Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

Task Distribution between Acetate and Acetoin Pathways To Prolong Growth in Lactococcus lactis under Respiration Conditions.

Lactococcus lactis is the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme under aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation, in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter-reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures. First, the acetate pathway contributes to biomass yield in respiration without affecting medium pH. Second, the acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways will lead to fine-tuning respiration growth, with improved yield and stability.IMPORTANCELactococcus lactis is used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications.

Effect of repeat unit structure and molecular mass of lactic acid bacteria hetero-exopolysaccharides on binding to milk proteins.

Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero-exopolysaccharides (HePSs) of 0.14-4.9MDa from lactic acid bacteria to different milk proteins (ß-casein, κ-casein, native and heat-treated ß-lactoglobulin) at pH 4.0-5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold, respectively, in the complexation with native ß-lactoglobulin at pH 4.0. Highest RUmax and KA,app were obtained with heat-treated ß-lactoglobulin and ß-casein, respectively. Overall, RUmax and KA,app decreased 6- and 20-fold, respectively, with increasing pH from 4.0 to 5.0. KA,app was influenced by ionic strength and temperature, indicating that polar interactions stabilize HePS-protein complexes. HePS size as well as oligosaccharide repeat structure, conferring chain flexibility and hydrogen bonding potential, influence the KA,app.

Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production.

Pangenomics--an avenue to improved industrial starter cultures and probiotics.

With the dramatic reductions in the cost and time involved in DNA sequencing, a new approach to characterisation of bacteria is emerging. It is based on a comparison of complete genome sequences of a number of members of the same species (pangenomics). Pangenomics opens an array of new opportunities for understanding and improving industrial starter cultures and probiotics. These include understanding the formation of texture and flavour in dairy products, understanding the functionality of probiotics as well as providing information that can be used for strain screening, strain improvement, safety assessments and process improvements.

Using heme as an energy boost for lactic acid bacteria.

Lactic acid bacteria (LAB) are a phylogenetically diverse group named for their main attribute in food fermentations, that is, production of lactic acid. However, several LAB are genetically equipped for aerobic respiration metabolism when provided with exogenous sources of heme (and menaquinones for some species). Respiration metabolism is energetically favorable and leads to less oxidative and acid stress during growth. As a consequence, the growth and survival of several LAB can be dramatically improved under respiration-permissive conditions. Respiration metabolism already has industrial applications for the production of dairy starter cultures. In view of the growth and survival advantages conferred by respiration, and the availability of heme and menaquinones in natural environments, we recommend that respiration be accepted as a part of the natural lifestyle of numerous LAB.

Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a model of their utilization.

Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacterium animalis subsp. lactis strains in particular. To elucidate the metabolism of XOS in the well-documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either XOS or glucose. The analyses show that 9 of the 10 genes that encode proteins predicted to play a role in XOS catabolism (i.e., XOS-degrading and -metabolizing enzymes, transport proteins, and a regulatory protein) were induced by XOS at the transcriptional level, and the proteins encoded by three of these (ß-d-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides) to bind XOS on the cell surface and transport them into the cell. XOS are then degraded intracellularly through the action of xylanases and xylosidases to d-xylose, which is subsequently metabolized by the d-fructose-6-P shunt. The findings obtained in this study may have implications for the design of a synbiotic application containing BB-12 and the XOS used in the present study.

Complete genome sequence of Bifidobacterium animalis subsp. lactis BB-12, a widely consumed probiotic strain.

Bifidobacterium animalis subsp. lactis BB-12 is a commercially available probiotic strain used throughout the world in a variety of functional foods and dietary supplements. The benefits of BB-12 have been documented in a number of independent clinical trials. Determination of the complete genome sequence reveals a single circular chromosome of 1,942,198 bp with 1,642 predicted protein-encoding genes, 4 rRNA operons, and 52 tRNA genes. Knowledge of this sequence will lead to insight into the specific features which give this strain its probiotic properties.

Bile-inducible efflux transporter from Bifidobacterium longum NCC2705, conferring bile resistance.

Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.

Streptococcus thermophilus core genome: comparative genome hybridization study of 47 strains.

A DNA microarray platform based on 2,200 genes from publicly available sequences was designed for Streptococcus thermophilus. We determined how single-nucleotide polymorphisms in the 65- to 75-mer oligonucleotide probe sequences affect the hybridization signals. The microarrays were then used for comparative genome hybridization (CGH) of 47 dairy S. thermophilus strains. An analysis of the exopolysaccharide genes in each strain confirmed previous findings that this class of genes is indeed highly variable. A phylogenetic tree based on the CGH data showed similar distances for most strains, indicating frequent recombination or gene transfer within S. thermophilus. By comparing genome sizes estimated from the microarrays and pulsed-field gel electrophoresis, the amount of unknown DNA in each strain was estimated. A core genome comprised of 1,271 genes detected in all 47 strains was identified. Likewise, a set of noncore genes detected in only some strains was identified. The concept of an industrial core genome is proposed. This is comprised of the genes in the core genome plus genes that are necessary in an applied industrial context.

Impact of aeration and heme-activated respiration on Lactococcus lactis gene expression: identification of a heme-responsive operon.

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O(2) and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.