臭氧水与副干酪乳杆菌Z21联合处理对绿豆芽中大肠杆菌O157:H7的抑制机制

为研究臭氧水联合副干酪乳杆菌Z21发酵上清液对绿豆芽中大肠杆菌O157:H7的杀菌效果、细胞结构影响和生物膜清除作用,本实验对人工污染大肠杆菌O157:H7的绿豆芽进行联合处理,选出最优的杀菌条件,采用流式细胞仪、扫描电镜、傅里叶红外光谱（Fourier-transform infrared spectroscopy,FT-IR）、拉曼光谱分析臭氧水联合Z21发酵上清液的杀菌机制;通过菌落计数及胞外聚合物分析,研究了臭氧水联合Z21发酵上清液对大肠杆菌O157:H7生物膜的清除效果。结果表明,1.5 mg/L臭氧水联合10%（v/v）Z21发酵上清液处理对大肠杆菌O157:H7杀菌效果最佳,菌落总数减少了2.81 lg CFU/g;与对照组相比,联合处理破坏了大肠杆菌O157:H7细胞壁和细胞膜中的多糖,脂质和蛋白质结构,增加了细胞膜的通透性,改变了菌体形态。联合处理对生物膜有良好的清除效果,显著降低了生物膜的胞外聚合物含量（P<0.05）。本研究为大肠杆菌生物膜的清除及农产品防腐保鲜提供了理论依据。     

乳酸菌对肠出血性大肠杆菌O157:H7抑制作用的研究

为探讨乳酸菌对肠出血性大肠杆菌O157：H7 ATCC43895(E．Coli O157：H7)的抑制作用，在培养基上进行了研究。将E．Coli O157：H7与干酪乳杆菌干酪亚种、植物乳杆菌、发酵乳杆菌、乳酸乳球菌和瑞士乳杆菌同时接种在培养基中，E．Coli O157：H7的活性不受影响；将E．Coli O157：H7接种到培养了24h的乳酸茵培养液中，E．Coli O157：H7活性显下降。以乳酸调整的低pH值对E．Coli O157：H7有一定的杀灭作用。本研究表明：乳酸菌的代谢产物乳酸对E．Coli O157：H7有杀灭作用。     

流式细胞术在超高压诱导大肠杆菌O157:H7亚致死研究中的应用

超高压作为一种应用日趋广泛的食品非热杀菌技术,对食源性大肠杆菌O157:H7有显著的致死作用,但对 其致死效应缺乏快速有效的评价手段,对其致死机制也需进一步证实。本研究运用流式细胞术结合传统培养方法, 评价了超高压对大肠杆菌O157:H7的致死效应,并通过扫描和透射电子显微镜技术观察分析了超高压处理后菌体细 胞微观结构的变化,初步探讨了超高压对大肠杆菌O157:H7的致死机制。结果表明：双染色流式细胞术检测方法能 够快速准确地对超高压处理后不同生理状态的大肠杆菌O157:H7细胞进行分析和分选,且具有较高灵敏度和精确 度;400 MPa的超高压处理对大肠杆菌O157:H7有明显的损伤和致死效果,菌体细胞出现严重变形,部分细胞的细 胞膜出现破裂;500 MPa处理时,致死效应更加明显,菌体细胞壁和细胞质膜严重分离,光透明区出现大量辐射丝 状物。菌体细胞膜完整性丧失很可能是超高压诱导大肠杆菌O157:H7致死的主要原因,即使经过500 MPa的超高压 处理,依然有不少大肠杆菌O157:H7菌体细胞处于亚致死状态,这部分亚致死状态的细菌是食源性疾病爆发的潜在 隐患。     

