Influence of food preservation parameters and associated microbiota on production rate,profile and stability of acylated homoserine lactones from food-derived Enterobacteriaceae

Quorum-dependent regulation is mediated by N-acyl-L-homoserine lactones (AHLs) in several Gram-negative bacteria. The production of AHLs has typically been studied using pure bacteria cultures grown in nutrient-rich media at optimal temperature. AHLs are produced in several chill-stored foods by Gram-negative bacteria participating in spoilage. As part of our investigation of the role of AHLs in food quality, we studied the AHL production in two Enterobacteriaceae isolated from cold-smoked salmon under growth conditions typical of those found in cold-smoked salmon. We tested the influence of carbon source (glucose, sucrose, xylose, arabinose, mannose, mannitol and sorbitol), temperature (5 and 25 degrees C), salt concentration (0-7%), pH (6, 7 and 8) and co-existing lactic acid bacteria microflora on the AHL profile and production rate from Serratia proteamaculans strain B5a and Enterobacter agglomerans strain B6a. The two strains produced the same types of AHLs under all conditions tested. The specific AHL concentrations (moles/liter/OD(450)) changed slightly for both strains at the various conditions. S. proteamaculans strain B5a produced approximately 150 nM/OD(450) N-3-oxo-hexanoyl homoserine lactone (OHHL) and E. agglomerans strain B6a produced two major signals, OHHL and N-3-oxo-octanoyl homoserine lactone (OOHL) in a 1:9 ratio with a total concentration of approximately 3000 nM/OD(450). The AHL signal molecules became unstable with increasing pH (>7.5). In cold-smoked salmon, pH is approximately 6 and therefore only a low degree of pH-induced turnover is expected to occur in this product. Overall, our study demonstrates that food-derived Enterobacteriaceae produce AHLs of the same type and in the same magnitude when grown under food-relevant conditions as when grown in laboratory media at high temperature. Also, the AHLs produced in foods will be relatively stable and their regulatory impact lasting during storage.     

The Predominance of Psychrotrophic Pseudomonads on Aerobically Stored Chilled Red Meat

Microbial spoilage of meat during chilled aerobic storage causes significant financial losses to the industry. Even with modern day preservation techniques, spoilage remains an unsolved problem. Spoilage of meat is a complex process that involves the activity of endogenous enzymes and microorganisms. Psychrotrophic Pseudomonas species are the key microorganisms that cause spoilage in aerobically stored chilled meat. Spoilage pseudomonads are highly robust and able to withstand stressful environmental conditions that would otherwise inhibit the growth of other spoilage organisms. In order to implement efficient control measures, and to minimize spoilage, a thorough understanding of the characteristics of spoilage pseudomonads is essential. This review focuses on the spoilage process and the key metabolic attributes of the main psychrotrophic spoilage Pseudomonas species to explain their predominance on meat over other psychrotrophic bacteria. This review also highlights less studied, but important, characteristics of psychrotrophic pseudomonads such as biofilm formation and quorum sensing in the context of meat spoilage. The importance of the use of model systems that are closely applicable to the food industry is also discussed in detail.     

基于细菌群体感应淬灭机制的食品保鲜应用研究进展

群体感应是细菌细胞与细胞之间的一种通讯机制,通过信号分子的不断积累,从而实现其特定基因的表达.群体感应淬灭可通过对信号分子的干扰和抑制,阻碍细菌群体感应进程,从而减缓特定食品的腐败速度,延长其货架期.因此,群体感应淬灭在食品保藏中的作用尤为重要.本文介绍了不同类型的群体感应系统及群体感应淬灭的机制,揭示了淬灭技术在食品...     

Insights into the role of quorum sensing in food spoilage

Food spoilage is a consequence of the degrading enzymatic activity of some food-associated bacteria. Several proteolytic, lipolytic, chitinolytic, and pectinolytic activities associated with the deterioration of goods are regulated by quorum sensing, suggesting a potential role of such cell-to-cell communication in food spoilage. Here we review quorum sensing signaling molecules and methods of their detection and quantification, and we provide insights into the role of quorum sensing in food spoilage and address potential quorum sensing inhibitors that might be used as biopreservatives.     

