Novel method for estimating viable Salmonella cell counts using real-time PCR

A novel method for estimating viable Salmonella Enteritidis cell counts with 5'-nuclease real-time PCR was developed in this study. Our method was based on the increase kinetics of the target DNA region (invA) of the microorganism growing in a food/clinical sample in a culture medium during incubation. The index of increase in the target DNA region studied here was threshold cycle, CT. A test Salmonella strain was grown in buffered peptone water at the optimal temperature (39 degrees C). As Salmonella cells were grown, the value of CT decreased with time, generating a downward sigmoidal curve. The slope of the curve was constant at various initial cell concentrations. With higher initial cell concentration, the CT value evaluated from the slope at a given time was lower. With this relationship, a novel method for estimating the initial viable cell concentration of a sample was developed. Dead Salmonella cells or bacteria other than the target cell caused deviation in the CT curve. Incubation in a selective media suppressed the deviation caused by other bacterial cells. We think that this method could be applied to many other microorganisms cultivable in a suitable medium.     

Estimation of bacterial concentrations in commercial foods by measuring ATP changes during incubation

Recently we have developed new kinetic methods of bacterial cell estimation in food products by measurement of the increase in bacterial adenosine-triphosphate (ATP) content during incubation using a conventional firefly luminometer [Fujikawa and Morozumi: Jpn. J. Food Hyg. (2002)]. The methods were the end point method and the delay method. We, in this study, evaluated these methods for bacterial concentration estimation in commercial foods. The methods were successfully applied to food products inoculated with Escherichia coli or Staphylococcus aureus. The methods were then used to estimate bacterial concentrations in commercial foods. For estimation, the kinetic parameter values were determined first from the data of bacterial strains studied previously. The bacterial concentrations estimated with these parameter values were slightly smaller than the measured concentrations for most of the food samples. The parameter values obtained with the least-squares method gave better estimations in both methods. The delay method predicted the bacterial concentrations in food products better than the end point method. This study showed that with appropriate parameter values, these methods could be useful tools for bacterial concentration estimation of commercial foods.     

大黄鱼源腐败菌的黏附特性与生物膜特性分析

以分离纯化自冰鲜大黄鱼的3 株希瓦氏菌（MA1-5、MA1-7、MA1-13）和3 株假单胞菌（R3-1、R3-2、R3-5）为供试菌株,研究鱼源腐败菌的表面疏水性、自凝聚能力、生物膜和腐败菌对鱼体（鱼肠、鱼鳃和体表）黏液的黏附能力,探明黏附特性与不同部位黏附能力的相关性,并进一步研究生物膜在不同因素下的形成特性。研究表明,希瓦氏菌具有更强的表面疏水性和自凝聚能力,对鱼体黏液的黏附性强,并具有更强的生物膜形成能力。主成分分析和聚类分析说明黏附能力在属间存在差异性,属内具有相似性。腐败菌对鱼肠和鱼鳃的黏附能力与自凝聚能力和生物膜形成能力具有较高的相关性,对体表黏附能力只与生物膜形成能力有关。腐败菌生物膜的形成受初始菌浓度、培养时间、温度、pH值、盐含量等多种环境因素影响。希瓦氏菌在初始菌浓度106 CFU/mL以上、37 ℃、pH 7～8、0.8% NaCl时形成的生物膜量最多;生物膜在12～24 h期间快速形成,并在36 h达到峰值后,随培养时间延长而逐渐下降。经鱼体黏液包被后,生物膜形成量受到显著影响,鱼鳃黏液促进生物膜形成,鱼肠黏液抑制生物膜形成。     

Modelling the growth parameters of lactic acid bacteria and total viable count in vacuum‐packaged Irish cooked sausages cooled by different methods

The growth kinetics of lactic acid bacteria (LAB) and total viable count (TVC) in cooled sausages as a function of storage time was studied. Cooked sausages were cooled either by commercial cooling or by immersion vacuum cooling (IVC), then vacuum‐packaged and stored for up to 71 days at 4 °C. Baranyi model was used to fit the growth of LAB and TVC. Growth curve, growth rate, lag time and initial and final cell concentrations for LAB and TVC were calculated through DMFit. The coefficient of determination and root‐mean‐squared error of the Baranyi models were used to evaluate its accuracy. Sausages cooled by IVC had a longer lag time and shelf life period than commercial cooled sausages. Accuracy analysis showed that the Baranyi model was adequate for representing the bacterial growth of vacuum‐packaged cooled sausages and had a potential for shelf life prediction.     

