Using metabolomics to understand stress responses in Lactic Acid Bacteria and their applications in the food industry.

BACKGROUND: Lactic Acid Bacteria (LAB) are commonly used as starter cultures, probiotics, to produce lactic acid and other useful compounds, and even as natural preservatives. For use in any food product however, LAB need to survive the various stresses they encounter in the environment and during processing. Understanding these mechanisms may enable direction of LAB biochemistry with potential beneficial impact for the food industry. AIM OF REVIEW: To give an overview of the use of LAB in the food industry and then generate a deeper biochemical understanding of LAB stress response mechanisms via metabolomics, and methods of screening for robust strains of LAB. KEY SCIENTIFIC CONCEPTS OF REVIEW: Uses of LAB in food products were assessed and factors which contribute to survival and tolerance in LAB investigated. Changes in the metabolic profiles of LAB exposed to stress were found to be associated with carbohydrates, amino acids and fatty acid levels and these changes were proposed to be a result of the bacteria trying to maintain cellular homeostasis in response to external conditions and minimise cellular damage from reactive oxygen species. This correlates with morphological analysis which shows that LAB can undergo cell elongation and shortening, as well as thinning and thickening of cell membranes, when exposed to stress. It is proposed that these innate strategies can be utilised to minimise negative effects caused by stress through selection of intrinsically robust strains, genetic modification and/or prior exposure to sublethal stress. This work demonstrates the utility of metabolomics to the food industry.

Novel cell wall polysaccharide genotypes and structures of lactococcal strains isolated from milk and fermented foods.

The biosynthetic machinery for cell wall polysaccharide (CWPS) formation in Lactococcus lactis and Lactococcus cremoris is encoded by the cwps locus. The CWPS of lactococci typically consists of a neutral rhamnan component, which is embedded in the peptidoglycan, and to which a surface-exposed side chain oligosaccharide or polysaccharide pellicle (PSP) component is attached. The rhamnan component has been shown for several lactococcal strains to consist of a repeating rhamnose trisaccharide subunit, while the side chain is diverse in glycan content, polymeric status and glycosidic linkage architecture. The observed structural diversity of the CWPS side chain among lactococcal strains is reflected in the genetic diversity within the variable 3' region of the corresponding cwps loci. To date, four distinct cwps genotypes (A, B, C, D) have been identified, while eight subtypes (C1 through to C8) have been recognized among C-genotype strains. In the present study, we report the identification of three novel subtypes of the lactococcal cwps C genotypes, named C9, C10 and C11. The CWPS of four isolates representing C7, C9, C10 and C11 genotypes were analysed using 2D NMR to reveal their unique CWPS structures. Through this analysis, the structure of one novel rhamnan, three distinct PSPs and three exopolysaccharides were elucidated. Results obtained in this study provide further insights into the complex nature and fascinating diversity of lactococcal CWPSs. This highlights the need for a holistic view of cell wall-associated glycan structures which may contribute to robustness of certain strains against infecting bacteriophages. This has clear implications for the fermented food industry that relies on the consistent application of lactococcal strains in mesophilic production systems.

Viromic and Metagenomic Analyses of Commercial Spirulina Fermentations Reveal Remarkable Microbial Diversity.

Commercially produced cyanobacteria preparations sold under the name spirulina are widely consumed, due to their traditional use as a nutrient-rich foodstuff and subsequent marketing as a superfood. Despite their popularity, the microbial composition of ponds used to cultivate these bacteria is understudied. A total of 19 pond samples were obtained from small-scale spirulina farms and subjected to metagenome and/or virome sequencing, and the results were analysed. A remarkable level of prokaryotic and viral diversity was found to be present in the ponds, with Limnospira sp. and Arthrospira sp. sometimes being notably scarce. A detailed breakdown of prokaryotic and viral components of 15 samples is presented. Twenty putative Limnospira sp.-infecting bacteriophage contigs were identified, though no correlation between the performance of these cultures and the presence of phages was found. The high diversity of these samples prevented the identification of clear trends in sample performance over time, between ponds or when comparing successful and failed fermentations.

Complete Genome Sequence of Floricoccus penangensis ML061-4 Isolated from Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.].

Floricoccus penangensis is a Gram-positive coccoid organism that is a member of the lactic acid bacteria. F. penangensis ML061-4 was originally isolated from the surface of an Assam tea leaf, and its genome is herein shown to contain gene clusters predicted to be involved in complex carbohydrate metabolism and biosynthesis of secondary metabolites.

Reduced synthesis of phospho-polysaccharide in Lactococcus as a strategy to evade phage infection.

