The gut microbiome of farmed Arctic char (Salvelinus alpinus) is shaped by feeding stage and nutrient presence.

The gut microbiome plays an important role in maintaining health and productivity of farmed fish. However, the functional role of most gut microorganisms remains unknown. Identifying the stable members of the gut microbiota and understanding their functional roles could aid in the selection of positive traits or act as a proxy for fish health in aquaculture. Here, we analyse the gut microbial community of farmed juvenile Arctic char (Salvelinus alpinus) and reconstruct the metabolic potential of its main symbionts. The gut microbiota of Arctic char undergoes a succession in community composition during the first weeks post-hatch, with a decrease in Shannon diversity and the establishment of three dominant bacterial taxa. The genome of the most abundant bacterium, a Mycoplasma sp., shows adaptation to rapid growth in the nutrient-rich gut environment. The second most abundant taxon, a Brevinema sp., has versatile metabolic potential, including genes involved in host mucin degradation and utilization. However, during periods of absent gut content, a Ruminococcaceae bacterium becomes dominant, possibly outgrowing all other bacteria through the production of secondary metabolites involved in quorum sensing and cross-inhibition while benefiting the host through short-chain fatty acid production. Whereas Mycoplasma is often present as a symbiont in farmed salmonids, we show that the Ruminococcaceae species is also detected in wild Arctic char, suggesting a close evolutionary relationship between the host and this symbiotic bacterium.

The Anti-Listeria Activity of Pseudomonas fluorescens Isolated from the Horticultural Environment in New Zealand.

Beneficial bacteria with antibacterial properties are attractive alternatives to chemical-based antibacterial or bactericidal agents. Our study sourced such bacteria from horticultural produce and environments to explore the mechanisms of their antimicrobial properties. Five strains of Pseudomonas fluorescens were studied that possessed antibacterial activity against the pathogen Listeria monocytogenes. The vegetative culture of these strains (Pseudomonas fluorescens-PFR46I06, Pseudomonas fluorescens-PFR46H06, Pseudomonas fluorescens-PFR46H07, Pseudomonas fluorescens-PFR46H08 and Pseudomonas fluorescens-PFR46H09) were tested against Listeria monocytogenes (n = 31), Listeria seeligeri (n = 1) and Listeria innocua (n = 1) isolated from seafood and horticultural sources and from clinical cases (n = 2) using solid media coculture and liquid media coculture. All Listeria strains were inhibited by all strains of P. fluorescens; however, P. fluorescens-PFR46H07, P. fluorescens-PFR46H08 and P. fluorescens-PFR46H09 on solid media showed good inhibition, with average zones of inhibition of 14.8 mm, 15.1 mm and 18.2 mm, respectively, and the other two strains and P. fluorescens-PFR46H09 had a significantly greater zone of inhibition than the others (p < 0.05). There was no inhibition observed in liquid media coculture or in P. fluorescens culture supernatants against Listeria spp. by any of the P. fluorescens strains. Therefore, we hypothesized that the structural apparatus that causes cell-to-cell contact may play a role in the ejection of ant-listeria molecules on solid media to inhibit Listeria isolates, and we investigated the structural protein differences using whole-cell lysate proteomics. We paid special attention to the type VI secretion system (TSS-T6SS) for the transfer of effector proteins or bacteriocins. We found significant differences in the peptide profiles and protein summaries between these isolates' lysates, and PFR46H06 and PFR46H07 possessed the fewest secretion system structural proteins (12 and 11, respectively), while PFR46H08 and PFR46H09 had 18 each. P. fluorescens-PFR46H09, which showed the highest antimicrobial effect, had nine tss-T6SS structural proteins compared to only four in the other three strains.

Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

Impact of Putative Probiotics on Growth, Behavior, and the Gut Microbiome of Farmed Arctic Char (Salvelinus alpinus).

