Inhibition of microbial growth in ready‐to‐eat food stored at ambient temperature by modified atmosphere packaging

This study was undertaken to develop a modified atmosphere package to control microbial growth in ready‐to‐eat (RTE) products stored at ambient temperature. Ethanol and/or limonene associated with modified atmosphere (CO₂ : O₂ : N₂ = 30% : 5% : 65%) was used to inhibit the growth of total air‐borne microorganisms and Escherichia coli in RTE products stored at 25°C. The results indicated that 0.05% ethanol vapour in the headspace was effective to inhibit the growth of air‐borne microorganisms and E. coli at 25°C for 72 h in a model study, and the effectiveness was related to ethanol content. Both 73 ppm limonene and 0.05% ethanol vapour enhanced the bacteriostatic effect of modified atmosphere in RTE sushi roll products, and no off‐flavour was detected using this formulated gas; however, no significant inhibitory effect was observed for RTE cold noodle products. This study concludes that combinations of carbon dioxide, ethanol or limonene vapours are effective to inhibit microbial growth in RTE food at ambient temperature, and the outcome may be due to the hurdle effect. Copyright © 2003 John Wiley & Sons, Ltd.     

Heat resistance of Escherichia coli O157:H7 in cook‐in‐bag ground beef as affected by pH and acidulant†

Summary The heat resistance of a four‐strain mixture of Escherichia coli O157:H7 was tested. The temperature range was 55–62.5 °C and the substrate was beef at pH 4.5 or 5.5, adjusted with either acetic or lactic acid. Inoculated meat, packaged in bags, was completely immersed in a circulating water bath and cooked to an internal temperature of 55, 58, 60, or 62.5 °C in 1 h, and then held for pre‐determined lengths of time. The surviving cell population was enumerated by spiral plating meat samples on tryptic soy agar overlaid with Sorbitol MacConkey agar. Regardless of the acidulant used to modify the pH, the D ‐values at all temperatures were significantly lower (P < 0.05) in ground beef at pH 4.5 as compared with the beef at pH 5.5. At the same pH levels, acetic acid rendered E. coli O157:H7 more sensitive to the lethal effect of heat. The analysis of covariance showed evidence of a significant acidulant and pH interaction on the slopes of the survivor curves at 55 °C. Based on the thermal‐death–time values, contaminated ground beef (pH 5.5/lactic acid) should be heated to an internal temperature of 55 °C for at least 116.3 min and beef (pH 4.5/acetic acid) for 64.8 min to achieve a 4‐log reduction of the pathogen. The heating time at 62.5 °C, to achieve the same level of reduction, was 4.4 and 2.6 min, respectively. Thermal‐death–time values from this study will assist the retail food processors in designing acceptance limits on critical control points that ensure safety of beef originally contaminated with E. coli O157:H7.     

密码子优化的牛精蛋白基因在大肠杆菌中的表达

以PCR的方法得到牛精蛋白基因的基因，去其内含子，得到牛精蛋白cDNA，克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PE，利用大肠杆菌偏好的编码精氨酸的密码子CGT将野生型牛精蛋白基因中编码精氨酸的稀有密码子（AGA或AGG）替换掉，通过基因合成得到密码子优化的牛精蛋白的基因，将其克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PS。将这两个表达载体分别转化入大肠杆菌表达菌株BL21中，经IPTG诱导，同样条件下，野生型牛精蛋白基因无法得到表达，密码子优化的牛精蛋白基因能够得到良好的表达，表达产物约占细菌总蛋白的18％，将表达蛋白纯化，进行DNA－蛋白结合实验，发现其能与DNA发生非特异性的结合。     

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

Application of artificial neural networks to the analysis of two‐dimensional fluorescence spectra in recombinant E coli fermentation processes

A two‐dimensional (2D) spectrofluorometer was used to monitor various fermentation processes with recombinant E coli for the production of 5‐aminolevulinic acid (ALA). The whole fluorescence spectral data obtained during a process were analyzed using artificial neural networks, ie self‐organizing map (SOM) and feedforward backpropagation neural network (BPNN). The SOM‐based classification of the whole spectral data has made it possible to qualitatively associate some process parameters with the normalized weights and variances, and to select some useful combinations of excitation and emission wavelengths. Based on the classified fluorescence spectra a supervised BPNN algorithm was used to predict some of the process parameters. It was also shown that the BPNN models could elucidate some sections of the process's performance, eg forecasting the process's performance. Copyright © 2005 Society of Chemical Industry     

