Identification of novel Carnobacterium maltaromaticum strains in bone marrow samples of patients with acute myeloid leukemia using a metagenomic binning approach.

The aim of this study aimed to examine the existence of a bacterial metagenome in the bone marrow of patients with acute myeloid leukemia (AML). We re-examined whole-genome sequencing data from the bone marrow samples of seven patients with AML, four of whom were remitted after treatment, for metagenomic analysis. After the removal of human reads, unmapped reads were used to profile the species-level composition. We used the metagenomic binning approach to confirm whether the identified taxon was a complete genome of known or novel strains. We observed a unique and novel microbial signature in which Carnobacterium maltaromaticum was the most abundant species in five patients with AML or remission. The complete genome of C. maltaromaticum "BMAML_KR01," which was observed in all samples, was 100% complete with 8.5% contamination and closely clustered with C. maltaromaticum strains DSM20730 and SF668 based on single nucleotide polymorphism variations. We identified five unique proteins that could contribute to cancer progression and 104 virulent factor proteins in the BMAML_KR01 genome. To our knowledge, this is the first report of a new strain of C. maltaromaticum in patients with AML. The presence of C. maltaromaticum and its new strain in patients indicates an urgent need to validate the existence of this bacterium and evaluate its pathophysiological role.

Characterization of novel bacteriocin produced by bacteriocinogenic Carnobacterium maltaromaticum isolated from raw milk.

This study was designed to assess newly isolated bacteriocin-producing strain as potential food preservative. A bacteriocin producing lactic acid bacterium, named Carnobacterium maltaromatium KCA018, was screened from raw milk using deferred and spot-on-the-lawn assays. The crude cell free supernatant (CFS) was purified to obtain proteinaceous bacteriocin by ammonium sulfate precipitation (assigned as bacteriocin KCA) and tested for bacteriocin production, physical stability, antimicrobial activity, and bacteriocin-encoding gene detection. The growth curves of C. maltaromatium KCA018 reached late exponential phase after 15 h of incubation at 25 °C and 30 °C (Fig. 2). The maximum production of bacteriocin KCA was reached after 12 h of incubation at 25 °C, showing the antimicrobial activity of more than 3000 AU/ml against Listeria monocytogenes. The purified bacteriocin KCA was stable up to 67 °C for 30 min of exposure and between pH 4 and 7, showing more than 6000 AU/ml. The antibacterial activity of bacteriocin KCA was lost in the presence of pronase, proteinase K, and trypsin. Purified bacteriocin KCA showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. The CFS and purified bacteriocin KCA effectively inhibited the growth of L. monocytogenes ATCC 1911, E. faecalis ATCC 19433, and E. feacium ATCC 11576. The molecular weight of purified bacteriocin KCA was estimated at approximately 5 kDa. The positive amplification was observed for pisA and cbnBM1 with approximately between 100 and 200 bp. The newly identified bacteriocin can be a promising preservative for application in food.

Characterization of bacterial communities in Coregonus peled fillets during chilled storage and interactions between selected bacterial strains.

AIM: Coregonus peled fillets were used as a model to evaluate the dominant bacterial growth of chilled fish during storage after shipping and interactions of selected bacterial strains. METHODS AND RESULTS: Coregonus peled fillets were transported by air and land in ice boxes about 48 h from aquatic products company in Xinjiang, China, to the laboratory located in Dalian, China. Both culture-dependent (plate counts on nonselective media) based on 16S rRNA gene sequencing and culture-independent (Illumina-MiSeq high-throughput sequencing) methods were used. To detect interactions among bacterial populations from chilled fish, the influence of 18 test strains on the growth of 12 indicator isolates was measured by a drop assay and in liquid culture medium broth. The results showed that bacterial counts exceeded 7.0 log CFU/g following storage for 4 days at 4 °C. When the bacterial counts exceeded 8.5 log CFU/g after 12 days, the predominant micro-organisms were Aeromonas, Pseudomonas, Carnobacterium, Psychrobacter and Shewanella, as measured by the culture-independent method. All test strains showed inhibiting effects on the growth of other strains in liquid culture. Pseudomonas isolates showed antibacterial activity for approximately 60% of the indicator strains on nutritional agar plates. The majority of test isolates enhancing indicator strain growth were the strains isolated on day 0. CONCLUSIONS: High-throughput sequencing approach gives whole picture of bacterial communities in chilled C. peled fillets during storage, while growth interferences between selected bacterial strains illustrate the complexity of microbial interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: We determined the bacterial communities and growth interferences in chilled Coregonus peled after shipping and these are the first data concerning microbiota in C. peled using a culture-independent analysis. The present study will be useful for manufacture and preservation of C. peled products by providing with valuable information regarding microbiological spoilage of C. peled.