Beneficial bacteria promise to promote the health and productivity of farmed fish species. However, the impact on host physiology is largely strain-dependent, and studies on Arctic char (Salvelinus alpinus), a commercially farmed salmonid species, are lacking. In this study, 10 candidate probiotic strains were subjected to in vitro assays, small-scale growth trials, and behavioral analysis with juvenile Arctic char to examine the impact of probiotic supplementation on fish growth, behavior and the gut microbiome. Most strains showed high tolerance to gastric juice and fish bile acid, as well as high auto-aggregation activity, which are important probiotic characteristics. However, they neither markedly altered the core gut microbiome, which was dominated by three bacterial species, nor detectably colonized the gut environment after the 4-week probiotic treatment. Despite a lack of long-term colonization, the presence of the bacterial strains showed either beneficial or detrimental effects on the host through growth rate enhancement or reduction, as well as changes in fish motility under confinement. This study offers insights into the effect of bacterial strains on a salmonid host and highlights three strains, Carnobacterium divergens V41, Pediococcus acidilactici ASG16, and Lactiplantibacillus plantarum ISCAR-07436, for future research into growth promotion of salmonid fish through probiotic supplementation.

Development of a rapid qPCR method to quantify lactic acid bacteria in cold-smoked salmon.

Quantification of lactic acid bacteria (LAB) is essential to control quality of seafood products like cold-smoked salmon (CSS). In the present study, we report the design and optimization of a dual-labelled TaqMan ™ probe targeting the V7 region of 16S rRNA gene for the detection of LAB in CSS. This quantitative PCR (qPCR) assays is useful for the simultaneous detection of the ten LAB genera communally encountered in CSS as Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Macrococcus, Streptococcus, Vagococcus and Weissella. The specificity of this method was demonstrated against 14 genera (44 isolates, 35 species) of Gram-positive bacteria and 19 genera of Gram-negative (40 isolates, 34 species). Calibration of the method was performed in CSS matrix using a mix of equimolar cultured solution of five LAB. Quantification with the qPCR method range from 3.5 to 8.5 Log CFU/g in CSS matrix, covering 5 orders of magnitude. On these artificially contaminated CSS slices, PCR method results correlated successfully (R2 = 0.9945) with the conventional enumeration on Elliker medium. In addition, the new method was successful on commercial CSS from five different origins with a quantification range from 3.7 Log CFU/g to 8.0 Log CFU/g. This one-step quantitative methodology is proposed as a rapid and complementary tool of the cultural methods to investigate the LAB microbiota and biodiversity of CSS.

Effect of the Manufacturing Process on the Microbiota, Organoleptic Properties and Volatilome of Three Salmon-Based Products.

Lightly preserved seafood products, such as cold-smoked fish and fish gravlax, are traditionally consumed in Europe and are of considerable economic importance. This work aimed to compare three products that were obtained from the same batch of fish: cold-smoked salmon (CSS) stored under vacuum packaging (VP) or a modified atmosphere packaging (MAP) and VP salmon dill gravlax (SG). Classical microbiological analyses and 16S rRNA metabarcoding, biochemical analyses (trimethylamine, total volatile basic nitrogen (TVBN), biogenic amines, pH, volatile organic compounds (VOCs)) and sensory analyses (quantitative descriptive analysis) were performed on each product throughout their storage at a chilled temperature. The three products shared the same initial microbiota, which were mainly dominated by Photobacterium, Lactococcus and Lactobacillus genera. On day 28, the VP CSS ecosystem was mainly composed of Photobacterium and, to a lesser extent, Lactococcus and Lactobacillus genera, while Lactobacillus was dominant in the MAP CSS. The diversity was higher in the SG, which was mainly dominated by Enterobacteriaceae, Photobacterium, Lactobacillus and Lactococcus. Although the sensory spoilage was generally weak, gravlax was the most perishable product (slight increase in amine and acidic off-odors and flavors, fatty appearance, slight discoloration and drop in firmness), followed by the VP CSS, while the MAP CSS did not spoil. Spoilage was associated with an increase in the TVBN, biogenic amines and spoilage associated VOCs, such as decanal, nonanal, hexadecanal, benzaldehyde, benzeneacetaldehyde, ethanol, 3-methyl-1-butanol, 2,3-butanediol, 1-octen-3-ol, 2-butanone and 1-octen-3-one. This study showed that the processing and packaging conditions both had an effect on the microbial composition and the quality of the final product.