超高压对大肠杆菌O157:H7细胞膜的损伤效应

本研究旨在揭示超高压对食源性致病微生物大肠杆菌O157:H7细胞膜的损伤。研究了200、400、500 MPa不同压力对大肠杆菌O157:H7的灭活作用,通过对菌体细胞核酸类物质、钾离子和镁离子泄漏量、碘化丙啶(propidium iodide,PI)摄入量、细胞膜Na+/K+-ATP酶和Ca~(2+)/Mg~(2+)-ATP酶活性变化的分析研究,评价不同超高压处理压力对大肠杆菌O157:H7膜损伤效应。结果表明,经200、400 MPa压力处理5 min后,大肠杆菌O157:H7菌落总数由初始8.8(lg(CFU/m L))分别下降至8.2(lg(CFU/m L))和6.3(lg(CFU/m L)),500 MPa压力处理后,大肠杆菌O157:H7全部死亡。压力升高,细菌细胞内核酸类物质、K+、Mg~(2+)离子泄漏量、PI摄入量均显著增加,细胞膜上Na+/K+-ATP酶和Ca~(2+)/Mg~(2+)-ATP酶活性显著降低。Ca~(2+)/Mg~(2+)-ATP酶对压力的敏感性更强,500 MPa处理组该酶活性几乎完全丧失。超高压处理引起大肠杆菌O157:H7细胞膜产生显著损伤,细胞膜上Ca~(2+)/Mg~(2+)-ATP酶的失活是导致大肠杆菌O157:H7死亡的主要原因。     

植物乳杆菌对食源性病原菌作用的研究

通过牛津杯法筛选得到几株高抑菌活性的植物乳杆菌,使用蛋白酶K和NaOH对这几株杆菌的发酵液上清进行处理,以大肠杆菌O157:H7和金黄色葡萄球菌为指示菌,发现有机酸为主要抑菌物质,但是调节pH值至6.5后的发酵上清液可以对大肠杆菌有较好的抑菌效果。其中植物乳杆菌QB3-3具有长达96 h的持续抑菌效果。电子显微镜观察显示,植物乳杆菌抗病原菌的主要机理为破坏细胞结构,导致病原菌菌体形态改变,内容物流出,致使细胞死亡。     

原料乳中大肠杆菌O157:H7的快速检测

建立了一种快速检测原料乳中大肠杆菌O157:H7的PCR技术.该方法利用过滤富集菌体后的PCR技术来检测原料乳中大肠杆菌O157:H7,先对人工污染大肠杆菌O157:H7的原料乳进行离心脱脂,然后添加EDTA-2Na获得澄清乳液,最后通过0.45 μm微膜过滤收集菌体,整个过程只需6 h左右即可完成.检测灵敏度高达10-mL-1.这种方法在传统检测方法的基础上做了有效改进,使得原料乳中的大肠杆菌O157:H7的检测能够快速、准确、灵敏的进行.     

干酪乳杆菌发酵液中的主要有机酸及其抑菌性

干酪乳杆菌在食品生物防腐方面起着重要的作用。为研究干酪乳杆菌发酵产生的有机酸的分离方法及其抑菌活性,采用乙酸乙酯萃取干酪乳杆菌的培养上清液,通过旋转蒸发仪对各萃取相进行浓缩,并采用spot-on-lawn改进法研究其抑菌活性,利用高效液相色谱(HPLC)分析萃取液中的主要有机酸成分。结果表明,乙酸乙酯相旋蒸余相对大肠杆菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特菌三种食源性致病菌均表现出很好的抑菌效果,且对大肠杆菌O157:H7和单核细胞增生李斯特菌的抑制最为明显,抑菌圈直径分别达到(20.00±0.82)、(21.67±0.94) mm,其主要的抑菌有机酸组分为乳酸、乙酸和苯乳酸。该研究为分离乳酸菌发酵液中有效活性抑菌物质提供了一种确实可行的方法。     

大肠杆菌O157:H7耐酸性及酸胁迫下菌体形态变化的初步研究

目的 对大肠杆菌O157:H7 耐酸性进行初步探讨。方法 通过稀释涂布平板计数法观察大肠杆菌O157:H7 在不同的pH 生长条件下的存活能力和利用电镜扫描法观察其在酸胁迫下菌体形态的变化。结果 当pH 为5.0 到7.0 之间时, 大肠杆菌O157:H7 生长状况良好, 当pH 小于4.0 时, 其生长受到抑制, 特别是pH 降到2.0 以下时, 大肠杆菌O157:H7 完全不能生长, 由此可以说明大肠杆菌O157:H7 耐酸性能力较强, 可以抵御酸性环境的影响。扫描电镜图显示大肠杆菌O157:H7 在不同pH 的环境条件下其菌体形态会作出相应的变化,随着培养基的酸性增强, 其细胞形态从长杆菌体形态变成了短杆状或钝圆形态。结论 本研究测定不同酸性条件下大肠杆菌O157:H7 的生存和繁殖能力的研究, 有利于帮助人们进一步了解其耐酸性, 从而为制定防治方案和措施提供参考。     

