Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1–10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry.

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bThe first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage YS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, YS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that YS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the 29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that YS61 also replicates by a protein-primed mechanism. Weissella phage YS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of YS61. Based on electron microscopy analysis, YS61 was classified as a member of the Podoviridae of morphotype C2, similar to the 29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of YS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage YS61 should represent a new subfamily within the family Podoviridae. Kimchi, a traditional Korean dish, is manufactured by fermentation of vegetables such as Chinese cabbage and radish. Hundreds of kimchi varieties are produced by the addition of different seasonings, such as scallions, powdered chili peppers, garlic, ginger, and fermented seafood. Lactic acid produced during fermentation contributes to preservation and gives kimchi its characteristic sour taste. Proper ripening and preservation are ensured by a 2 to 5% (wt/vol) salt content and anaerobic fermentation at low temperatures. Kimchi is traditionally prepared by spontaneous fermentation by lactic acid bacteria (LAB) indigenous to the vegetable ingredients. However, starter cultures have been developed in order to better control the fermentation and thus improve quality, safety, and shelf life of the fermented product (15,16,20,24). Several LAB species have been identified as likely contributors in kimchi fermentations, including species of the genera Leuconostoc, Lactobacillus, Lactococcus, Pediococcus, and Weissella (6,18,19,32,35,36,[38][39][40][41]51,53,57). Weissella species isolated from kimchi include Weissella confusa, Weissella kimchii, and Weissella koreensis. These bacteria are abundant late in the fermentation process and can continue to grow during storage at low temperatures (Ϫ1°C). Weissella species have thus been associated with the excessive acidic taste of overripened kimchi products. The species Weissella kimchii, first described in 2002 (19), was reclassified as Weissella cibaria in 2004 (9, 23).The kimchi fermentation process is ch...

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 subsp and 28 subsp. lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using , a core gene, and, present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture.