Analysis of Microbial Diversity and Dynamics During Bacon Storage Inoculated With Potential Spoilage Bacteria by High-Throughput Sequencing

Staphylococcus xylosus, Leuconostoc mesenteroides, Carnobacterium maltaromaticum, Leuconostoc gelidum, and Serratia liquefaciens were investigated for their roles in in the spoilage of sterilized smoked bacon. These five strains, individually and in combination, were applied as starters on sliced bacon at 4–5 log10 CFU/g using a hand-operated spraying bottle and stored for 45 days at 0–4°C. Dynamics, diversity, and succession of microbial community during storage of samples were studied by high-throughput sequencing (HTS) of the V3–V4 region of the 16S rRNA gene. A total of 367 bacterial genera belonging to 21 phyla were identified. Bacterial counts in all the inoculated specimens increased significantly within the first 15 days while the microbiota developed into more similar communities with increasing storage time. At the end of the storage time, the highest abundance of Serratia (96.46%) was found in samples inoculated with S. liquefaciens. Similarly, for samples inoculated with C. maltaromaticum and L. mesenteroides, a sharp increase in Carnobacterium and Leuconostoc abundance was observed as they reached a maximum relative abundance of 97.95 and 81.6%, respectively. Hence, these species were not only the predominant ones but could also have been the more competitive ones, potentially inhibiting the growth of other microorganisms. By analyzing the bacterial load of meat products using the SSO model, the relationships between the microbial communities involved in spoilage can be understood to assist further research.

Rejection of the reclassification of Leuconostoc gasicomitatum as Leuconostoc gelidum subsp. gasicomitatum based on whole genome analysis

In 2014, Rahkila et al. transferred Leuconostoc gasicomitatum to Leuconostoc gelidum as L. gelidum subsp. gasicomitatum comb. nov. based on a 75 % DNA–DNA hybridization value. In the present study, the taxonomic status of L. gelidum subsp. gasicomitatum is re-evaluated by a polyphasic approach, including 16S rRNA, pheS, rpoA, recA, and atpA gene sequence analyses, phylogenomic treeing, analyses of ANI (average nucleotide identity) and dDDH (digital DNA–DNA hybridization), fatty acid methyl ester analysis and a phenotypic characterization. On the basis of the ANI and dDDH values, we propose to reject the proposal of Rahkila et al. to reclassify L. gasicomitatum as L. gelidum subsp. gasicomitatum.

Characterization of the Psychrotrophic Lactic Acid Bacterium Leuconostoc gelidum subsp. aenigmaticum LS4 Isolated from Kimchi Based on Comparative Analyses of Its Genomic and Phenotypic Properties

With the aim of developing a new food starter culture, twenty-three psychrotrophic lactic acid bacteria (LAB) were isolated from 16 kimchi samples. One strain, Leuconostoc gelidum subsp. aenigmaticum LS4, which had typical psychrotrophic characteristics, was selected, and its phenotypic and genomic properties as a starter culture were investigated. The complete genome of L. aenigmaticum LS4 consisted of one circular chromosome (1,988,425 bp) and two plasmids (19,308 bp and 11,283 bp), with a guanine–cytosine content of 36.8%. L. aenigmaticum LS4 could grow at 5 °C but not at 37 °C, and maximum cell growth was obtained at 15~25 °C. L. aenigmaticum LS4 did not show any harmful characteristics such as hemolysis, undesirable enzyme activities, biogenic amine production, or antibiotic resistance. L. aenigmaticum LS4 was investigated for its suitability for technological processes (pH, temperature and NaCl treatment). L. aenigmaticum LS4 exhibited strong antimicrobial activity caused by the production of organic acids and bacteriocin, and it produced an exopolysaccharide composed of glucose with a molecular weight of 3.7 × 106 Da. Furthermore, L. aenigmaticum LS4 improved the organoleptic qualities of kimchi juice. Our results indicate that L. aenigmaticum LS4 could be used as a functional starter culture for food (vegetable or fruit) fermentation at low temperatures.

Genomic profiling of bacterial and fungal communities and their predictive functionality during pulque fermentation by whole-genome shotgun sequencing