Quorum Sensing Regulation and Inhibition of Exoenzyme Production and Biofilm Formation in the Food Spoilage Bacteria Pseudomonas psychrophila PSPF19

Gram-negative bacteria use the mechanism of acylated homoserine lactone (AHL) mediated quorum-sensing to regulate gene expression in response to population density. The present work aimed to detect signal molecule production and its influence on the production of food spoilage phenotypes such as exoenzyme (lipases and proteases) synthesis and biofilm formation in Pseudomonas psychrophila PSPF19, isolated from freshwater fish stored in refrigerated conditions. The C4-HSL signaling molecule produced by the isolate induced exoenzyme production by twofold and increased attachment and biofilm formation. Further, studies on the effect of a synthetic quorum sensing inhibitor, furanone C-30 on protease and lipase production and biofilm formation, revealed that it inhibited exoenzyme production and bacterial attachment. This study adds to our understanding that quorum sensing plays a role in the low temperature spoilage of foods by psychrotrophic Pseudomonads and quorum sensing inhibitors such as furanones can be developed as new intervention techniques and food preservatives.     

Food spoilage--interactions between food spoilage bacteria

Food spoilage is a complex process and excessive amounts of foods are lost due to microbial spoilage even with modern day preservation techniques. Despite the heterogeneity in raw materials and processing conditions, the microflora that develops during storage and in spoiling foods can be predicted based on knowledge of the origin of the food, the substrate base and a few central preservation parameters such as temperature, atmosphere, a(w) and pH. Based on such knowledge, more detailed sensory, chemical and microbiological analysis can be carried out on the individual products to determine the actual specific spoilage organism. Whilst the chemical and physical parameters are the main determining factors for selection of spoilage microorganisms, a level of refinement may be found in some products in which the interactive behavior of microorganisms may contribute to their growth and/or spoilage activity. This review gives three such examples. We describe the competitive advantage of Pseudomonas spp. due to the production of iron-chelating siderophores, the generation of substrates for spoilage reactions by one organism from another microorganism (so-called metabiosis) and the up-regulation of phenotypes potentially involved in spoilage through cell-to-cell communication. In particular, we report for the first time the widespread occurrence of N-acyl homoserine lactones (AHL) in stored and spoiling fresh foods and we discuss the potential implications for spoilage and food preservation.     

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.     

Behaviour of Brochothrix thermosphacta in presence of other meat spoilage microbial groups

The microbial flora of fresh meat stored aerobically at 5 degrees C up to spoilage was enumerated and collected in order to have mixed spoilage bacterial groups to be used in competition tests against Brochothrix thermosphacta. The bacterial groups collected as bulk colonies were identified by PCR-DGGE followed by partial 16S rDNA sequencing. The predominant bacteria associated with the spoilage of the refrigerated beef were B. thermosphacta, Pseudomonas spp, Enterobacteriaceae and lactic acid bacteria (LAB). The interactions between B. thermosphacta and the other spoilage microbial groups were studied in vitro at 5 degrees C. The results showed that a decrease of the growth of B. thermosphacta was evidenced in presence of LAB at 5 degrees C while the bacterium is the dominant organism when inoculated with mixtures of Pseudomonas spp., LAB and Enterobacteriaceae. A better understanding of bacterial meat spoilage interactions may lead to improved quality of fresh meat stored in refrigerated conditions.     

The ecology of bacterial spoilage of fresh meat at chill temperatures

At chill temperatures the spoilage flora of meat is composed of psychrotrophs originating largely from the hides of slaughtered animals. Under humid conditions, aerobic floras are usually dominated by pseudomonads while anaerobic floras are dominated by lactobacilli. In both cases growth occurs on low molecular weight soluble components of meat which are attacked in the order glucose, glucose-6-phosphate (Enterobacteriaceae only) and amino acids. Under aerobic conditions spoilage becomes detectable when the bacteria begin to degrade amino acids which remain abundant at the meat surface when growth ceases, probably because of limited availability of oxygen. Under anaerobic conditions growth ceases because the diffusion of fermentable substrates to the surface is not rapid enough to support further growth. Aerobically, there is no interaction between different bacterial species until the maximum cell density is approached; anaerobically, however, lactobacilli produce an antimicrobial agent which inhibits growth of competing species. The composition of spoilage floras can be affected by changes in water activity and the storage atmosphere.     

食品防腐中群体感应及其抑制剂的研究进展

食品的腐败变质是食品工业长期以来一直面对的重大问题。近年来研究表明细菌群体感应(quorum sensing,QS)现象与食品腐败变质密切相关。本文简要介绍了群体感应系统和信号分子的分类,重点阐述了群体感应抑制剂(quorum sensing inhibitors,QSIs)的研究进展,并讨论了利用群体感应抑制剂控制食品腐败变质的应用前景。      

Autoinducer-2-like activity associated with foods and its interaction with food additives

The autoinducer-2 (AI-2) molecule produced by bacteria as part of quorum sensing is considered to be a universal inducer signal in bacteria because it reportedly influences gene expression in a variety of both gram-negative and gram-positive bacteria. The objective of this study was to determine whether selected fresh produce and processed foods have AI-2-like activity and whether specific food additives can act as AI-2 mimics and result in AI-2-like activity. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used as the basis for determining AI-2 activity in the selected foods and food ingredients. Maximum AI-2 activity was seen on the frozen fish sample (203-fold, compared with the negative control) followed by tomato, cantaloupe, carrots, tofu, and milk samples. Interestingly, some samples were capable of inhibiting AI-2 activity. Turkey patties showed the highest inhibition (99.8% compared with the positive control) followed by chicken breast (97.5%), homemade cheeses (93.7%), beef steak (90.6%), and beef patties (84.4%). AI-2 activity was almost totally inhibited by sodium propionate, whereas sodium benzoate caused 93.3% inhibition, compared with 75% inhibition by sodium acetate. Sodium nitrate did not have any appreciable effect, even at 200 ppm. Understanding the relationships that exist between AI-2 activity on foods and the ecology of pathogens and food spoilage bacteria on foods could yield clues about factors controlling food spoilage and pathogen virulence.     

降解温和气单胞菌AHLs乳酸菌的筛选及在三文鱼保鲜中的应用

从传统发酵食品中筛选对温和气单胞菌（Aeromonas sobria）N-酰基高丝氨酸内酯（N-acyl-homoserine lactones,AHLs）信号分子具有降解活性的乳酸菌,对其进行菌种鉴定,探究乳酸菌产生的群体感应淬灭作用酶存在位置与类型。以三文鱼为载体,通过测定细菌菌落总数、持水力、硫代巴比妥酸（thiobarbituric acid,TBA）值以及挥发性盐基氮（total volatile basic nitrogen,TVB-N）值等指标评价乳酸菌抑制温和气单胞菌致腐能力的效果。结果表明：采用96孔板法结合牛津杯法筛选获得1株对温和气单胞菌AHLs降解活性接近100%的菌株YF-8,经鉴定为戊糖片球菌（Pediococcus pentosaceus）。菌株YF-8淬灭酶存在于胞外上清液中,且在酸性、中性条件下均具有降解活性,初步判定为AHLs酰基转移酶。通过生长曲线确定YF-8淬灭酶粗提物在亚抑菌质量浓度2.0、4.0 mg/mL和6.0 mg/mL时不影响温和气单胞菌生长。此外,感染温和气单胞菌的三文鱼经YF-8淬灭酶粗提物处理过后,细菌菌落总数、持水力、TBA值...     

Identities of the Pseudomonas spp. in flora from chilled chicken.

Nine broilers from each of two different broiler farms were collected at the slaughterhouse. Microbiological samples were collected from broiler chicken carcasses which were stored aerobically at 3+/-0.5 degrees C for 0, 3 or 8 days. By characterizing 40 colonies per broiler it was possible to evaluate the shift in psychrotrophic bacteria on the skin during cold storage. Most of these bacteria belong to the pseudomonads. The Shewan scheme was used in order to distinguish between four groups of pseudomonads. On fresh poultry group II pseudomonads were most abundantly represented, followed by group IV; group I and III strains were present in lower amounts. Non-fluorescing group II pseudomonads always predominated as spoilage became obvious (day 8). By including 36 reference strains, numerical analysis based on the simple matching coefficient was performed on 180 representatively selected strains. This revealed that Pseudomonas species indeed predominated when spoilage was obvious. Non-fluorescing species were identified mainly as P. fragi, but also as other strains belonging to P. fluorescens biovars A, B, C and F, P. lundensis and cluster 7 strains (unidentified). Microorganisms already substantially present on the fresh poultry were found in the highest numbers at the time of spoilage.     

Development of the aerobic spoilage flora of chilled rabbit meat

Even though worldwide production of rabbit meat is over 1,000,000ton, little information is available on rabbit meat microbiology. This paper reports on the microflora developing on chill-stored rabbit carcasses. Four different lots of 24h post-mortem rabbit carcasses dressed and kept at 0°C in a medium-size abattoir were collected and evaluated for sensory, physicochemical and microbiological changes during aerobic storage at 3±1°C. Mean initial pH value (pH(24)), extract-release volume (ERV) and lactate content of Biceps femoris muscle, were 6.26±0.20, 13.50±3.50ml and 0.70±0.07%, respectively. As with other muscle foods kept chilled in air, pH increased and ERV and lactate decreased as storage progressed. Initial levels (logcfu/g) of aerobes (APC), psychrotrophic flora, Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae and yeasts were 4.76±0.31, 4.81±0.81, 3.39±1.12, 2.01±0.92, 2.76±0.51, 0.49±0.45 and 3.46±0.32, respectively. Pseudomonads, most of them fluorescent, and to a lesser extent B. thermosphacta and yeasts grew faster than the remaining microorganisms and became predominant at the end of the shelf life. Carcasses spoiled when mean APC, psychrotrophic and pseudomonads numbers were ca. 8logcfu/g, their mean shelf life being estimated at 6.8 days. A lot of DFD-like rabbit carcasses, with higher pH and lower ERV values but similar microbial loads to normal meat, developed a strong putrid odour after 4 days.     

Microbial community analysis of food-spoilage bacteria in commercial custard creams using culture-dependent and independent methods

Custard cream is made from highly nutritive raw materials such as milk and sugar and is easily spoiled by the multiplication of specific microbial contaminants or residents. However, this spoilage microbial community has not been studied. We determined the spoilage microbiota in commercial custard creams using culture-dependent and independent methods. Using the culture-dependent analysis with various agar media, 185 bacterial colonies and 43 eukaryal colonies were isolated from 7 commercial custard cream products. All bacterial isolates were morphologically, physiologically, and genetically identified as bacilli, staphylococci, lactic acid bacteria, and psychrotrophic gram-negative rods. Using culture-independent molecular analysis, the PCR-denaturing gradient gel electrophoresis technique, spoilage of the commercial custard creams was found to be caused by bacilli, staphylococci, lactic acid bacteria, psychrotrophic gram-negative rods, Anoxybacillus sp., Caurobacter sp., and Streptococcus sp. bacteria. The detected spoilage bacteria were the same species as previously detected in spoiled milk products and shown in other reports, suggesting that spoilage bacteria in a raw material easily grow in processed foods made from milk. We determined the spoilage microbial communities in commercial custard creams, and these are the first data concerning spoilage microbiota in nonfermented processed foods using a culture-independent analysis. Our study will be useful for the manufacture and safe preservation of dairy products because the first step toward safe food preservation by food manufacturers is to understand the spoilage microbiota in a target food to select optimal preservatives and to reduce the use of food additives.     