乳酸和过氧乙酸对大肠杆菌O157:H7生物膜的抑制作用及其机制

研究乳酸和过氧乙酸对大肠杆菌O157:H7生物膜的抑制作用,并从代谢活性、胞外聚合物质量浓度、生物膜的微观结构以及相关功能基因的表达等方面探究乳酸和过氧乙酸对大肠杆菌O157:H7生物膜的抑制机理。结果发现：乳酸和过氧乙酸的最小抑菌剂量（minimum inhibitory concentrations,MIC）分别为0.25%和0.002 5%;通过活菌计数测定分析不同剂量乳酸和过氧乙酸对大肠杆菌O157:H7生物膜抑制效果,发现在亚MIC（1/2 MIC和1/4 MIC）下乳酸和过氧乙酸的生物膜抑制作用较弱,而MIC下两种有机酸均可显著抑制大肠杆菌O157:H7生物膜的形成（P＜0.05）,2 MIC可完全抑制大肠杆菌O157:H7生物膜形成;经MIC的乳酸和过氧乙酸处理后,大肠杆菌O157:H7生物膜的代谢活性分别降低了84.25%和43.49%,而胞外聚合物质量浓度显著减少（P＜0.05）;乳酸和过氧乙酸均有效抑制了大肠杆菌O157:H7的黏附相关基因（csgA、flhC）、群体感应相关基因（luxS、sdiA）、胞外多糖合成相关基因（csrA、adrB、adrA）以及双组分调控系统相关基因（phoQ、phoP、envZ、ompR）。综上所述,乳酸和过氧乙酸可以作为食品工业中有效的杀菌剂,降低食品生产和加工过程中相关微生物污染的风险。     

等离子体活化水-苯乳酸协同杀灭大肠杆菌O157:H7作用及机制研究

拟研究等离子体活化水（Plasma-activated water,PAW）与苯乳酸（Phenyllactic acid,PLA）协同处理对大肠杆菌O157:H7的杀灭效果及其作用机制。采用平板计数、扫描电镜、荧光染色等方法研究PAW与PLA协同处理对大肠杆菌O157:H7的杀灭作用及其对细胞形态、细胞膜完整性和胞内活性氧水平等的影响。结果表明,经终浓度为0.125～1.0 mg/mL的PLA处理8 min后,大肠杆菌O157:H7活细胞数未发生显著变化（P>0.05）。经PAW与PLA（终浓度为1.0 mg/mL）协同处理8 min后,大肠杆菌O157:H7降低了5.65 lg CFU/mL,显著高于PAW单独处理组（降低了1.06 lg CFU/mL） （P<0.05）。扫描电镜结果表明,PAW与PLA协同处理可造成细胞形态发生明显变化。与对照组细胞相比,经PAW-PLA（1.0 mg/mL）协同处理8 min后,胞外蛋白含量、细胞膜电位和胞内活性氧分别升高了25.6、0.75和9.53倍（P<0.05）。综上所述,PAW与PLA协同处理能够有效杀灭大肠杆菌O157:H7,这可能与其破坏细胞膜及诱导氧化损伤等有关。本研究为PAW与PLA协同处理在食品保鲜中的应用提供了理论依据。     

Survival of Salmonella spp. and Escherichia coli O157:H7 on fresh and frozen strawberries

For maximum shelf life, fresh strawberries are harvested directly without washing into retail containers. Frozen berries are usually hulled in the field and washed prior to freezing, sometimes with the addition of sucrose. To determine survival of potential bacterial contaminants, cut or intact surfaces of fresh strawberries were spot inoculated with five- or six-strain cocktails of Salmonella or Escherichia coli O157:H7 (log 7.0 CFU/sample). Inoculated strawberries were dried for 1 h at 24 degrees C and were stored in closed containers at 5 or 24 degrees C. Sliced strawberries with or without added 20% sucrose were inoculated with one of two strains of E. coli O157:H7 and frozen at -20 degrees C. An initial population reduction of approximately 0.5-log cycles was observed on intact but not cut berries after the 1-h drying period. During storage at 24 degrees C for up to 48 h, populations of Salmonella and E. coli O157:H7 did not decline further. When strawberries were stored at 5 degrees C for up to 7 days, populations of both pathogens remained constant on cut surfaces but decreased by 1 - to 2-log cycles on intact surfaces. After 30 days of frozen storage, the population of E. coli O157:H7 had declined by 0.7- to 2.2-log cycles (with and without sucrose, respectively). Results of this study indicate that E. coli O157:H7 and Salmonella are capable of survival but not growth on the surface of fresh strawberries throughout the expected shelf life of the fruit and can survive in frozen strawberries for periods of greater than 1 month.     