A new logistic model for Escherichia coli growth at constant and dynamic temperatures

A new logistic model for bacterial growth was developed in this study. The model, which is based on the logistic model, contains an additional term for expression of the very low rate of growth during a lag phase, in its differential equation. The model successfully described sigmoidal growth curves of Escherichia coli at various initial cell concentrations and constant temperatures. The model predicted well the bacterial growth curves, similar to the Baranyi model and better than the modified Gompertz model, especially in terms of the rate constant and the lag period of the growth curves. Using the experimental data obtained at the constant temperatures, the new logistic model was studied for growth prediction at a dynamic temperature. The model accurately described E. coli growth curves at various patterns of dynamic temperature. It also well described other bacterial growth curves reported by other investigators. These results showed that this model could be a useful tool for bacterial growth prediction from the temperature history of a tested food.     

以豆粕为基质纳豆芽孢杆菌生长条件优化

研究纳豆芽孢杆菌在以豆粕为基质的发酵培养基中的生长条件。利用全自动生长曲线测定仪,以菌液浊度OD660nm为指标,对纳豆芽孢杆菌液体发酵培养基中的氮源(豆粕)和碳源(葡萄糖)配比进行研究,确定豆粕质量浓度50g/L,葡萄糖质量浓度10g/L为最佳配比,并通过单因素及正交试验筛选出影响纳豆芽孢杆菌生长的主要条件。再根据Box-Behnken试验优化,结果表明,采用功率80W超声波处理豆粕4.32min,且培养基初始pH6.16,发酵温度35.5℃时纳豆芽孢杆菌在发酵生长13h后达到最大生物量,此时OD660nm为1.635。     

纳豆芽孢杆菌Bacillus natto 16-1对小麦粉中呕吐毒素的脱毒机制

采用酶联免疫吸附法(ELISA)及高效液相色谱—质谱(HPLC-mass spectrometry,HPLC-MS)检测纳豆芽孢杆菌Bacillus natto 16-1各细胞成分对小麦粉中呕吐毒素(deoxynivalenol,DON)的清除效果及降解产物,探讨纳豆芽孢杆菌清除呕吐毒素的效果及降解机制。结果显示,培养5h后不同浓度的菌悬液、发酵上清液、全菌裂解液、粗提酶液以及细胞壁悬浮液,对DON清除率均在53%以上,其中不同浓度的菌悬液处理间存在显著差异,高浓度细胞壁悬浮液处理对DON清除率高达62.85%;培养10h后不同菌体成分对DON的清除率在57%以上,其中不同浓度发酵上清液、菌体裂解液以及细胞壁悬浮液处理间存在显著差异。除细胞壁处理组外,其他各组均可将DON降解为分子质量较小的物质。结果表明,纳豆芽孢杆菌Bacillus natto 16-1各细胞成分对DON均有清除作用,且这种作用在一定程度与细胞成分的浓度及作用时间有关;各细胞成分对DON的清除机制主要是生物降解作用,细胞壁可能是通过生物吸附作用来清除DON。     

Fungal lipids. II. Effects of varying concentration of nitrogen in the medium and incubation period on the chemical composition of lipids of Pythium irregulare Buisman

Effects of varying the concentration of nitrogen in the medium and the incubation period on the chemical composition of lipids of Pythium irregulare Buisman were studied. In general, with low C/N ratios, the growth of mycelium and the lipid synthesis were found to increase up to the eleventh day of incubation after an initial lag period of five days. Thereafter only slight changes were observed. The fatty acid composition with low C/N ratios showed a considerable variation throughout the growth period. With high C/N ratios, the lipid content continued to increase during the whole period of growth. During this phase of growth, a rapid synthesis of triglycerides along with a marginal increase in the polar fraction was observed. In all the non-polar lipid classes, oleic and palmitic were the major fatty acid components. Free fatty acid fractions comprised mainly of palmitic, oleic and arachidic acids. In all polar lipid fractions, except phosphatidyl choline and phosphatidic acids, oleic and palmitic were the principal acids. By argentation thin-layer chromatography, eight mixed triglyceride classes were observed.     

