传统泡菜腐败过程中膜醭和盐卤的 微生物区系分析

目的研究以Lactobacillus plantarum为菌剂发酵的传统泡菜在腐败过程中微生物的区系变化。方法通过提取腐败前、中、后期3个阶段腐败膜醭及盐卤中微生物总基因组并构建16S r RNA基因文库,分析其细菌群落构成和动态变化,并通过丰富度指数SChao1和SACE、覆盖率及多样性指数H等揭示其群落多样性。结果从不同阶段膜醭和盐卤样品中共检测到Lactobacillus等15个属,盐卤及膜醭不同腐败阶段的微生物区系差异较大。其中L.plantarum为腐败前期膜醭及盐卤样品中的优势菌。腐败中期膜醭中的优势菌群为L.plantarum、Propionbacterium acidipropionici,腐败中期盐卤中的优势菌为L.plantarum、Enterococcus malodoratus、P.acidipropionici。而腐败后期样品中,膜醭的优势菌为E.malodoratus、Providencia rettgeri和Shewanella algae,盐卤的优势菌为Morganella morganii、S.algae、E.malodoratus和Proteus mirabilis。结论盐卤及膜醭中的微生物区系在腐败前、中、后期都以细菌为主并细菌种类有差异较大。腐败前期以革兰氏阳性为主,腐败后期以革兰氏阴性为主。     

Growth Characteristics and Biofilm Formation of Various Spoilage Bacteria Isolated from Fresh Produce

This study investigated the characteristics of spoilage bacteria isolated from fresh produce including growth at various temperatures, biofilm formation, cell hydrophobicity, and colony spreading. The number of spoilage bacteria present when stored at 35 °C was significantly greater than when stored at lower temperatures, and maximum population size was achieved after 10 h. However, Bacillus pumilus, Dickeya zeae, Pectobacterium carotovorum subsp. Carotovorum Pcc21, and Bacillus pumilus (RDA‐R) did not grow at the storage temperature of 5 °C. The biofilm formation by Clavibacter michiganensis, Acinetobacter calcoaceticus, and A. calcoaceticus (RDA‐R) are higher than other spoilage bacteria. Biofilm formation showed low correlation between hydrophobicity, and no significant correlation with colony spreading. These results might be used for developing safe storage guidelines for fresh produce at various storage temperatures, and could be basic information on the growth characteristics and biofilm formation properties of spoilage bacteria from fresh produce.     

D-核糖及肉桂醛对四川泡菜菌群多样性和生物被膜的影响

选择2 种群体感应抑制剂D-核糖与肉桂醛,通过高通量测序技术检测其对发酵泡菜菌群结构的调控作用,并进一步分析二者对特定腐败菌生理行为的影响。结果表明D-核糖处理后,泡菜发酵液中Lactobacillus的相对丰度由第2位（19.38%）上升至第1位（80.10%）,同时不含有Staphylococcus equorum（OTU4）、Lactobacillus ginsenosidimutans（OTU6）、Propionibacterium acnes subsp. acnes（OTU8）、Lactococcus lactis subsp. lactis（OTU9）;肉桂醛对菌群结构的影响不明显。此外,通过绘制生长曲线发现D-核糖对特定腐败菌的生长无明显作用,肉桂醛对其生长有抑制作用,但是二者均可显著抑制腐败菌的生物被膜形成。D-核糖和肉桂醛处理后Pantoea calida的生物被膜形成量分别降低至对照的11%和10%;Pantoea ananatis的生物被膜形成量分别降低至对照的21%和17%,Pantoea anthophila的生物被膜量分别降低至对照的11%和9%,Staphylococcus saprophyticus的生物被膜量分别降低至对照的11%和15%,作用效果很明显。以上结果对进一步研究发酵泡菜菌群调控机制具有参考价值,同时为开发发酵食品的新型防腐保鲜技术提供新的思路。     