Pulque is a culturally important 4,000-year-old traditional Mexican fermented drink. Pulque is produced by adding fresh aguamiel (agave sap) to mature pulque, resulting in a mixture of microbial communities and chemical compositions. We performed shotgun metagenomic sequencing of five stages of pulque fermentation to characterize organismal and functional diversity. We identified 6 genera (Acinetobacter, Lactobacillus, Lactococcus, Leuconostoc, Saccharomyces and Zymomonas) and 10 species (Acinetobacter boissieri, Acinetobacter nectaris, Lactobacillus sanfranciscensis, Lactococcus lactis, Lactococcus piscium, Lactococcus plantarum, Leuconostoc citreum, Leuconostoc gelidum, Zymomonas mobilis and Saccharomyces cerevisiae) that were present ≥ 1% in at least one stage of pulque fermentation. The abundance of genera and species changed during fermentation and was associated with a decrease in sucrose and increases in ethanol and lactic acid, suggesting that resource competition shapes organismal diversity. We also predicted functional profiles, based on organismal gene content, for each fermentation stage and identified an abundance of genes associated with the biosynthesis of folate, an essential B-vitamin. Additionally, we investigated the evolutionary relationships of S. cerevisiae and Z. mobilis, two of the major microbial species found in pulque. For S. cerevisiae, we used a metagenomics assembly approach to identify S. cerevisiae scaffolds from pulque, and performed phylogenetic analysis of these sequences along with a collection of 158 S. cerevisiae strains. This analysis suggests that S. cerevisiae from pulque is most closely related to Asian strains isolated from sake and bioethanol. Lastly, we isolated and sequenced the whole-genomes of three strains of Z. mobilis from pulque and compared their relationship to seven previously sequenced isolates. Our results suggest pulque strains may represent a distinct lineage of Z. mobilis.

Draft Genome Sequences of Nine Strains of Brochothrix thermosphacta, Carnobacterium divergens, Lactobacillus algidus, Lactobacillus fuchuensis, Lactococcus piscium, Leuconostoc gelidum subsp. gasicomitatum, Pseudomonas lundensis, and Weissella viridescens, a Collection of Psychrotrophic Species Involved in Meat and Seafood Spoilage

ABSTRACT In this study, we present the draft genome sequences of nine strains from various psychrotrophic species identified in meat products and being recognized as important emerging food spoilers. Many of these species have only one or few strains being sequenced, and this work will contribute to the improvement of the overall genomic knowledge about them.

Microbiota of regular sodium and sodium-reduced ready-to-eat meat products obtained from the retail market

The aim of this study was to assess the influence of sodium content on the microbiota on the surface of ready-to-eat (RTE) meat products purchased from the retail market in Canada. Products, including sliced and sausage-type deli meats, were analysed with culture-dependent and culture-independent methods. Bacteria were identified from 23 brands of products from different meat processors with claims of sodium content ranging from 390 to 1200 mg per 100 g of product. Out of 150 bacterial isolates, the most common were identified as Leuconostoc gelidum, Carnobacterium maltaromaticum, Brochothrix thermosphacta, and Leuconostoc gasicomitatum. Vacuum-packaged RTE deli sliced meat products had the largest population of bacteria. Leuconostocci were the most common isolates in this group of products, while carnobacteria were prevalent on products with moderate loads of bacteria. A higher incidence of carnobacteria and lower incidence of B. thermosphacta were detected on sodium-reduced products. Simpson’s and Shannon–Wiener indices showed that low sodium products (25%–50% less sodium) had an overall higher bacterial diversity. This was also observed when individual low sodium products were compared with their regular sodium counterpart.

Multilocus Sequence Typing of Leuconostoc gelidum subsp. gasicomitatum, a Psychrotrophic Lactic Acid Bacterium Causing Spoilage of Packaged Perishable Foods

ABSTRACTLeuconostoc gelidumsubsp.gasicomitatumis a psychrotrophic lactic acid bacterium (LAB) that causes spoilage of a variety of modified-atmosphere-packaged (MAP) cold-stored food products. During the past 10 years, this spoilage organism has been increasingly reported in MAP meat and vegetable products in northern Europe. In the present study, the population structure within 252L. gelidumsubsp.gasicomitatumstrains was determined based on a novel multilocus sequence-typing (MLST) scheme employing seven housekeeping genes. These strains had been isolated from meat and vegetable sources over a time span of 15 years, and all 68 previously detected pulsed-field gel electrophoresis (PFGE) genotypes were represented. A total of 46 sequence types (STs) were identified, with a majority of the strains (>60%) belonging to three major STs, which were grouped into three clonal complexes (CCs) and 17 singletons by Global Optimal eBURST (goeBURST). The results by Bayesian analysis of population structure (BAPS) mostly correlated with the grouping by goeBURST. Admixture analysis by BAPS indicated a very low level of exchange of genetic material between the subpopulations. Niche specificity was observed within the subpopulations: CC1 and BAPS cluster 1 consisted mostly of strains from a variety of MAP meats, whereas vegetable strains grouped together with strains from MAP poultry within CC2 and BAPS cluster 2. The MLST scheme presented in this study provides a shareable and continuously growing sequence database enabling global comparison of strains associated with spoilage cases. This will further advance our understanding of the microbial ecology of this industrially important LAB.