野葛细胞固定化培养及异黄酮化合物的生产

以海藻酸钠固定化处理的野葛细胞为试材,研究了固定化细胞培养周期中细胞生长、糖源消耗和异黄酮含量的变化。结果表明,野葛细胞生长曲线呈“S”形,可分为3个主要阶段,即迟滞期(0~8 d)、指数生长期(8~14 d)和稳定增长期(14~18 d)。在培养的第18天达到最大生物量(48.56 g(FW)/L),约为初始接种量的3.04倍。同时,糖源随细胞生长量的增加而不断消耗。在野葛细胞培养周期中,固定化细胞胞内黄酮和培养液黄酮逐渐累积,培养体系中的总黄酮含量与细胞生长量呈同步增长的关系,第16天,总黄酮含量约为初始培养时的17.28倍。试验结果为采用野葛细胞固定化培养的方法进行异黄酮化合物的生产提供了实验依据。     

巨大芽孢杆菌发酵降解亚硝酸盐动力学研究

研究了在不同初始亚硝酸钠和葡萄糖浓度条件下,巨大芽孢杆菌B.megaterium MPF-906细胞生长和降解亚硝酸盐的发酵动力学.结果发现,初始亚硝酸盐浓度对细胞生长有重要影响,初始亚硝酸盐浓度为2mmol/L的环境最利于细胞增殖,此时亚硝酸盐的消耗速率也最大,发酵液中亚硝酸盐消耗曲线和细胞生长曲线表现出互为对应的关系;低浓度诱导物亚硝酸钠的环境也更有利于细胞生产亚硝酸还原酶(NiR).初始葡萄糖浓度对细胞生长也有重要影响,初始葡萄糖浓度为2.0％的环境最利于细胞增殖,此时细胞生成速率最大,初始较高的葡萄糖浓度时,亚硝酸盐的降解速率减小,但亚硝酸盐降解率仍可在14h内达到100％;初始葡萄糖浓度为2.0％时,糖耗速率最快;较高浓度葡萄糖的环境也有利于细胞生产NiR.     

红豆皮多酚提取物对两种致病菌的抑菌活性及作用机理

多酚类化合物具有一定的抑菌活性，为探究红豆皮多酚对两种致病菌的抑菌机理，本实验采用高效液相色谱法确定红豆皮多酚的组成。通过最小抑菌浓度（minimum inhibitory concentration，MIC）和抑菌生长曲线分析红豆皮多酚提取物对李斯特菌ATCC19119和沙门氏菌ATCC14028的抑菌效果，并从菌体细胞蛋白和核酸含量、细胞膜电位、细胞内ATP含量、细胞外碱性磷酸酶活力和菌体细胞形态变化的角度来揭示其抑菌机理。结果表明，红豆皮多酚组成成分中含有13 种酚类化合物，红豆皮多酚提取物对李斯特菌ATCC19119和沙门氏菌ATCC14028的MIC分别为625 μg/mL和2 500 μg/mL，红豆皮多酚提取物的添加对菌体细胞蛋白和核酸含量、细胞膜电位、细胞内ATP含量、细胞外碱性磷酸酶含量和菌体细胞形态变化等有明显影响。综上，本研究结果可为粮食源天然抑菌产品的开发和粮食加工副产物的综合利用提供理论参考。     

Flow Characteristics of Soybean Constituents Controlled by Ratio of Total to Imbibed Water

The objective was to quantify the relationship between water imbibing capacity of soybean components and their rheological characteristics in suspension. These components were: full fat soy flour, desludged full fat soy flour, defatted soy flour, soybean cell wall material and soybean sodium proteinate. It was found that the relationship between the ratio of the total to imbibed water (T/I) and consistency coefficient for all components at all concentrations could be expressed by a single curve. Flow behavior index vs T/I for all components also showed a single curve. A plot of apparent viscosity against T/I at the maximum shear rate showed that the data for all components at all concentrations fell on the same curve at a given shear rate.     