Investigation of the microbiota associated with ungerminated and germinated Norwegian barley cultivars with focus on lactic acid bacteria.

The microbial community of ungerminated and germinated barley grains from three different cultivars grown at four different locations in Norway was investigated by culture dependent and culture independent methods. Lactic acid bacteria (LAB) was focused in this study and was isolated from germinated barley. The number of LAB ranged between 2.8 and 4.6 log cfu/g in ungerminated grains and between 4.9 and 6.3 log cfu/g in germinated grains. In total 66 out of 190 isolates were Gram+, catalase-negative and presumptive LAB. The LAB isolates were by 16S rRNA sequencing identified to be Carnobacterium maltaromaticum (6), Lactococcus lactis (2), Enterococcus sp. (1) and Leuconostoc sp. (57). Germination significantly influenced the bacterial composition. Regarding the different cultivars and growth places no significant difference in bacterial composition was seen. The most abundant bacterial genus was Pantoea (18.5% of the total sequences), followed by Rhizobium (10.1%) and Sphingomonas (9.9%). Fungal composition was significantly influenced by the germination process and the cultivation place, but no significant difference in fungal composition was detected between the 3 cultivars. The most abundant fungal genera were Cryptococcus (43.8% of all the sequences), Cladosporium (8.2%), Pyrenophora (7.4%) and Vagicola (6.3%). This study revealed knowledge of barley grain associated microbes of Norwegian barley that can be useful to control the malt quality. Germination affected both bacterial and fungal microbiota composition. No difference in bacterial microbiota composition was seen regarding cultivars and cultivation place, however, the fungal microbiota composition was significantly influenced by the cultivation place. Differences in fungal community of ungerminated and germinated barley samples of different geographical locations were more pronounced than differences in bacterial communities.

Genome-wide comparison of Carnobacterium maltaromaticum derived from diseased fish harbouring important virulence-related genes.

Although Carnobacterium maltaromaticum has been used as a probiotic in fish, it was reported to cause disease for the first time in Korea. The objective of this study was to understand the differences between pathogenic and non-pathogenic strains. Pathogenicity was tested by challenging rainbow trout with C. maltaromaticum ATCC35586 and 18ISCm isolated from diseased fish, and DSM20342 isolated from a dairy product. We also compared 24 genomes of C. maltaromaticum strains plus the genome of our isolate 18ISCm sequenced in this study. Only the strains from diseased fish caused high mortality with severe histopathological changes. Although all strains shared more than 90% of Ko_id, wecC and xtmA were found only in strains from diseased fish. Interestingly, only strains from diseased fish harboured two wecC paralogs involved in the production of D-mannosaminuronic acid which is a major component of a well-known virulence factor, teichuronic acid. Two wecC paralogs of 18ISCm were increased when they were co-cultured with trout blood cells, suggesting that wecC genes might play a role in virulence. The results of this study show that strains isolated from diseased fish are different from strains derived from food in terms of pathogenicity to fish and the presence of virulence-related genes.

The membrane topology of immunity proteins for the two-peptide bacteriocins carnobacteriocin XY, lactococcin G, and lactococcin MN shows structural diversity.

The two-peptide bacteriocins produced by Gram-positive bacteria require two different peptides, present in equimolar amounts, to elicit optimal antimicrobial activity. Producer organisms are protected from their bacteriocin by a dedicated immunity protein. The immunity proteins for two-peptide bacteriocins contain putative transmembrane domains (TMDs) and might therefore be associated with the membrane. The immunity protein CbnZ for the two-peptide bacteriocin carnobacteriocin XY (CbnXY) was identified by heterologously expressing the cbnZ gene in sensitive host strains. Using protein topology prediction methods and the dual pho-lac reporter system, we mapped out the membrane topology of CbnZ, along with those of the immunity proteins LagC and LciM for the two-peptide bacteriocins lactococcin G and lactococcin MN, respectively. Our results reveal wide structural variety between these immunity proteins that can contain as little as one TMD or as many as four TMDs.