食源性细菌关键小分子信号通路研究进展

食源性细菌引起的食品腐败和生物被膜形成是食品科学领域关注的热点。研究表明这些现象与细菌群体感应（quorum sensing,QS）系统和第二信使环二鸟苷酸（cyclic di-guanosine monophosphate,c-di-GMP）系统密切相关,深入研究发现细菌可通过利用胞外自诱导物和胞内c-di-GMP分别感知细胞密度和外界刺激来调控一系列生理活性。鉴于此,本文主要围绕食品腐败和生物被膜形成关联的QS和c-di-GMP这两个系统,阐述食源性细菌关键小分子信号通路,探讨QS和c-di-GMP信号通路之间的调控网络,旨在更好地理解食品体系中细菌引起的腐败和生物被膜形成现象,提供保障食品品质与安全的控制靶点。     

冷却猪肉及托盘表面细菌生物被膜分析和肉源荧光假单胞菌的鉴定与被膜研究

为研究冷却猪肉及其接触面中细菌生物被膜能力并探讨肉类特定腐败菌的生物被膜特征,分析了腐败的冷却肉和销售托盘表面的细菌生物被膜形成能力;利用形态学观察、16S r DNA分析和VITEK2微生物鉴定系统鉴定了冷却肉中一株生物被膜能力较强的荧光假单胞菌(Pseudomonas fluorescens);测定了该菌在培养基和猪肉浸提液中的生物被膜能力;用激光共聚焦和扫描电子显微镜观测了其生物被膜的结构特征。结果发现:冷却肉中及其销售托盘表面的细菌能够形成生物被膜的比例较高,37%和45%的细菌具有较强生物被膜形成能力。肉源性荧光假单胞菌能够在培养基和肉液中形成大量生物被膜,具有较强的生物被膜能力。显微成像结果表明荧光假单胞菌在培养6 h后有明显黏附,18 h后多糖分泌增多、菌体堆积并形成具有生物功能的立体生物被膜。这些特点可能有利于荧光假单胞菌在冷却肉表面黏附并成为优势腐败菌。     

Beer enemy number one: genetic diversity,physiology and biofilm formation of Lactobacillus brevis

Lactobacillus brevis is the most significant beer spoilage bacteria worldwide. It is found as a contaminant at all stages of brewing, including during primary and secondary fermentation, storage, filtration and the packaging process. In production with flash pasteurisation and subsequent hygienic filling, avoiding and tracing secondary contaminations is the key to a microbiologically stable product. However, L. brevis strains vary in their spoilage potential and can grow in many different beer types. This study presents a physiological test scheme for growth potential and biofilm formation in various media. It was determined that a large number of L. brevis strains can form biofilms as a first coloniser. The identification of the species alone is therefore not enough to be sure of the spoilage risk, which shows the need for a more in depth differentiation. DNA fingerprint techniques are crucial to differentiate isolates of this species at strain level. The rep‐PCR fingerprint system (GTG)₅ was used to differentiate a selected collection of 20 isolates, which were characterised in growth and biofilm formation in various media. The data showed a high variation within the selected isolates. As second step, generated fingerprint clusters of L. brevis were traced back to contamination sources in a German brewery, revealing a high number of isolates with potentially varying growth, spoilage and biofilm potential. L. brevis being the demonstrator species, the PCR system used is a powerful and compatible tracing and troubleshooting tool for all kinds of spoilage bacteria in the brewing industry. © 2019 The Institute of Brewing & Distilling     

生鲜牛肉中的腐败微生物概述

生鲜牛肉腐败是一个涉及多种特定微生物相互作用并受多种因素影响的复杂的微生物生态现象。了解牛 肉中微生物的污染源及微生物的多样性对于控制其腐败进程、延长牛肉货架期十分重要。因此,本文对肉牛从 下刀宰杀到包装前所有可能的污染源、污染微生物在种的水平上的分类和多样性、单个菌属及多种微生物作用的 腐败机制等方面对生鲜牛肉中腐败微生物的相关信息进行了系统性分析,初步总结得出活体动物的皮毛及皮毛上 的污染物、去皮用的刀具和剥皮操作工人的手、分割时的工作接触面为生鲜牛肉中微生物的主要污染源;主要 的微生物种类为假单胞菌（Pseudomonas）、肠杆菌（Enterobacteriaceae）、乳酸杆菌及热杀索丝菌（Brochothrix thermosphacta）;不同种类的微生物因利用肉中营养代谢底物的顺序不同而产生不同的腐败现象。期望通过本文的 总结和概述能够为生鲜牛肉腐败微生物的控制提供理论指导。     