Isolation,selection, and characterization of lactic acid bacteria for a competitive exclusion product to reduce shedding of Escherichia coli O157:H7 in cattle

Lactic acid bacteria (LAB) were selected on the basis of characteristics indicating that they would be good candidates for a competitive exclusion product (CEP) that would inhibit Escherichia coli O157:H7 in the intestinal tract of live cattle. Fecal samples from cattle that were culture negative for E. coli O157:H7 were collected. LAB were isolated from cattle feces by repeated plating on deMan Rogosa Sharpe agar and lactobacillus selection agar. Six hundred eighty-six pure colonies were isolated, and an agar spot test was used to test each isolate for its inhibition of a four-strain mixture of E. coli O157:H7. Three hundred fifty-five isolates (52%) showed significant inhibition. Seventy-five isolates showing maximum inhibition were screened for acid and bile tolerance. Most isolates were tolerant of acid at pH levels of 2, 4, 5, and 7 and at bile levels of 0.05, 0.15, and 0.3% (oxgall) and were subsequently identified with the API system. Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus delbreukii, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus cellobiosus, Leuconostoc spp., and Pediococcus acidilactici were the most commonly identified LAB. Nineteen strains were further tested for antibiotic resistance and inhibition of E. coli O157:H7 in manure and rumen fluid. Four of these 19 strains showed susceptibility to all of the antibiotics, 13 significantly reduced E. coli counts in manure, and 15 significantly reduced E. coli counts in rumen fluid (P < 0.05) during at least one of the sampling periods. One of the strains, M35, was selected as the best candidate for a CEP. A 16S rRNA sequence analysis of M35 revealed its close homology to Lactobacillus crispatus. The CEP developed will be used in cattle-feeding trials.     

INHIBITION OF LISTERIA MONOCYTOGENES AND OTHER BACTERIA BY A PLANT ESSENTIAL OIL (DMC) IN SPANISH SOFT CHEESE

The inhibitory effects of a mixture of plant essential oils (DMC) were tested in culture media and Spanish soft cheese. The minimum inhibitory concentration (MIC) was determined in plate count agar and tryptose broth, with pH adjusted to 6.5, against Listeria monocytogenes, Listeria innocua, Staphylococcus aureus, Lactobacillus brevis, Micrococcus luteus, Salmonella cholerasuis, Escherichia coli O157:H7 , Enterobacter cloacae, Pseudomonas aeruginosa and Pseudomonas fluorescens. The mixture of essential oils inhibited all Gram-positive bacteria tested at 40 ppm, but higher concentrations were needed to inhibit Gram-negative bacteria, and no inhibitory effect was found against Ps. fluorescens. The effects of DMC against L. monocytogenes and E. coli O157:H7 were evaluated in Spanish soft cheese (Queso Fresco, pH=6.5) stored at 7C. DMC had a bacteriostatic effect against L. monocytogenes at concentrations of 2500 ppm but was ineffective to control the growth of E. coli O157:H7 .     

Evaluation of a polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.     