Complementary Antibacterial Effects of Bacteriocins and Organic Acids as Revealed by Comparative Analysis of Carnobacterium spp. from Meat.

Carnobacterium maltaromaticum and Carnobacterium divergens are often predominant in the microbiota of vacuum-packaged (VP) meats after prolonged storage at chiller temperatures, and more so in recent studies. We investigated the antibacterial activities of C. maltaromaticum and C. divergens (n = 31) from VP meats by phenotypic characterization and genomic analysis. Five strains showed antibacterial activities against Gram-positive bacteria in a spot-lawn assay, with C. maltaromaticum strains having an intergeneric and C. divergens strains an intrageneric inhibition spectrum. This inhibitory activity is correlated with the production of predicted bacteriocins, including carnobacteriocin B2 and carnolysin for C. maltaromaticum and divergicin A for C. divergens The supernatants of both species cultured in meat juice medium under anaerobic conditions retarded the growth of most Gram-positive and Gram-negative bacteria in broth assay in a strain-dependent manner. C. maltaromaticum and C. divergens produced formate and acetate but not lactate under VP meat-relevant conditions. The relative inhibitory activity by Carnobacterium strains was significantly correlated (P < 0.05) to the production of both acids. Genomic analysis revealed the presence of genes required for respiration in both species. In addition, two clusters of C. divergens have an average nucleotide identity below the cutoff value for species delineation and thus should be considered to be two subspecies. In conclusion, both bacteriocins and organic acids are factors contributing significantly to the antibacterial activity of C. maltaromaticum and C. divergens under VP meat-relevant conditions. A few Carnobacterium strains can be explored as protective cultures to extend the shelf life and improve the safety of VP meats.IMPORTANCE The results of this study demonstrated that both bacteriocins and organic acids are important factors contributing to the antibacterial activities of Carnobacterium from vacuum-packaged (VP) meats. This study demonstrated that formate and acetate are the key organic acids produced by Carnobacterium and demonstrated their association with the inhibitory activity of carnobacteria under VP meat-relevant storage conditions. The role of lactate, on the other hand, may not be as important as previously believed in the antimicrobial activities of Carnobacterium spp. on chilled VP meats. These findings advance our understanding of the physiology of Carnobacterium spp. to better explore their biopreservative properties for chilled VP meats.

Microbiota encompassing putative spoilage bacteria in retail packaged broiler meat and commercial broiler abattoir.

It is well established, that certain bacteria within the Brochothrix, Carnobacterium, Lactobacillus, Lactococcus, and Leuconostoc genera have an important role in the spoilage of chill stored poultry meat packaged in modified atmosphere. However, little is known about the role of microorganisms that are difficult to culture and the microbiota during poultry spoilage. We combined traditional cultivation and culture-independent 16S rRNA amplicon sequencing to investigate the microbiota encompassing putative bacteria of whole broiler meat, packaged in modified atmosphere, during and exceeding shelf-life. Samples were taken from 6 flocks during independent slaughter days. Additional samples were analysed from the production line. There was a significant difference in the microbial community structure of 80%O2/20%CO2 retail packaged broiler meat during different times of shelf-life, mainly due to an increase of species within the Brochothrix, Carnobacterium, Vagococcus, and Janthinobacterium genera. These genera were already detected four to eight days after slaughter. However, no significant difference between flocks with respect to the microbiota encompassing putative spoilage bacteria was observed when examined in retail packaged broilers, slaughtered at the same abattoir on different days. Our study also showed that lactic acid bacteria within the Vagococcus genus can constitute a dominating part of the later shelf-life microbiota in fresh whole broiler meat packaged in 80%O2/20%CO2 modified atmosphere. A single operational taxonomic unit (OTU) assigned as Janthinobacterium lividum, an occasional spoiler of meat products, was identified as a major part of the microbiota in late shelf life broiler meat and swab samples in the cooling facility at the slaughter house production line. The combination of traditional cultivation and culture-independent methods provided a great insight into the microbiota of broiler meat during shelf-life and identified a potential point of contamination in the production line for cold tolerant Janthinobacterium.

Dynamics of bacterial communities and interaction networks in thawed fish fillets during chilled storage in air.