小米粥油的制备及抑菌和吸附内毒素作用研究

本文以抑菌圈大小为评价指标,采用正交法优化小米粥油制备工艺;真空冷冻干燥技术制备小米粥油粉。采用牛津杯法测定小米粥油对金黄色葡萄球菌、伤寒沙门氏菌、大肠埃希菌、痢疾志贺菌、肺炎克雷伯菌的抑制活性。平板涂布法确定其对供试菌种的最低抑菌浓度(MIC),评估其抑菌能力,同时通过测定生长曲线、核酸含量分析、电导率变化,探究了其抑菌机理;使用鲎试剂显色基质法检验小米粥油粉吸附细菌内毒素作用。结果表明：小米粥油在浸泡时间30 min,料液比1:20(g/mL),熬煮时间25 min条件下,小米粥油对致病菌的抑菌圈最大;小米粥油对金黄色葡萄球菌、痢疾志贺菌和肺炎克雷伯菌有一定的抑菌作用,小米粥油对金黄色葡萄球菌、痢疾志贺菌和肺炎克雷伯菌的最低抑菌浓度(MIC)即料液比均为1:20;小米粥油对供试菌体生长具有抑制作用,且粥油会使细菌胞体内物质外泄导致菌悬液核酸含量增加、菌悬液电导率增加、菌体死亡;因此小米粥油具有良好的抑菌能力。在最佳工艺条件与真空冷冻干燥技术下制备的小米粥油粉具有良好的吸附内毒素效果。     

Autolytic methods for the reduction of the purine content of baker's yeast,a form of single-cell protein

When a suspension of baker's yeast was incubated at 50°C, the cells became permeable to H⁺, a process completed in about 4 h. Cellular constituents of low molecular weight then diffused into the medium, but only after a long lag period were cell nucleic acids and proteins degraded by intracellular enzymes. The lag period was shorter at higher temperatures, when RNase and proteinases suffered some thermal denaturation. It was abolished if, before autolysis at 50°C, yeast was plasmolysed by ethyl acetate, or heated to 70°C for 1 min, treatments which, it is thought, disorganised the membrane of the yeast cell vacuole, within which the enzymes are located. Heat treatment, when effective, was sufficiently severe to disorganise the yeast cell plasma-lemma, with the consequence that a single adjustment of pH immediately following heat treatment served to maintain a constant pH, without addition of buffer, during the subsequent incubation at 50°C. Autolysis was not found to be an entirely suitable procedure for the reduction of the nucleic acid content of baker's yeast, because of the concomitant and severe loss of protein.     

Prolonged incubation period of salmonellosis associated with low bacterial doses

In gastroenteritis outbreaks caused by Salmonella-contaminated lunches at elementary, junior high, and nursery schools. outbreaks with long median incubation periods (i.e., 60 to 120 h) were observed frequently between 1990 and 1999 in Japan. We analyzed epidemiological data on 185 outbreaks of Salmonella Enteritidis infection to study the factors underlying the long incubation period. These survey results showed that the median incubation period for Salmonella Enteritidis infection from contaminated school and nursery school lunches was significantly longer than that from other types of cooking facilities. In addition, we analyzed the relationship between the median incubation period and the bacterial dose ingested per person in nine outbreaks of Salmonella Enteritidis infection; the bacterial dose was estimated with reference to the bacterial concentration in the causative foods. A significant negative correlation between the bacterial dose ingested per person and the median incubation period is clearly shown. The time elapsed from the start of the cooking process to the consumption of school and nursery school lunches was significantly shorter than at other cooking facilities, suggesting limited bacterial growth, which in turn is thought to lead to a long incubation period.     