肠道杆菌的扫描电镜观察

我们用扫描电镜观察肠道杆菌种(Enterobacteriaceae)中的四种细菌,大肠埃希氏杆菌(Escherichiacoli),普通变形杆菌(Proteus vulgaris),伤寒沙门氏菌(Salmonella typhi)和福氏志贺氏菌(Shigella Flexneri)。未处理的菌落标本,菌落表面都可形成一层厚薄不同的表膜(Surface film)。四种细菌菌落的表膜,形状不一,千姿百态。适当处理后的菌落标本,则可显示菌体的本来面目。四种细菌菌体在菌落表面的分布和排列,也是各不相同,千差万别。本文根据扫描电镜的观察,对四种不同菌落进行了讨论。     

Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest minimal hemolysis concentration (MHC, 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time–kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 μg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.     

人Leptin基因的克隆及其在大肠杆菌中的表达

目的克隆Leptin基因,构建其重组表达菌株,并对表达产物进行免疫学鉴定。方法利用RT-PCR方法从脂肪细胞RNA中扩增出人瘦素前体(pre-Leptin)的cDNA片段和去信号肽序列的Leptin成熟基因片段,并插入pET32a中,转化大肠杆菌BL21(DE3),获得表达重组人瘦素(rh-Leptin)的菌株。结果DNA序列分析显示获得的pre-Leptin和成熟基因片段的序列与预计序列一致,菌株诱导后经SDS-PAGE检测,rh-Leptin表达量达菌体总蛋白的40%以上。Western blot分析显示重组Lep-tin具有免疫学活性。结论已获得高效表达Leptin的重组菌株。     

大肠杆菌肉碱脱水酶基因的克隆、测序及高效表达

［摘 要］目的 克隆并表达大肠杆菌肉碱脱水酶基因。方法 用聚合酶链反应（PCR）从大肠杆菌K12s中扩增大肠杆菌肉碱脱水酶基因caiB，测定其DNA序列，将caiB基因重组到T7启动子控制下的表达载体pET－28a（＋）中，构建表达质粒pETCaiB，转化大肠杆菌BL21（DE3），用1mmol/L异两基硫代-β-D半乳糖苷（IPTG）诱导表达。结果 克隆得到的肉碱脱水酶编码基因 caiB长度为 1221bp，与文献报道值相比,DNA序列有26个不同，氨基酸序列只有一个不同，即302位的丙氨酸变为苏氨酸。重组菌经IPTG诱导，可高效表达，SDS－PAGE分析表达蛋白相对分子质量约43000，表达产物含量占菌体总蛋白45％以上。结论 得到了高效表达肉碱脱水酶的重组菌，为制备L-肉碱创造了条件。     

耐药大肠杆菌的致病性与免疫原性

目的了解大肠杆菌耐药株的致病性与免疫原性。方法临床分离耐药大肠杆菌,经适宜条件培养后接种小鼠,观察耐药菌株对小鼠的致病性;并选择已知血清型的不同耐药谱和不同耐药水平的菌,分别培养至对数生长期,经甲醛灭活后免疫小鼠,2周后分别用致死剂量的原菌株和同期分离菌株进行攻毒,考察耐药菌株免疫原性。结果24株耐药菌普通肉汤培养物分别感染小鼠,在接种后18h,存活率仅为4%。只有2株菌在1周内仍不能将小鼠全部致死,经小鼠体内传3代后,可在18h内致死小鼠。耐药谱广的菌株感染的小鼠,心肌发生颗粒变性、局灶性出血、坏死;肝脏糖元溶解,脂肪变性;脾脏轻度淤血,淋巴细胞减少;肾脏出血,肾小球肾炎,上皮细胞颗粒变性,水泡变性。而耐药种类少的菌株与对照敏感菌株感染的小鼠主要特征为脾脏出血、淤血,坏死,脾小体消失;肠上皮细胞坏死、脱落、卡他性肠炎。免疫小鼠以免疫用菌株攻毒,均可得较高的保护率,最低为75%,最高为100%。免疫小鼠用非免疫菌株攻毒,小鼠感染后症状出现较缓慢,在18h后出现死亡高峰。耐药种类少的菌株免疫小鼠,对同期分离的非免疫用的致病性大肠杆菌攻击的保护率偏低,而对受试的11种抗生素耐受7种以上的菌株免疫小鼠后,对致病大肠杆菌攻击的保护率显著提高,多数达到75%以上,经统计学分析差异显著。结论耐药大肠杆菌具有较强致病性,且耐药特性与其免疫保护效果相关,多重耐药株对当前流行菌株具有更好的免疫保护作用。