Thawed hake (Merluccius capensis and M. paradoxus) and plaice (Pleuronectes platessa) fillets were used as a model to evaluate the effect of storage temperature (0 or 10 °C) and biological variability (fish species, lot to lot) on bacterial growth kinetics and microbial successions. Both culture dependent methods (plate counts on non-selective and selective media) and culture independent methods (qPCR and 16S rRNA gene metabarcoding) were used. Bacterial counts exceeded 107 cfu/g within 2-3 days at 10 °C and 7-8 days at 0 °C. Plate counts on three media (Plate Count Agar +0.5% NaCl, Iron Agar Lyngby and Pseudomonas Selective medium) and 16S rRNA gene counts estimated by qPCR were highly correlated. Growth was modelled using the D-model and specific growth rate ranged between 0.97 and 1.24 d-1 and 3.54 and 5.90 d-1 at 0 and 10 °C, respectively. The initial composition of the microbiota showed lot-to-lot variation, but significant differences between the two fish species were detected. Alpha diversity significantly decreased during storage. When bacterial counts exceeded 107 cfu/g, the microbiota was dominated by members of the genera Pseudomonas, Psychrobacter, Acinetobacter, Serratia, Flavobacterium, Acinetobacter, Carnobacterium, Brochothrix and Vagococcus. However, Photobacterium and Shewanella, two genera frequently associated with fish spoilage, were either absent or minor components of the microbiota. As expected, storage temperature significantly affected the abundance of several species. The inference of microbial association networks with three different approaches (an ensemble approach using the CoNet app, Sparse Correlations for Compositional data, and SParse InversE Covariance Estimation for Ecological Association Inference) allowed the detection of both a core microbiota, which was present throughout storage, and a number of taxa, which became dominant at the end of spoilage and were characterized by a disproportionate amount of negative interactions.

Multilocus sequence typing of Carnobacterium maltaromaticum strains associated with fish disease and dairy products.

AIMS: Carnobacterium maltaromaticum is a lactic acid bacterium of technological interest in the field of dairy ripening and food bioprotection and is generally recognized as safe in the United States. As it is associated with fish infections, the European Food Safety Agency did not include this species in the qualified presumption safety list of micro-organisms. This implies that the risk assessment for the species has to be performed at the strain level. METHODS AND RESULTS: Multilocus sequence typing (MLST) is a tool that (i) potentially allows to discriminate strains isolated from diseased fish from apathogenic strains and (ii) to assess the genetic relatedness between both groups of strains. In this study, we characterized by MLST 21 C. maltaromaticum strains including 16 strains isolated from diseased fish and 5 apathogenic dairy strains isolated from cheese. The resulting population structure was investigated by integrating these new data to the previously published population structure (available at http://pubmlst.org), which represents an overall of 71 strains. CONCLUSIONS: This analysis revealed that none of the strains isolated from diseased fish is assigned to a clonal complex containing cheese isolates, and that 11 strains exhibit singleton genotypes suggesting that the population of diseased fish isolates is not clonal. SIGNIFICANCE AND IMPACT OF THE STUDY: This study thus provides a population structure of C. maltaromaticum that could serve in the future as a reference that could contribute to the risk assessment of C. maltaromaticum strains intended to be used in the food chain.

Growth of Carnobacterium spp. isolated from chilled vacuum-packaged meat under relevant acidic conditions.

Carnobacterium spp. are frequently isolated from vacuum-packaged (VP) meat. Specific strains of Carnobacterium and their growth characteristics may be associated with the storage life of such products. This study investigated the growth of 44 Carnobacterium isolates obtained from VP meat cuts produced at three Canadian abattoirs (A, B and C) under the following conditions: pH 5.4, 6.2 and 7.4; lactic acid at 60 and 90 mM; acetic acid at 33.6 mM. Whole genome sequencing was performed for all 44 isolates and a core genome phylogenetic tree was created to identify strain variability among isolates from different abattoirs. The isolates were clustered into 11 groups. All isolates from abattoirs B and C were identified as C. divergens, while the isolates from abattoir A included both C. maltaromaticum and C. divergens at equal proportions. C. divergens isolates from abattoir A belonged to two phylogenetic groups and none of them was found in the phylogenetic groups containing isolates from abattoirs B or C. Whole genome sequencing revealed that identical strains were isolated from different samples obtained at the same abattoir. The mean growth rate and maximum population density of the C. maltaromaticum isolates were lower than those of the C. divergens isolates. C. divergens isolates from abattoir A had higher growth rates and maximum population density than those from abattoirs B and C. In conclusion, growth characteristic and whole genome analysis both demonstrated strain variability of Carnobacterium among abattoirs, which could be a result of the difference in the antimicrobial interventions used for carcasses at different abattoirs, and may be associated with different storage lives of VP meats produced from different abattoirs.

