能力验证考核可以反映出我国实验室之间的差异，有助于进一步提高实验室检验水平。     

Arsenic content of some edible mushroom species

The arsenic contents of 162 fruit body samples of 37 common edible mushroom taxa were analyzed. The samples were gathered from different habitats of Hungary (mainly from mountains) between 1984 and 1999. The arsenic content of the samples was measured by the inductively coupled plasma spectrometry method. Very low [lower than 0.05 mg/kg dry matter (DM)] concentrations were found in the samples of 13 taxa, while higher (or very high) contents were quantified in other common taxa (the highest arsenic content was recorded in the fruit body of Laccaria amethysthea at 146.9 mg/kg DM). The species of eight genera (Agaricus, Calvatia, Collybia, Laccaria, Langermannia, Lepista, Lycoperdon, Macrolepiota) belong to the so-called accumulating taxa, and this tendency is evident on all habitats. This arsenic accumulation capability is found in two orders of Basidiomycetes (Agaricales and Gasteromycetales), which is to say this phenomenon occurs in the families Agaricaceae, Tricholomataceae and Gasteromycetaceae. The accumulating taxa found all have a saprotrophic type of nutrition; arsenic accumulation is not detectable in xilophagous or in mycorrhizal species. The consumption of the accumulating species found has only a low toxicological risk for three reasons: the consumed fresh fruit bodies contain about a tenfold lower arsenic level than the dried ones, the majority of arsenic occurs not in poisonous inorganic, but in less dangerous (or not poisonous) organic forms, and the frequency of consumption is low.     

Organization and localization of the dnaA and dnaK gene regions on the multichromosomal genome of Burkholderia multivorans ATCC 17616

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for fundamental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH-dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating sequence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE-orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the sigma32-dependent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome.     

Susceptibility of stored product insects to high concentrations of ozone at different exposure intervals

Ozone is a highly reactive gas with insecticidal activity. Past studies have indicated that ozone technology has potential as a management tool to control insect pests in bulk grain storage facilities. The objective of this study was to determine the efficacy of short periods of exposure to high ozone concentrations to kill all life stages of red flour beetle (Tribolium castaneum (Herbst)) (Coleoptera: Tenebrionidae), and Indianmeal moth (Plodia interpunctella (Hübner)) (Lepidoptera: Pyralidae), adult maize weevil (Sitophilus zeamais (Motsch.)) (Coleoptera: Curculionidae) and adult rice weevil (S. oryzae (L)) (Coleoptera: Curculionidae). Insects were treated with six ozone concentrations between 50 and 1800 ppm. The specific objective was to determine minimal time needed to attain 100% mortality. The most ozone-tolerant stages of T. castaneum were pupae and eggs, which required a treatment of 180 min at 1800 ppm ozone to reach 100% mortality. Eggs of P. interpunctella also required 180 min at 1800 ppm ozone to reach 100% mortality. Ozone treatments of 1800 ppm for 120 min and 1800 ppm for 60 min were required to kill all adult S. zeamais and adult S. oryzae, respectively. The results indicate that high ozone concentrations reduce the treatment times significantly over previously described results. Our results also provide new baseline information about insect tolerance to ozone treatment.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

Formation of cereulide and enterotoxins by Bacillus cereus in fermented African locust beans

Afitin, iru and sonru are three spontaneously fermented African locust bean Benin condiments. The fermentation processes are exothermic, with temperatures mostly being above 40 °C. A total of 19 predominant Bacillus cereus isolates from afitin, iru and sonru, were investigated. The enterotoxin genes nhe (A, B, C) were present in all 19 isolates, the hbl (A, C, D) in one (afitin), and the cytK gene in three isolates (afitin). Levels of cytotoxicity to Vero cells and NheA production in BHI-broth was within the range of known diarrheal outbreak strains. Autoclaved cooked African locust beans inoculated with emetic (cereulide producing) B. cereus Ba18H2/RIF supported growth at 25, 30 and 40 °C with highly different maximum cereulide productions of 6 ± 5, 97 ± 3 and 0.04 ± 0.02 μg/g beans, respectively (48 h). For non-autoclaved cooked beans inoculated with 2, 4 and 6 log₁₀B. cereus Ba18H2/RIF spores/g beans, cereulide production was 5 ± 4, 64 ± 8 and 69 ± 34 μg/g beans, respectively at 24 h, while it was 70 ± 43, 92 ± 53 and 99 ± 31 μg/g at 48 h of fermentation at 30 °C. Even though high toxin levels were observed, to date there are no known reports on diarrhea or vomiting due to the consumption or afitin, iru and sonru in Benin, which also according to the present study is likely to be expected from the low levels of cereulide produced at 40 °C.     

Microbial profiles of frozen trimmings and silver sides prepared at Indian buffalo meat packing plants

To assess microbiological quality of buffalo meat trimmings (TT = 114) and silver sides (SS = 41), samples were collected from four different Indian meat packing plants. The aim of this study was: (i) to evaluate standard plate count (SPC), psychrotrophic count (PTC), Enterococcus feacalis count (EFC), Staphylococcus aureus count (SAC) and Escherichia coli count (ECC) and the presence of Salmonella spp. and Listeria monocytogenes; and (ii) also to determine vero toxic E. coli (VTEC) by polymerase chain reaction (PCR). TT samples had significantly higher (P < 0.001) SPC, PTC, EFC, and SAC than SS, while across the meat types there was no difference (P > 0.05) in ECC. E. coli was recovered from 32.4% TT and 19.5% SS samples. The prevalence rate of Salmonella spp. and L. monocytogenes in TT was 1.75% and 0.87%, respectively. But no SS sample was found to be positive for any of these two pathogens. VTEC was found in 2.58% of all the tested samples. This finding suggests that TT contain higher microbes but only small numbers of pathogens of latent zoonotic importance. The present study confirmed the importance of maintaining good process hygiene at meat plants for microbiological status of buffalo meat.     

Feeding deterrent activity of terpenoid lactones with a p-menthane system against stored-product pests

Feeding deterrent activity of eight enantiomeric pairs and one optically inactive terpenoid lactone with a p-menthane system against three storage pests was determined. The lactones were tested on adults of Sitophilus granarius, adults and larvae of Tribolium confusum and larvae of Trogoderma granarium. The isomeric starting natural compounds, (+) and (−) pulegones and (+) and (−) isopulegols, were also tested. The results showed that the introduction of the lactone moiety into the p-menthane system produced antifeedant activity in the lactones obtained. The lactones with a spiro arrangement of lactone and cyclohexane rings were more active than those with condensed rings. The configuration of chiral centres present in the molecule significantly influenced the activity of the compounds studied. In most cases, lactones obtained from R-(+)-pulegone were more active antifeedants than those obtained from S-(−)-pulegone.     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.