EMA-PCR法快速检测啤酒中腐败短乳杆菌

将叠氮溴乙锭(ethidium bromide monoazide,EMA)与聚合酶链式反应(polymerase chain reaction,PCR)技术相结合,以酒花耐受基因hor C为靶基因,用啤酒腐败短乳杆菌基因组DNA作为模板进行扩增。结果发现,在前处理过程中加入EMA,当终质量浓度小于20μg/m L时,对活的短乳杆菌中靶基因的扩增没有明显抑制作用;而当EMA终质量浓度为1.0μg/m L时可有效抑制10~5 CFU/m L短乳杆菌死细胞的扩增。本实验建立的EMA-PCR检测方法的灵敏度为10~4活细胞/m L酒液样品。验证实验结果表明,在13株乳酸菌中,建立的hor C特异性EMA-PCR能有效检测到其中的全部5株啤酒污染菌,同时可区分这5株菌的活/死细胞混合体系,降低检测过程中的假阳性。     

一株从泡菜中分离的产细菌素乳杆菌的鉴定及细菌素特性研究

从泡菜中分离的329 株乳酸菌中,筛选出一株产细菌素的乳酸菌,编号为SD-22。经生理生化实验鉴定为短乳杆菌(Lactobacillus brevis)。在排除过氧化氢干扰和有机酸抑菌作用后,brevicin SD-22 对革兰氏阳性细菌和革兰氏阴性细菌具有较好的抑制作用,对部分真菌也有抑制作用,但对酵母菌和青霉无抑制作用,具有良好的热稳定性和pH 值稳定性,经胰蛋白酶、胃蛋白酶和蛋白酶K 处理后抑菌活性完全消失,对α- 淀粉酶不敏感。因此初步认为该菌株是一株产广谱细菌素的乳酸菌。     

乳源短乳杆菌M8 S- 层蛋白的提纯及其生物学特征分析

通过利用原子力显微镜(AFM)来观察凝胶过滤层析法提纯短乳杆菌M8菌株的S-层蛋白的表面形貌,同时探讨S-层蛋白的再生特性及黏附特性。结果表明：凝胶过滤层析法能够获得纯度较高的S-层蛋白;该蛋白在纯水中可自我组装成纳米级“团簇”结构;去除S-层蛋白的菌体细胞仍然具有生命活性,适当培养后可重新表达该蛋白;短乳杆菌M8可黏附到Caco-2细胞上,其S-层蛋白介导此过程。     

Comparison of Enrichment Media for Routine Detection of Beer Spoiling Lactic Acid Bacteria and Development of Trouble‐shooting Medium for Lactobacillus backi

The beer spoiling lactic acid bacteria (LAB) are known to have a substantial financial impact in the brewing industry and their rapid detection is essential. Thus more effective media for the cultivation of LAB in both routine quality control and special trouble‐shooting situations are needed. In this study, different media were tested for the routine detection of LAB at a commercial brewery. The results showed that the use of an enzyme controlled glucose delivery system, in combination with beer‐MRS medium, can significantly decrease the total analysis time. For more effective trouble‐shooting in contamination incidents a trouble‐shooting media, including the reducing agents L‐cysteine‐HCl and sodium bicarbonate, was developed. The presented medium was shown to improve the growth of beer spoiling L. backi and L. brevis, and is thereby suggested for faster detection of these strains at the breweries.     

Short communication: Roles of outer membrane protein W on survival,cellular morphology,and biofilm formation of Cronobacter sakazakii in response to oxidative stress

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H₂O₂. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.     

Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance,motility, and biofilm formation of Cronobacter sakazakii

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.     

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene

BACKGROUND: To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species‐specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. RESULTS: The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species‐specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231‐bp species‐specific band was found only in L. delbrueckii. A SNaPshot mini‐sequencing assay using recA as a target gene was also developed. The specificity of the mini‐sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. CONCLUSION: The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species‐specific polymerase chain reaction combined with SNaPshot mini‐sequencing. Copyright © 2012 Society of Chemical Industry     

Purification,characterization, and mode of action of a novel bacteriocin BM173 from Lactobacillus crustorum MN047 and its effect on biofilm formation of Escherichia coli and Staphylococcus aureus

There is an increasing demand for dairy products, but the presence of food-spoilage bacteria seriously affects the development of the dairy industry. Bacteriocins are considered to be a potential antibacterial or antibiofilm agent that can be applied as a preservative. In this study, bacteriocin BM173 was successfully expressed in the Escherichia coli expression system and purified by a 2-step method. Furthermore, it exhibited a broad-spectrum antibacterial activity, high thermal stability (121°C, 20 min), and broad pH stability (pH 3–11). Moreover, the minimum inhibitory concentration values of BM173 against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were 14.8 μg/mL and 29.6 μg/mL, respectively. Growth and time-kill curves showed that BM173 exhibited antibacterial and bactericidal activity. The results of scanning electron microscopy and transmission electron microscopy demonstrated that BM173 increased membrane permeability, facilitated pore formation, and even promoted cell lysis. The disruption of cell membrane integrity was further verified by propidium iodide uptake and lactic dehydrogenase release. In addition, BM173 exhibited high efficiency in inhibiting biofilm formation. Therefore, BM173 has promising potential as a preservative used in the dairy industry.     

Dry surface biofilms in the food processing industry: An overview on surface characteristics,adhesion and biofilm formation,detection of biofilms,and dry sanitization methods

Bacterial biofilm formation in low moisture food processing (LMF) plants is related to matters of food safety, production efficiency, economic loss, and reduced consumer trust. Dry surfaces may appear dry to the naked eye, however, it is common to find a coverage of thin liquid films and microdroplets, known as microscopic surface wetness (MSW). The MSW may favor dry surface biofilm (DSB) formation. DSB formation is similar in other industries, it occurs through the processes of adhesion, production of extracellular polymeric substances, development of microcolonies and maturation, it is mediated by a quorum sensing (QS) system and is followed by dispersal, leading to disaggregation. Species that survive on dry surfaces develop tolerance to different stresses. DSB are recalcitrant and contribute to higher resistance to sanitation, becoming potential sources of contamination, related to the spoilage of processed products and foodborne disease outbreaks. In LMF industries, sanitization is performed using physical methods without the presence of water. Although alternative dry sanitizing methods can be efficiently used, additional studies are still required to develop and assess the effect of emerging technologies, and to propose possible combinations with traditional methods to enhance their effects on the sanitization process. Overall, more information about the different technologies can help to find the most appropriate method/s, contributing to the development of new sanitization protocols. Thus, this review aimed to identify the main characteristics and challenges of biofilm management in low moisture food industries, and summarizes the mechanisms of action of different dry sanitizing methods (alcohol, hot air, UV-C light, pulsed light, gaseous ozone, and cold plasma) and their effects on microbial metabolism.