Genomic diversity of Serratia proteamaculans and Serratia liquefaciens predominant in seafood products and spoilage potential analyses.

Serratia sp. cause food losses and waste due to spoilage; it is noteworthy that they represent a dominant population in seafood. The main spoilage associated species comprise S. liquefaciens, S. grimesii, S. proteamaculans and S. quinivorans, also known as S. liquefaciens-like strains. These species are difficult to discriminate since classical 16S rRNA gene-based sequences do not possess sufficient resolution. In this study, a phylogeny based on the short-length luxS gene was able to speciate 47 Serratia isolates from seafood, with S. proteamaculans being the main species from fresh salmon and tuna, cold-smoked salmon, and cooked shrimp while S. liquefaciens was only found in cold-smoked salmon. The genome of the first S. proteamaculans strain isolated from the seafood matrix (CD3406 strain) was sequenced. Pangenome analyses of S. proteamaculans and S. liquefaciens indicated high adaptation potential. Biosynthetic pathways involved in antimicrobial compounds production and in the main seafood spoilage compounds were also identified. The genetic equipment highlighted in this study contributed to gain further insights into the predominance of Serratia in seafood products and their capacity to spoil.

New Insight into Antimicrobial Compounds from Food and Marine-Sourced Carnobacterium Species through Phenotype and Genome Analyses.

Carnobacterium maltaromaticum and Carnobacterium divergens, isolated from food products, are lactic acid bacteria known to produce active and efficient bacteriocins. Other species, particularly those originating from marine sources, are less studied. The aim of the study is to select promising strains with antimicrobial potential by combining genomic and phenotypic approaches on large datasets comprising 12 Carnobacterium species. The biosynthetic gene cluster (BGCs) diversity of 39 publicly available Carnobacterium spp. genomes revealed 67 BGCs, distributed according to the species and ecological niches. From zero to six BGCs were predicted per strain and classified into four classes: terpene, NRPS (non-ribosomal peptide synthetase), NRPS-PKS (hybrid non-ribosomal peptide synthetase-polyketide synthase), RiPP (ribosomally synthesized and post-translationally modified peptide). In parallel, the antimicrobial activity of 260 strains from seafood products was evaluated. Among the 60% of active strains, three genomes were sequenced and submitted to a dereplication process. C. inhibens MIP2551 produced a high amountof H2O2, probably thanks to the presence of four oxidase-encoding genes. C. maltaromaticum EBP3019 and SF668 strains were highly efficient against Listeria monocytogenes. A new extracellular 16 kDa unmodified bacteriocin in the EBP3019 strain and five different bacteriocins in SF668 were highlighted. In this study, the overview of antimicrobial BGC and inhibitory activities of Carnobacterium spp. allowed the prediction of potential innovative natural products that could be relevant for biotechnological applications.

Salmon Gravlax Biopreservation With Lactic Acid Bacteria: A Polyphasic Approach to Assessing the Impact on Organoleptic Properties, Microbial Ecosystem and Volatilome Composition.