Cell age, suspending medium and metal ion influence the susceptibility of Escherichia coli O157:H7 to water-soluble maltose chitosan derivative

The effects of cell age, suspending medium and metal ions on the susceptibility of Escherichia coli O157:H7 to the water-soluble maltose chitosan derivative were investigated. In addition, the leakage of glucose, protein (absorbance at 280 nm) and lactate dehydrogenase (LDH) induced by maltose chitosan derivative in saline solution and deionized water, was examined. Cells of E. coli O157:H7 in the mid-exponential phase (6 h) were most susceptible to the chitosan derivative followed by cells in the late-exponential phase (12 h) and stationary phase (24 h). In addition, it was found that the susceptibility of the test organism to the maltose chitosan derivative was less in saline solution than in deionized water. The viable population of E. coli O157:H7 in deionized water containing the maltose chitosan derivative (500 ppm), was reduced from ca 7.6 log cfu/ml to a non-detectable level after 10 h of incubation at 37 degrees C compared to a viable population of ca 6.2 log cfu/ml noted in the chitosan derivative-containing-saline solution. After cells of E. coli O157:H7 were exposed to the chitosan derivative in deionized water, a marked increase in the levels of glucose, protein and LDH activity was observed in the supernatant of cell suspension compared to cells of test organism exposed to the saline solution containing chitosan derivative. Metal ions were also found to reduce the antibacterial activity of chitosan derivative. Their effectiveness increased at greater concentrations and varied with the kinds of metal ions with Ba(2+) the most effective and Mg(2+) the least effective.     

Effects of recovery, plating, and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries

Effects of different recovery and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries were studied. Strawberries were spot or dip inoculated with 7 to 8 log CFU per strawberry of each pathogen, air dried for 2 h, and stored for 1, 3, and 7 days at 4 degrees C. The inoculated samples were stomached or washed with phosphate-buffered saline (PBS; pH 7.2) or with modified PBS (pH 8.4). Bacterial levels were determined using a direct selective plating, thin agar layer plating, or membrane-transferring plating (MTP) with tryptic soy agar and sorbital MacConkey agar (E. coli O157:H7) or modified Oxford agar (L. monocytogenes). Under most test conditions, washing with PBS followed by MTP had significantly higher (P < 0.05) recovery for both bacteria compared with other tested methods. Within a 7-day storage period for spot-inoculated strawberries, a stomaching step resulted in an injury of 0.9 to 1.4 log CFU for E. coli O157: H7 and 1.4 to 1.7 log CFU for L. monocytogenes. When a washing step was used instead, this resulted in an injury of only 0.2 to 0.6 log CFU for E. coli O157:H7 and 0.2 to 0.7 log CFU for L. monocytogenes. Both bacteria could survive on strawberry surfaces, but their recovered levels decreased with the increase of storage time at 4 degrees C for both spot and dip inoculation methods. Dip inoculation generally had a lower recovery than spot inoculation. An ideal protocol to recover and enumerate E. coli O157:H7 and L. monocytogenes from strawberries involved shaking and washing samples with 100 ml of PBS for 15 min at 22 degrees C coupled with a MTP enumeration method.     

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.     

Antimicrobial activity of lactoferrin against foodborne pathogenic bacteria incorporated into edible chitosan film

The objectives of this research were to develop and characterize edible chitosan film containing lactoferrin as a natural antimicrobial agent, and to investigate the combination effects of lactoferrin with lysozyme in chitosan film against the growth of Escherichia coli O157:H7 and Listeria monocytogenes. Chitosan films containing lactoferrin, lysozyme, or nisin were fabricated, and the antimicrobial concentrations were 0.5, 1, or 2 mg in a circular disc of chitosan film. Three concentrations of lactoferrin or EDTA (0.28, 0.56, or 1.12 mg per disc) were also incorporated into the chitosan film containing lysozyme to investigate the combination effects of lactoferrin. The water barrier properties of the chitosan films containing lactoferrin were characterized. The antimicrobial activities against E. coli O157:H7 and L. monocytogenes were determined using the agar diffusion assay and cell count assay. The chitosan films containing lactoferrin less than 1 mg per disc did not alter the water vapor permeability of the chitosan film. Although the film containing lysozyme exhibited significant antimicrobial activity, the incorporation of lactoferrin alone into chitosan film did not exhibit significant antimicrobial activity against both E. coli O157:H7 and L. monocytogenes. However, the combination of lactoferrin with lysozyme-containing chitosan film significantly decreased the growth of E. coli O157:H7, exhibiting a comparable effect to that of the combination of EDTA with lysozyme (P < 0.05). Furthermore, the combination of lactoferrin with lysozyme in chitosan film exhibited greater reduction in the growth of L. monocytogenes than did the combination EDTA with lysozyme, resulting in an approximate 3-log reduction.