基于高通量测序分析虾夷扇贝柱菌群结构及腐败优势菌

为揭示虾夷扇贝柱的菌群结构及腐败优势菌,通过对宏基因组DNA进行高通量测序,分析虾夷扇贝柱新鲜和腐败样品的菌群结构,同时对可培养细菌菌群基因组DNA进行高通量测序,分析新鲜和腐败样品可培养细菌菌群的结构特征。结果表明,对宏基因组测序后获得的新鲜虾夷扇贝柱中细菌菌群多样性丰富,其中相对丰度较高的有Candidatus kuenenia（10.73%）、弧菌属（Vibrio,8.15%）、别弧菌属（Aliivibrio,7.38%）、芽孢杆菌属（Bacillus,7.20%）和未分类菌（unclassified,16.08%）;4?℃的腐败优势菌为发光杆菌属（Photobacterium,46.91%）、别弧菌属（36.82%）和假交替单胞菌属（Pseudoalteromonas,11.01%）,15?℃的腐败优势菌为发光杆菌属（46.97%）和别弧菌属（43.33%）,25?℃的腐败优势菌为发光杆菌属（41.94%）、别弧菌属（23.10%）、梭杆菌属（Fusobacterium,18.61%）和乳酸菌属（Lactobacillus,10.60%）。对可培养细菌菌群基因组测序后发现,新鲜样品可培养细菌菌群多样性大大降低,大部分为假交替单胞菌属（89.46%）,在25?℃腐败后,可培养的腐败优势菌为弧菌属（50.98%）、希瓦氏菌属（Shewanella,17.43%）和乳酸菌属（10.68%）。本研究揭示了虾夷扇贝柱新鲜及腐败样品的菌群结构特征及腐败优势菌,为进一步研究微生物对虾夷扇贝柱品质劣化的作用机制提供一定参考。     

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat

Biofilms formations of spoilage and pathogenic bacteria on food or food contact surfaces have attracted increasing attention. These events may lead to a higher risk of food spoilage and foodborne disease transmission. While Pseudomonas lundensis is one of the most important bacteria that cause spoilage in chilled meat, its capability for biofilm formation has been seldom reported. Here, we investigated biofilm formation characteristics of P. lundensis mainly by using crystal violet staining, and confocal laser scanning microscopy (CLSM). The swarming and swimming motility, biofilm formation in different temperatures (30, 10, and 4 °C) and the protease activity of the target strain were also assessed. The results showed that P. lundensis showed a typical surface‐associated motility and was quite capable of forming biofilms in different temperatures (30, 10, and 4 °C). The strain began to adhere to the contact surfaces and form biofilms early in the 4 to 6 h. The biofilms began to be formed in massive amounts after 12 h at 30 °C, and the extracellular polysaccharides increased as the biofilm structure developed. Compared with at 30 °C, more biofilms were formed at 4 and 10 °C even by a low bacterial density. The protease activity in the biofilm was significantly correlated with the biofilm formation. Moreover, the protease activity in biofilm was significantly higher than that of the corresponding planktonic cultures after cultured 12 h at 30 °C.     

Incidence of Moraxella and other gram-negative,oxidase-positive bacteria in fresh and spoiled ground beef

A total of 1535 bacterial isolates were examined from 29 retail samples of ground beef when fresh and after aerobic spoilage at 5–7°C. Of these, 1354 (88%) were gram-negative and oxidase-positive, but only 34 were identified by API 20E as Moraxella-26 from fresh and eight from spoiled samples. Ninety-five percent of the oxidase-positive isolates were Pseudomonas spp. and 66% of these were recovered from the meats after spoilage. The findings bring into question the widely-held view that moraxellae along with Acinetobacter are rather abundant in ground beef.     