Seafood and fishery products are very perishable commodities with short shelf-lives owing to rapid deterioration of their organoleptic and microbiological quality. Microbial growth and activity are responsible for up to 25% of food losses in the fishery industry. In this context and to meet consumer demand for minimally processed food, developing mild preservation technologies such as biopreservation represents a major challenge. In this work, we studied the use of six lactic acid bacteria (LAB), previously selected for their properties as bioprotective agents, for salmon dill gravlax biopreservation. Naturally contaminated salmon dill gravlax slices, with a commercial shelf-life of 21 days, were purchased from a French industrial company and inoculated by spraying with the protective cultures (PCs) to reach an initial concentration of 106 log CFU/g. PC impact on gravlax microbial ecosystem (cultural and acultural methods), sensory properties (sensory profiling test), biochemical parameters (pH, TMA, TVBN, biogenic amines) and volatilome was followed for 25 days of storage at 8°C in vacuum packaging. PC antimicrobial activity was also assessed in situ against Listeria monocytogenes. This polyphasic approach underlined two scenarios depending on the protective strain. Carnobacterium maltaromaticum SF1944, Lactococcus piscium EU2229 and Leuconostoc gelidum EU2249, were very competitive in the product, dominated the microbial ecosystem, and displayed antimicrobial activity against the spoilage microbiota and L. monocytogenes. The strains also expressed their own sensory and volatilome signatures. However, of these three strains, C. maltaromaticum SF1944 did not induce strong spoilage and was the most efficient for L. monocytogenes growth control. By contrast, Vagococcus fluvialis CD264, Carnobacterium inhibens MIP2551 and Aerococcus viridans SF1044 were not competitive, did not express strong antimicrobial activity and produced only few organic volatile compounds (VOCs). However, V. fluvialis CD264 was the only strain to extend the sensory quality, even beyond 25 days. This study shows that C. maltaromaticum SF1944 and V. fluvialis CD264 both have a promising potential as bioprotective cultures to ensure salmon gravlax microbial safety and sensorial quality, respectively.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes.

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow's milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.

Bioprotective Effect of Lactococcus piscium CNCM I-4031 Against Listeria monocytogenes Growth and Virulence.

Listeria monocytogenes is a Gram-positive pathogen occurring in many refrigerated ready-to-eat foods. It is responsible for foodborne listeriosis, a rare but severe disease with a high mortality rate (20-30%). Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp and in a chemically defined medium using a cell-to-cell contact-dependent mechanism. To characterize this inhibition further, the effect of L. piscium was tested on a collection of 42 L. monocytogenes strains. All strains were inhibited but had different sensitivities. The effect of the initial concentration of the protective and the target bacteria revealed that the inhibition always occurred when L. piscium had reached its maximum population density, whatever the initial concentration of the protective bacteria. Viewed by scanning electron microscopy, L. monocytogenes cell shape and surface appeared modified in co-culture with L. piscium CNCM I-4031. Lastly, L. monocytogenes virulence, evaluated by a plaque-forming assay on the HT-29 cell line, was reduced after cell pre-treatment by the protective bacteria. In conclusion, the bioprotective effect of L. piscium toward L. monocytogenes growth and virulence was demonstrated, and a hypothesis for the inhibition mechanism is put forward.

Effect of vacuum and modified atmosphere packaging on the microbiological, chemical and sensory properties of tropical red drum (Sciaenops ocellatus) fillets stored at 4°C.

AIMS: The effect of vacuum (VP - 4°C) and CO2/N2-atmosphere (MAP - 4°C) packaging on the quality of red drum fillets compared with whole gutted iced fish was investigated. METHODS AND RESULTS: A metagenomic approach, bacterial enumeration and isolation, biochemical and sensory analyses were carried out. The organoleptic rejection of whole fish was observed at day 15 whereas VP and MAP fillets appeared unacceptable only after 29days. At these dates, total mesophilic counts reached 107-108CFU g-1. According to Illumina MiSeq sequencing, Arthrobacter, Chryseobacterium, Brevibacterium, Staphylococcus and Kocuria were the main genera of the fresh red drum fillets. At the sensory rejection time, lactic acid bacteria (LAB), particularly Carnobacterium sp., dominated the microbiota of both types of packaging. The pH value of fresh samples was between 5.96 and 6.37 and did not vary greatly in all trials. Total volatile basic nitrogen (TVBN) and trimethylamine (TMA) concentrations were low and not represent reliable indicators of the spoilage, contrary to some biogenic amines (cadaverine, putrescine and tyramine). CONCLUSION: Chilled packed fillets of red drum have an extended shelf-life compared to whole gutted iced fish. Overall, few differences in sensory and microbial quality were observed between the VP and MAP samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Next-Generation Sequencing (NGS) provided data on the microbiota of a tropical fish.