不同贮藏条件下罗氏沼虾细菌总数生长数学模型的研究

本实验主要研究了贮藏在不同温度、初始pH和初始NaCl%浓度条件下罗氏沼虾细菌总数的生长情况。实验结果表明:其中建立的Gompertz方程拟合的相关系数R2>0.95,偏差度Bf和准确度Af在0.70～0.90及1.06～1.15之间。依据F值和显著性判断,环境因子对细菌总数生长特征参数的影响大小顺序为:温度>初始NaCl%浓度>初始pH值。在延滞期的响应面方程模型中,温度与初始NaCl%浓度的一次、二次效应和它们之间的交互效应对延滞期都有显著性的影响。     

醋酸对巴氏醋杆菌生长和代谢活性的影响

醋酸对微生物具有较高的毒性,醋酸菌具有特殊的机制能够耐受较高浓度的醋酸,常用于醋酸的发酵生产。本论文以巴氏醋杆菌AC2005为研究对象,研究了醋酸对菌体生长、葡萄糖代谢和胞内ATP浓度的影响。与不含醋酸条件下相比,培养基中加入1%(V/V)的醋酸,菌体的生物量增加了92%,单位菌体葡萄糖消耗速率增加了13%,细胞代谢活性增加了120%,代谢产物中丙酮酸浓度增加了约46%,柠檬酸和琥珀酸的浓度分别降低了80%和46%,胞内ATP浓度增加了171%。进一步利用液相色谱串联质谱法(LC-MS/MS)的方法分析了蛋白表达情况,发现加入醋酸后细胞内与三羧酸循环相关的蛋白明显上调。结果表明,醋酸菌可以通过提高菌体代谢活性,增加胞内ATP浓度,从而耐受较高浓度的醋酸。     

微酸性电解水对肠炎沙门氏菌的杀菌机制初探

为研究微酸性电解水(Slightly Acidic Electrolyzed Water,SAEW)对肠炎沙门氏菌的杀菌机制,本文探讨了SAEW对肠炎沙门氏菌细胞膜通透性、细胞膜完整性、胞内活性氧(Reactive Oxygen Species,ROS)及胞内ATP等的影响。试验结果显示,有效氯浓度为10 mg/L的SAEW处理1 min后,可杀灭鸡蛋表面肠炎沙门氏菌2.04 lg(CFU/g);与对照组相比,SAEW处理组菌悬液的电导率升高,由13.78±0.13 ms/cm显著增加到16.32±0.10 ms/cm(P<0.05),蛋白质泄漏量增加,由0.07±0.04 g prot/L显著增长至0.30±0.03 g prot/L(P<0.05),核酸泄漏量增加,ROS积累量增加,胞内ATP含量降低。扫描电镜(Scanning Electron Microscope,SEM)显示,经SAEW处理后,肠炎沙门氏菌的细胞壁结构不完整,遭到破坏,有明显的穿孔现象,且许多细菌表面凹陷变形或破碎粘连。试验结果表明,SAEW能够显著提高肠炎沙门氏菌细胞膜的通透性并严重破坏细胞膜的...     

香芹酚对阴沟肠杆菌的抑菌作用及机理

香芹酚具有较好的抑菌效果,符合人们对食品防腐剂安全无毒、无副作用的要求。本研究通过最小抑菌浓度和杀菌实验分析了香芹酚对阴沟肠杆菌（Enterobacter cloacae C4）的抑菌活性,并检测其对阴沟肠杆菌细胞膜电势、胞外ATP化学发光值的影响,使用激光共聚焦显微镜观察细胞渗透性,并通过扫描电子显微镜观察香芹酚对阴沟肠杆菌细胞形态的影响,从而探究香芹酚对阴沟肠杆菌的抑菌机理。结果表明：香芹酚对阴沟肠杆菌的最小抑菌浓度为0.5 μL/mL;阴沟肠杆菌细胞膜通透性随着香芹酚含量（0.25～2 μL/mL）的增加而逐渐增加。经香芹酚处理的阴沟肠杆菌细胞膜内外电势改变,ATP大量漏出。通过扫描电子显微镜观察,发现经香芹酚处理的菌体细胞形态干瘪且明显被破坏,含量高于1 μL/mL的香芹酚几乎能够溶解菌体。推测香芹酚通过作用于细胞膜改变膜内外电势,造成细胞膜通透性改变,引起细胞ATP等内容物泄露,从而影响细胞功能,达到抑菌的作用。