Carnobacterium antarcticum sp. nov., a psychrotolerant, alkaliphilic bacterium isolated from sandy soil in Antarctica.

A novel, alkaliphilic, psychrotolerant, facultative anaerobe, designated CP1T, was isolated from sandy soil near the Davis Station in Antarctica. The short-rod-shaped cells displayed Gram-positive staining and did not form spores. Strain CP1T was able to grow at temperatures between 4 and 36 °C, pH 6.0-9.5, and in the presence of up to 5.0 % (w/v) NaCl. 16S rRNA gene and multilocus (pheS, rpoA, and atpA) sequence analysis revealed Carnobacterium mobile DSM 4848T and Carnobacterium iners LMG 26642T as the closest relatives (97.4 and 97.1 % 16S rRNA gene sequence similarity, respectively). The genomic G+C content was 38.1 mol%, and DNA-DNA hybridization with DSM 4848T revealed 32.4±3.4 % similarity. The major fatty acid components were C14 : 0 and C16 : 1ω9c. The cell wall contained meso-diaminopimelic acid and was of peptidoglycan type A1γ. Based on physiological, genotypic and biochemical characteristics, strain CP1T represents a novel species of the genus Carnobacterium for which the name Carnobacterium antarcticum sp. nov. is proposed. The type strain is CP1T (=DSM 103363T=CGMCC 1.15643T).

Diversity of the dominant bacterial species on sliced cooked pork products at expiration date in the Belgian retail.

Pork-based cooked products, such as cooked hams, are economically valuable foods that are vulnerable to bacterial spoilage, even when applying cooling and modified atmosphere packaging (MAP). Besides a common presence of Brochothrix thermosphacta, their microbiota are usually dominated by lactic acid bacteria (LAB). Yet, the exact LAB species diversity can differ considerably among products. In this study, 42 sliced cooked pork samples were acquired from three different Belgian supermarkets to map their bacterial heterogeneity. The community compositions of the dominant bacterial species were established by analysing a total of 702 isolates from selective agar media by (GTG)5-PCR fingerprinting followed by gene sequencing. Most of the isolates belonged to the genera Carnobacterium, Lactobacillus, and Leuconostoc, with Leuconostoc carnosum and Leuconostoc gelidum subsp. gelidum being the most dominant members. The diversity of the dominant bacterial species varied when comparing samples from different production facilities and, in some cases, even within the same product types. Although LAB consistently dominated the microbiota of sliced cooked pork products in the Belgian market, results indicated that bacterial diversity needs to be addressed on the level of product composition and batch variation. Dedicated studies will be needed to substantiate potential links between such variability and microbial composition. For instance, the fact that higher levels of lactobacilli were associated with the presence of potassium lactate (E326) may be suggestive of selective pressure but needs to be validated, as this finding referred to a single product only.

Exploring lot-to-lot variation in spoilage bacterial communities on commercial modified atmosphere packaged beef.

Understanding the factors influencing meat bacterial communities is important as these communities are largely responsible for meat spoilage. The composition and structure of a bacterial community on a high-O2 modified-atmosphere packaged beef product were examined after packaging, on the use-by date and two days after, to determine whether the communities at each stage were similar to those in samples taken from different production lots. Furthermore, we examined whether the taxa associated with product spoilage were distributed across production lots. Results from 16S rRNA amplicon sequencing showed that while the early samples harbored distinct bacterial communities, after 8-12 days storage at 6 °C the communities were similar to those in samples from different lots, comprising mainly of common meat spoilage bacteria Carnobacterium spp., Brochothrix spp., Leuconostoc spp. and Lactococcus spp. Interestingly, abundant operational taxonomic units associated with product spoilage were shared between the production lots, suggesting that the bacteria enable to spoil the product were constant contaminants in the production chain. A characteristic succession pattern and the distribution of common spoilage bacteria between lots suggest that both the packaging type and the initial community structure influenced the development of the spoilage bacterial community.