Complete Genome Sequence of Lactococcus piscium CNCM I-4031, a Bioprotective Strain for Seafood Products.

Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria.

Improving simultaneously the quality and safety of cooked and peeled shrimp using a cocktail of bioprotective lactic acid bacteria.

Tropical shrimp is of considerable economic importance in the world but is highly perishable due to microbial and chemical degradation. Biopreservation is a food preservation technology based on the addition of "positive" bacteria able to kill or prevent the growth of undesirable microorganisms. Two strains of lactic acid bacteria (LAB) have previously been selected for a biopreservation strategy: Lactococcus piscium CNCM I-4031, for its ability to prevent the sensory deterioration of seafood and Carnobacterium divergens V41, which inhibits growth of Listeria monocytogenes. The objective was to test the association of the two strains to improve both the quality and safety of shrimp. In a first trial, the two LAB were inoculated alone or in a cocktail in cooked and peeled shrimp (CPS) Penaeus vannamei at 5×105CFU/g. Chemical, sensory and microbiological analyses by culture-dependent and -independent methods were performed during storage under modified atmosphere packaging (MAP) at 8°C. The results were compared to a non-inoculated batch. In a second trial, the same experiments were repeated in the presence of 102CFU/g of L. monocytogenes RF191. The microbiota of CPS was composed of LAB, Shewanella spp. and Enterobacteriaceae. Brochothrix thermosphacta was not detected. L. piscium and C. divergens reached 108 and 109CFU/g, respectively, in 7days and did not inhibit each other when co-inoculated. L. piscium reduced L. monocytogenes by 1Log (CFU/g) for 28days. C. divergens had an immediate listericidal effect lasting 7days. A regrowth of L. monocytogenes was then observed but the count was always 2 to 5Log (CFU/g) lower than in the control. No additional or synergic effect between protective strains was observed and the cocktail had the same inhibitory effect as C. divergens alone. C. divergens was very effective at preventing the sensory deterioration of CPS. This may be related to the inhibition of Shewanella and Enterobacteriaceae. However, the panelists could detect the presence of C. divergens during the first 10days of storage, with slight unpleasant odors and flavors. L. piscium improved the sensory quality of CPS for 14days only. In co-culture, L. piscium eliminated the off-odors and flavors released by C. divergens in the early stage of storage and the co-culture allowed maintaining a good quality of CPS throughout the storage. Therefore, the use of a cocktail of L. piscium CNCM I-4031 and C. divergens V41 is recommended in a strategy of biopreservation of shrimp.

Quality assessment of ice-stored tropical yellowfin tuna (Thunnus albacares) and influence of vacuum and modified atmosphere packaging.

Metagenomic, microbial, chemical and sensory analyses of Thunnus albacares from Martinique stored in ice (AIR - 0 °C), vacuum (VP - 4/8 °C) and modified atmosphere packaging (MAP - 4/8 °C) (70% CO2 - 30% O2) were carried out. The organoleptic rejection of AIR tuna was observed at day 13 when total bacterial counts equaled 10(6)-10(7) CFU g(-1). No extension of shelf-life was provided by VP and MAP. According to 16S rRNA gene sequence analyzed by Illumina MiSeq and PCR-TTGE, Rhodanobacter terrae was the main species of the freshly caught tuna. At the sensory rejection time, Brochothrix thermosphacta and Pseudomonas dominated the AIR products while B. thermosphacta alone or a mix of B. thermosphacta, Enterobacteriaceae and lactic acid bacteria (LAB) dominated the microbiota of MAP and VP products, respectively. The pH value remained stable in all trials, ranging from 5.77 to 5.97. Total volatile basic nitrogen (TVBN) and trimethylamine (TMA-N) concentrations were weak and not significantly different between batches. Lipid oxidation increased in the samples containing O2 (MAP > AIR). The initial concentration of histamine was high (75-78 mg kg(-1)) and stable up to 8 days but then significantly decreased in all trials to reach 25-30 mg kg(-1), probably due to the presence of histamine-decomposing bacteria.