Lactococcus spp. are applied routinely in dairy fermentations and their consistent growth and associated acidification activity is critical to ensure the quality and safety of fermented dairy foods. Bacteriophages pose a significant threat to such fermentations and thus it is imperative to study how these bacteria may evade their viral predators in the relevant confined settings. Many lactococcal phages are known to specifically recognise and bind to cell wall polysaccharides (CWPSs) and particularly the phospho-polysaccharide (PSP) side chain component that is exposed on the host cell surface. In the present study, we generated derivatives of a lactococcal strain with reduced phage sensitivity to establish the mode of phage evasion. The resulting mutants were characterized using a combination of comparative genome analysis, microbiological and chemical analyses. Using these approaches, it was established that the phage-resistant derivatives incorporated mutations in genes within the cluster associated with CWPS biosynthesis resulting in growth and morphological defects that could revert when the selective pressure of phages was removed. Furthermore, the cell wall extracts of selected mutants revealed that the phage-resistant strains produced intact PSP but in significantly reduced amounts. The reduced availability of the PSP and the ability of lactococcal strains to revert rapidly to wild type growth and activity in the absence of phage pressure provides Lactococcus with the means to survive and evade phage attack.

Lactic Acid Bacteria Diversity and Characterization of Probiotic Candidates in Fermented Meats.

Probiotics are defined as live microorganisms which confer health benefits to the host when administered in adequate amounts. Many lactic acid bacteria (LAB) strains have been classified as probiotics and fermented foods are an excellent source of such LAB. In this study, novel probiotic candidates from two fermented meats (pancetta and prosciutto) were isolated and characterized. LAB populations present in pancetta and prosciutto were evaluated and Lactiplantibacillus plantarum was found to be the dominant species. The antagonistic ability of selected isolates against LAB and non-LAB strains was investigated, in particular, the ability to produce anti-microbial compounds including organic acids and bacteriocins. Probiotic characteristics including antibiotic susceptibility, hydrophobicity and autoaggregation capacity; and ability to withstand simulated gastric juice, bile salt, phenol and NaCl were assessed. Among the characterized strains, L. plantarum 41G isolated from prosciutto was identified as the most robust probiotic candidate compared. Results from this study demonstrate that artisanal fermented meat is a rich source of novel strains with probiotic potential.

Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.].

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75-100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

Lysogenization of a Lactococcal Host with Three Distinct Temperate Phages Provides Homologous and Heterologous Phage Resistance.

Lactococcus lactis is the most widely exploited microorganism in global dairy fermentations. Lactococcal strains are described as typically harboring a number of prophages in their chromosomes. The presence of such prophages may provide both advantages and disadvantages to the carrying host. Here, we describe the deliberate generation of three distinct lysogens of the model lactococcal strain 3107 and the impact of additional prophage carriage on phage-resistance and anti-microbial susceptibility. Lysogen-specific responses were observed, highlighting the unique relationship and impact of each lysogenic phage on its host. Both homologous and heterologous phage-resistance profiles were observed, highlighting the presence of possible prophage-encoded phage-resistance factors. Superinfection exclusion was among the most notable causes of heterologous phage-resistance profiles with resistance observed against members of the Skunavirus, P335, P087, and 949 lactococcal phage groups. Through these analyses, it is now possible to identify phages that may pursue similar DNA injection pathways. The generated lysogenic strains exhibited increased sensitivity to the antimicrobial compounds, nisin and lysozyme, relative to the parent strain, although it is noteworthy that the degree of sensitivity was specific to the individual (pro)phages. Overall, the findings highlight the unique impact of each prophage on a given strain and the requirement for strain-level analysis when considering the implications of lysogeny.

Metabolic Insights Into the Effects of Nutrient Stress on Lactobacillus plantarum B21.

Lactobacillus plantarum B21 is a strain of lactic acid bacteria first isolated from a fermented meat product from Vietnam. It is also a promising biopreservative with potential use in the food industry as it is a source of a novel bacteriocin (Plantacyclin B21AG) which has inhibitory effects against a wide range of species, including several pathogenic and spoilage strains. Nutrient stress is known to increase the survivability, storage stability, and bacteriocin production capability of L. plantarum B21 during industrial processing. It is however, unknown what the underlying biochemical responses that control these abilities are. This study therefore investigates the metabolite profiles of L. plantarum B21 using NMR spectroscopy and GC-MS to further understand the biochemical responses of this strain to various stress events. Unstressed cells were found to use glucose as their primary energy source with high concentrations of metabolites involved in glycolysis and organic acid synthesis, such as lactic acid, acetic acid, propanoic acid, malic acid, and 2-butenedioic acid being present in these cells. In contrast, large numbers of metabolites involved in amino acid metabolism including alanine, glutamic acid, aspartic acid, valine, proline, and norleucine were upregulated in glucose stressed cells, indicating that they were using amino acids as their main source of energy. Differences in levels of fatty acids, particularly octadecenoic acid, tetracosanoic acid, and 7-hexadecenoic acid were also observed between stressed and unstressed cells. The results from this study provide insight on the biochemical response of this bacterial strain to stresses commonly found during industrial processing.