Isolation and characterisation of lactic acid bacteria from donkey milk.

During the last years the interest in donkey milk has increased significantly mainly because of its compelling functional elements. Even if the composition and nutritional properties of donkey milk are known, its microbiota is less studied. This Research Communication aimed to provide a comprehensive characterisation of the lactic acid bacteria in raw donkey milk. RAPD-PCR assay combined with 16S rDNA sequencing analysis were used to describe the microbial diversity of several donkey farms in the North West part of Italy. The more frequently detected species were: Lactobacillus paracasei, Lactococcus lactis and Carnobacterium maltaromaticum. Less abundant genera were Leuconostoc, Enterococcus and Streptococcus. The yeast Kluyveromyces marxianus was also isolated. The bacterial and biotype distribution notably diverged among the farms. Several of the found species, not previously detected in donkey milk, could have an important probiotic activity and biotechnological potential. This study represents an important insight to the ample diversity of the microorganisms present in the highly selective ecosystem of raw donkey milk.

Genes associated to lactose metabolism illustrate the high diversity of Carnobacterium maltaromaticum.

The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative genomic analysis revealed that the species C. maltaromaticum exhibits genes related to the Leloir and the tagatose-6-phosphate (Tagatose-6P) pathways. More precisely, strains can bear genes related to one or both pathways and several strains apparently do not contain homologs related to these pathways. Analysis at the population scale revealed that the Tagatose-6P and the Leloir encoding genes are disseminated in multiple phylogenetic lineages of C. maltaromaticum: genes of the Tagatose-6P pathway are present in the lineages I, II and III, and genes of the Leloir pathway are present in the lineages I, III and IV. These data suggest that these genes evolved thanks to horizontal transfer, genetic duplication and translocation. We hypothesize that the lac and gal genes evolved in C. maltaromaticum according to a complex scenario that mirrors the high population diversity.

Repeat-based Sequence Typing of Carnobacterium maltaromaticum.

Carnobacterium maltaromaticum is a Lactic Acid Bacterium (LAB) of technological interest for the food industry, especially the dairy as bioprotection and ripening flora. The industrial use of this LAB requires accurate and resolutive typing tools. A new typing method for C. maltaromaticum inspired from MLVA analysis and called Repeat-based Sequence Typing (RST) is described. Rather than electrophoresis analysis, our RST method is based on sequence analysis of multiple loci containing Variable-Number Tandem-Repeats (VNTRs). The method described here for C. maltaromaticum relies on the analysis of three VNTR loci, and was applied to a collection of 24 strains. For each strain, a PCR product corresponding to the amplification of each VNTR loci was sequenced. Sequence analysis allowed delineating 11, 11, and 12 alleles for loci VNTR-A, VNTR-B, and VNTR-C, respectively. Considering the allele combination exhibited by each strain allowed defining 15 genotypes, ending in a discriminatory index of 0.94. Comparison with MLST revealed that both methods were complementary for strain typing in C. maltaromaticum.

Volatile organic compounds and Photobacterium phosphoreum associated with spoilage of modified-atmosphere-packaged raw pork.

Accumulation of volatile organic compounds was monitored in association with sensory quality, bacterial concentrations and culture-independent microbial community analyses in raw pork loin and pork collar during storage under high-oxygen modified atmosphere at +4 °C. Of the 48 volatile compounds detected in the pork samples, the levels of acetoin, diacetyl and 3-methyl-1-butanol had the highest correlations with the sensory scores and bacterial concentrations. These compounds accumulated in all of the four monitored lots of non-sterile pork but not in the sterilized pork during chilled storage. According to the culture-dependent and culture-independent characterization of bacterial communities, Brochothrix thermosphacta, lactic acid bacteria (Carnobacterium, Lactobacillus, Lactococcus, Leuconostoc, Weissella) and Photobacterium spp. predominated in pork samples. Photobacterium spp., typically not associated with spoilage of meat, were detected also in 8 of the 11 retail packages of pork investigated subsequently. Eleven isolates from the pork samples were shown to belong to Photobacterium phosphoreum by phenotypic tests and sequencing of the 16S rRNA and gyrB gene fragments. Off-odors in pork samples with high proportion of Photobacterium spp. were associated with accumulation of acetoin, diacetyl and 3-methyl-1-butanol in meat, but these compounds did not explain all the off-odors reported in sensory analyses.

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.