食源假单胞菌16S rRNA鉴定及其食品腐败特性的研究

从市售鱼中分离的三株革兰氏阴性菌FML05-1、FML05-2、FML05-3,经16s rRNA鉴定为假单胞菌属。该菌是一种导致食品腐败的重要腐败细菌。选取代表菌株的16S rRNA序列,利用phylip软件网上构建系统进化树,同时对其蛋白水解活性、嗜铁素产生和生物膜形成等与食品腐败相关的特性进行检测,发现FML05-2具有很强的蛋白水解活性和生物膜的形成能力,并且在脱脂牛奶中生长还产生大量的嗜铁素,菌株FML05-1具有很强的生物膜形成能力,但蛋白水解活性较弱,在LB及脱脂牛奶中生长均不产生嗜铁素,而菌株FML05-3不具有上述三种特性。     

Effect of industrial and natural biocides on spoilage, pathogenic and technological strains grown in biofilm

This study aimed at investigating bactericide solutions effective on spoilage and pathogenic bacteria while preserving technological bacteria. Two compounds of essential oil (thymol and eugenol), one essential oil of Satureja thymbra and two industrial biocides (PE 270-30, Brillo) were tested on technological strains (Staphylococcus equorum, Staphylococcus succinus and Lactobacillus sakei) grown in monoculture biofilm and on a mixed biofilm of pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes) and spoilage bacteria (Pseudomonas fragi, Escherichia coli). Biofilm cultures were performed in glass fibre filters for 24h at 20 degrees C before application of biocides. Thymol and eugenol had no effect on the mixed biofilm. S. thymbra (2%) was highly effective on spoilage strains (5 log reduction), and S. equorum (4 log reduction) was moderately effective on pathogens (2.3 log reduction) and not effective on S. succinus and L. sakei (0.5 log reduction). PE-270-30 with 10% Na((2))SO((4)) decreased spoilage bacteria (5.1 log reduction), maintained the technological bacteria, but did not reduce the pathogens. The disinfectant Brillo (3%) killed all the strains. These results showed the difficulty in obtaining a biocide that is effective in destroying spoilage and pathogenic bacteria while preserving technological bacteria. Essential oils could be a good alternative for eradicating spoilage bacteria in the food environment where they are often found at high levels.     

Bacterial Quorum Sensing and Food Industry

Abstract: Food spoilage and biofilm formation by food‐related bacteria are significant problems in the food industry. Even with the application of modern‐day food preservative techniques, excessive amounts of food are lost due to microbial spoilage. A number of studies have indicated that quorum sensing plays a major role in food spoilage, biofilm formation, and food‐related pathogenesis. Understanding bacterial quorum‐sensing signaling systems can help in controlling the growth of undesirable food‐related bacteria. This review focusses on the various signaling molecules produced by Gram‐negative and Gram‐positive bacteria and the mechanism of their quorum‐sensing systems, types of signaling molecules that have been detected in different food systems using biosensors, the role of signaling molecules in biofilm formation, and significance of biofilms in the food industry. As quorum‐sensing signaling molecules are implicated in food spoilage, based on these molecules potential, quorum‐sensing inhibitors/antagonists can be developed to be used as novel food preservatives for maintaining food integrity and enhancing food safety. Practical Application: Bacteria use signaling molecules for inter‐ and intracellular communication. This phenomenon of bacterial cell‐to‐cell communication is known as quorum sensing. Quorum‐sensing signals are implicated in bacterial pathogenicity and food spoilage. Therefore, blocking the quorum‐sensing signaling molecules in food‐related bacteria may possibly prevent quorum‐sensing‐regulated phenotypes responsible for food spoilage. Quorum‐sensing inhibitors/antagonists could be used as food preservatives to enhance the shelf life and also increase food safety.     