Inhibition mechanism of Listeria monocytogenes by a bioprotective bacteria Lactococcus piscium CNCM I-4031.

Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3-4 log CFU g(-1) after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent.

Selection of bioprotective cultures for preventing cold-smoked salmon spoilage.

Biopreservation is a natural technology of food preservation, which consists of inoculating food with microorganisms selected for their antibacterial properties. The objective of this study was to select lactic acid bacteria (LAB) to improve the quality of cold-smoked salmon (CSS). In this work, different strains representative of the 4 dominant species, identified in a previous study by pyrosequencing the 16S rRNA gene, were isolated and their spoiling potential in CSS blocks, sterilized by ionization, was assessed by twelve trained panelists along the vacuum storage at 8°C. Photobacterium phosphoreum, Brochothrix thermosphacta and Serratia proteamaculans released strong off-odors whereas the spoiling potential of Carnobacterium divergens was weaker. The spoiling capacity of Lactococcus piscium EU2241, Leuconostoc gelidum EU2247, Lactobacillus sakei EU2885, Staphylococcus equorum S030674 and 4 commercial starters was tested by the same method and 2 strains were eliminated due to off-odor production. The effect of the 6 selected LAB against the 4 specific spoiling organisms (SSOs) selected was tested by challenge tests in sterile CSS blocks. The protective effect of the LAB differed from one SSO to another and no correlation could be established between the sensory improvement, SSO inhibition, and the implantation or acidification of protective cultures (PCs). All the PCs except L. piscium reduced the off-odors released by P. phosphoreum although some of them had no effect on its growth. S. equorum, which did not grow in CSS, favored the implantation of P. phosphoreum but prevented its off-odor formation. L. piscium was the only strain that prevented the spoilage of B. thermosphacta and S. proteamaculans although it did not grow very well and did not acidify the product. L. gelidum EU2247 inhibited the growth of these 2 SSOs and lowered the pH but had no effect on the sensory quality. Finally, L. piscium was tested in 2 naturally contaminated products, with a positive effect on 1 batch. This effect was not correlated with the microbial ecosystem as determined by acultural and cultural techniques. Based on these results, the selection strategy is discussed.

Development of a real-time PCR method coupled with a selective pre-enrichment step for quantification of Morganella morganii and Morganella psychrotolerans in fish products.

Histamine fish poisoning is common and due to toxic concentrations of histamine often produced by Gram-negative bacteria in fin-fish products with a high content of the free amino acid histidine. The genus Morganella includes two species previously reported to cause incidents of histamine fish poisoning. Morganella morganii and Morganella psychrotolerans are both strong producer of histamine. However, little is known about the occurrence and critical stages for fish contamination with these bacteria. To elucidate contamination routes of Morganella, specific real-time quantitative PCR (RTi qPCR) methods for quantification of M. morganii and M. psychrotolerans have been developed. Selective primers amplified a 110 bp region of the vasD gene for M. psychrotolerans and a 171 bp region of the galactokinase gene for M. morganii. These primer-sets showed high specificity as demonstrated by using purified DNA from 23 other histamine producing bacteria and 26 isolates with no or limited histamine production. The efficiency of the qPCR reactions on artificially contaminated fish samples were 100.8% and 96.3% respectively. The limit of quantification (LOQ) without enrichment was 4 log CFU/g. A quantitative enrichment step with a selective medium was included and improved the sensitivity of the methods to a LOQ of below 50 CFU/g in seafood. RTi qPCR methods with or without enrichment were evaluated for enumeration of Morganella species in naturally contaminated fresh fish and lightly preserved seafood from Denmark. These new methods will contribute to a better understanding of the occurrence and histamine production by Morganella species in fish products, information that is essential to reduce the unacceptably high frequency of histamine fish poisoning.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.