MICROBIOLOGICAL STUDY OF ARZÚA CHEESE (NW SPAIN) THROUGHOUT CHEESEMAKING AND RIPENING

Changes during production and ripening in the microbial flora of 11 batches of Arzúa, a soft cheese made from raw cow's milk, were investigated. The following microbial groups were counted on the surface and interior of the cheese: total viable count (TVC), lactic acid bacteria (LAB), halotolerant flora, enterococci, proteolytic enterococci, staphylococci, Staphylococcus aureus, Enterobacteriaceae, faecal coliforms, molds, yeasts, Listeria spp. and (in milk) Brucella spp. pH and water activity were also determined. TVC and LAB were, generally, more than 9 log (cfu/g). Enterococci counts increased gradually, reaching values in excess of 6 log units. Halotolerant flora and staphylococci remained practically constant throughout ripening, at 6–8 and 5–7 log units, respectively. Maximum Enterobacteriaceae and faecal coliform counts exceeded 7 and 6 log units, respectively. Brucella spp. were not detected in any of the milk samples. Listeria spp. were detected in four batches, and Listeria monocytogenes in two.     

Possible involvement of psychrotolerant Enterobacteriaceae in blown pack spoilage of vacuum-packaged raw meats

Recent investigations of blown pack spoilage in New Zealand chilled vacuum-packaged meats have found moderate to high numbers of Enterobacteriaceae in the spoilage flora, but no clostridia, such as C. estertheticum and C. gasigenes, that are usually associated with blown pack spoilage. This study showed that pyschrotolerant Enterobacteriaceae produced gas in a lamb homogenate model under anaerobic conditions and that these organisms could cause blown pack spoilage of vacuum-packaged chilled meats. Significant gas production was observed with the majority of the psychrotolerant Enterobacteriaceae strains tested including presumptive species of Enterobacter, Serratia, Hafnia and Rahnella. However, no gas was produced in lamb homogenates inoculated with presumptive species of Ewingella americana or Yersinia enterocolitica. Gas production was also confirmed in vacuum-packaged lamb shoulders stored at 4 degrees C for 21 days after being inoculated with individual representative Enterobacteriaceae isolates. Biochemical characterisation proved to be more useful than genotype-based typing of 16S rRNA genes for discriminating different psychrotolerant Enterobacteriaceae from naturally contaminated meat microflora.     

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.     

Isolation of lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms associated with fresh meat

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.     

The spoilage microflora of cured, cooked turkey breasts prepared commercially with or without smoking

Lactobacillus sakei subsp. carnosus was predominant in the spoilage flora of sliced, vacuum-packed, smoked, oven-cooked turkey breast fillets which developed mild, sour spoilage flavors after 4 weeks storage at 4 degrees C. In contrast, Leuconostoc mesenteroides subsp. mesenteroides predominated in the spoilage flora of sliced, vacuum-packed, unsmoked, boiled turkey breast fillets from the same plant which were also stored at 4 degrees C. The spoilage flora of the unsmoked breasts grew faster than that of the smoked breasts and was more diverse. Lactobacillus sakei, Weissella viridescens and an atypical group of leuconostoc-like bacteria were also members of the unsmoked turkey breasts flora. Consequently, the unsmoked breasts spoiled after 2 weeks at 4 degrees C: the packs swelled and the meat developed strong sour odors and flavors and abundant slime. Except for the unidentified leuconostocs, which apparently survived boiling of the unsmoked turkey, all the spoilage organisms contaminated the meats during the slicing and vacuum packaging operations. From their biochemical reactions and cellular fatty acid profiles, the atypical leuconostocs were more closely related to Leuconostoc carnosum than W. viridescens. Carnobacteria and Brochothrix thermosphacta were present in relatively large numbers on the raw turkey, but were not numerous in the spoilage flora of the cooked, vacuum-packed meat products.