Effect of single or sequential hot water and lactic acid decontamination treatments on the survival and growth of listeria monocytogenes and spoilage microflora during aerobic storage of fresh beef at 4, 10, and 25 degrees C

The survival and growth of Listeria monocytogenes and spoilage microflora during storage of fresh beef subjected to different decontamination treatments was studied. Fresh beef inoculated with a five-strain mixture of L. monocytogenes (5.18 log CFU/cm2) was left untreated (control) or was immersed (30 s) in hot water (HW; 75 degrees C), 2% lactic acid (LA; 55 degrees C), hot water followed by lactic acid (HW-LA), or lactic acid followed by hot water (LA-HW) and then stored aerobically at 4, 10, and 25 degrees C for 25, 17, and 5 days, respectively. Initial populations of L. monocytogenes were reduced by 0.82 (HW), 1.43 (LA), 2.73 (HW-LA), and 2.68 (LA-HW) log CFU/cm2. During storage, the pathogen grew at higher rates in HW than in control samples at all storage temperatures. Acid decontamination treatments (LA. HW-LA, and LA-HW) resulted in a weaker inhibition of L. monocytogenes (P < 0.05) at 25 degrees C than at 4 and 10 degrees C. In general, the order of effectiveness of treatments was HW-LA > LA > LA-HW > HW > control at all storage temperatures tested. In untreated samples, the spoilage microflora was dominated by pseudomonads, while lactic acid bacteria, Enterobacteriaceae, and yeasts remained at lower concentrations during storage. Brochothrix thermosphacta was detected periodically in only a limited number of samples. Although decontamination with HW did not affect the above spoilage microbial profile, acid treatments shifted the predominant microflora in the direction of yeasts and gram-positive bacteria (lactic acid bacteria). Overall, the results of the present study indicate that decontamination with LA and combinations of LA and HW could limit growth of L. monocytogenes and inhibit pseudomonads, which are the main spoilage bacteria of fresh beef stored under aerobic conditions. However, to optimize the efficacy of such treatments, they must be applied in the appropriate sequence and followed by effective temperature control.     

紫薯腐败菌的分离与鉴定

目的 从自然腐败的紫薯上分离纯化紫薯腐败菌, 通过反接种确定其致腐性, 并将其鉴定到属。方法 通过多次稀释平板涂布培养, 进行单菌落形态学观察, 将菌种反接种到新鲜紫薯上, 筛选出腐败菌, 以真菌ITS序列、细菌16S rDNA序列构建发育树, 鉴定到属, 再将鉴定结果与分离的菌种的形态学鉴定结果对比, 验证菌种鉴定结果。结果 从样品中分离出6种典型腐败菌, 有4种属, 分别为枝孢霉属、青霉属、曲霉属、芽孢杆菌属。结论 确定紫薯的腐败菌为哥氏枝孢霉(Cladosporium gossypiicola)、Penicillium christenseniae、热带青霉(Penicillium tropicum)、黄曲霉(Aspergillus flavus)、贝莱斯芽孢杆菌(Bacillus velezensis)和阿氏芽孢杆菌(Bacillus aryabhattai)。     

大菱鲆荧光假单胞菌的群体感应现象及不同碳源培养下的腐败特性研究

本文以大菱鲆分离的荧光假单胞菌作为研究对象,紫色杆菌CVO26平行划线法检测信号分子。不同碳源(葡萄糖、蔗糖、果糖、木糖、乳糖、麦芽糖)的AB培养基培养并检测其生物被膜、嗜铁素和胞外蛋白酶的产生量,同时添加外源的信号分子标准品,研究AHLs与腐败因子之间的相关性。研究表明:大菱鲆分离的荧光假单胞菌能够发生群体感应现象,经不同碳源培养,其腐败因子产生情况有明显差异,碳源的添加对生物被膜的产生有促进作用,对胞外蛋白酶的产生没有显著影响;以葡萄糖、麦芽糖为碳源,生物被膜的产生量较高;以蔗糖和乳糖为碳源,嗜铁素的产量较高。添加外源信号分子标准品,生物被膜、嗜铁素和胞外蛋白酶的产生量有显著提高。因此,生物被膜、嗜铁素和胞外蛋白酶的产生与AHLs有关,群体感应现象可调控腐败特性的表达,并在水产品腐败过程中发挥作用。     

Study of the microbial ecology of wild and aquacultured Tunisian fresh fish

Eighty samples of fresh fish were collected in Tunisia and analyzed for microbial load. Quality and hygienic safety of the meat and intestines of wild and aquacultured fresh fish were determined. The mesophilic aerobic plate count and populations of psychrotrophic lactic acid bacteria (LAB) and other psychrotrophic bacteria ranged from 5.67 to 7.29, 4.51 to 6, and 5.07 to 6.21 log CFU/g, respectively. For all microbiological determinations, bacterial counts were lower in meat than in the intestines of fresh fish. For all samples lower microbial populations were found in most of the wild fish than in the aquacultured fish. No isolates of the pathogenic genera Salmonella and Listeria were detected in any sample. Among the 160 strains of biopreservative psychrotrophic LAB and the 150 strains of spoilage psychrotrophic gram-negative bacteria identified by biochemical and molecular methods, Lactobacillus (six species) and Pseudomonas (six species) predominated. Lactococcus, Leuconostoc, Carnobacterium (C. piscicola and C. divergens), Aeromonas, and Photobacterium were the most common genera, and Lactococcus lactis, Lactobacillus plantarum, Pseudomonas fluorescens, and Aeromonas hydrophila were the most common species. These findings indicate that the microbiological quality of fresh fish in Tunisia can be preserved by controlling pathogenic and psychrotrophic bacteria.     

Role of biogenic amines as index of freshness in beef meat packed with different biopolymeric materials

The main objectives of this work were to evaluate the chemical and microbiological fresh beef meat quality packed in aerobic atmosphere with biopolymers, to investigate the possible role of biogenic amines (BAs) as indicators of spoilage in fresh beef meat stored at 4 °C for 8 days. The results of this research highlighted that for fresh meat packaging it could be possible to replace the PS tray/PVC film system, with an expanded PLA biopolymeric tray heat-sealed with a biopolymeric film, characterized by a negligible environmental impact in comparison with the use of synthetic plastic materials. The storage time differentiated the meat samples on the basis of pH and microbiological characteristics. With regard to BAs, tyramine and cadaverine resulted strongly influenced by the storage time, and to a less extent putrescine and spermidine. Tyramine and cadaverine could be used as spoilage indexes of fresh beef meat chilled and packed in aerobic atmosphere with biopolymers.     

Sensory and Microbial Quality of Irradiated Crab Meat Products

Low dose gamma irradiation has proved effective in reducing pathogenic and spoilage microorganisms in a variety of seafood products. However, little information is available on the effect of irradiation on the sensory quality and consumer acceptance of such products. We compared microbial and sensory quality of irradiated (2 kGy or less) crab products (white lump, claw, and fingers) through 14-days of ice-storage. Irradiation effectively reduced spoilage bacteria extending shelf-life by more than 3 days beyond control samples. During storage, fresh crab odor and flavor were similar for treated and control samples, while off-flavors and odors developed more rapidly in controls. Overall acceptability scores for irradiated crab samples were higher than for control samples throughout 14-days ice storage.     

Antibiofilm mechanism of dielectric barrier discharge cold plasma against Pichia manshurica

Biofilm spoilage has become one of the most common concerns in fermented foods. The objective of the present study was to explore the biofilm-inhibitory effect and antibiofilm mechanism of dielectric barrier discharge (DBD) cold plasma on Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods). After plasma treatment (80 kV, 50 Hz) for 4.5 min and 7.5 min, viable counts decreased by 2.38 and 5.11 lg CFU/mL, respectively, and the biofilm-forming rate decreased to 73% and 48% of the control group, respectively, in microbiological media. In addition, DBD caused severe morphological damage, cell membrane permeabilization and metabolic changes. A number of amino acid metabolic pathways, the tricarboxylic acid cycle (TCA), and the synthesis of extracellular polymeric substance (EPS) were destroyed. The cell damage and changes in multiple metabolic activities led to significant inhibitory effects on Pichia manshurica and its biofilm-forming ability.Industrial relevanceThis study found that DBD can significantly inhibit the biofilm-forming ability of Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods) by causing severe cell damage and metabolic changes. The exploration of the biofilm inhibitory effect of DBD on Pichia manshurica and its mechanism can provide a theoretical basis for the application of DBD technology in the inhibition of biofilm spoilage in foods.