Phenotypic, Technological, Safety, and Genomic Profiles of Gamma-Aminobutyric Acid-Producing Lactococcus lactis and Streptococcus thermophilus Strains Isolated from Cow's Milk.

Gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) can be used as starters in the development of GABA-enriched functional fermented foods. In this work, four GABA-producing strains each of Lactococcus lactis and Streptococcus thermophilus species were isolated from cow's milk, and their phenotypic, technological, and safety profiles determined. Genome analysis provided genetic support for the majority of the analyzed traits, namely, GABA production, growth in milk, and the absence of genes of concern. The operon harboring the glutamate decarboxylase gene (gadB) was chromosomally encoded in all strains and showed the same gene content and gene order as those reported, respectively, for L. lactis and S. thermophilus. In the latter species, the operon was flanked (as in most strains of this species) by complete or truncated copies of insertion sequences (IS), suggesting recent acquisition through horizontal gene transfer. The genomes of three L. lactis and two S. thermophilus strains showed a gene encoding a caseinolytic proteinase (PrtP in L. lactis and PrtS in S. thermophilus). Of these, all but one grew in milk, forming a coagulum of good appearance and an appealing acidic flavor and taste. They also produced GABA in milk supplemented with monosodium glutamate. Two L. lactis strains were identified as belonging to the biovar. diacetylactis, utilized citrate from milk, and produced significant amounts of acetoin. None of the strains showed any noticeable antibiotic resistance, nor did their genomes harbor transferable antibiotic resistance genes or genes involved in toxicity, virulence, or pathogenicity. Altogether these results suggest that all eight strains may be considered candidates for use as starters or components of mixed LAB cultures for the manufacture of GABA-enriched fermented dairy products.

Probiotic Supplementation During Human Pregnancy Affects the Gut Microbiota and Immune Status.

The consumption of probiotics and fermented foods has been very popular in recent decades. The primary aim of our study was to evaluate the effect of probiotics on the gut microbiota and the changes in inflammatory cytokines after an average of 6.7 weeks of probiotic administration among normal pregnant women. Thirty-two healthy pregnant women at 32 weeks of gestation were recruited and divided into two groups. The probiotic group ingested combined probiotics until after birth. The base characteristics of the probiotics and control groups showed no significant differences. The structure of the fecal microbiota at the genus level varied during the third trimester, and administration of probiotics had no influence on the composition of the fecal microbiota however, many highly abundant taxa and core microbiota at the genus level changed in the probiotic group when compared to the control group. The analysis of cytokines showed that IL-5, IL-6, TNF-α, and GM-CSF had equal levels between the baseline and control groups but were significantly increased after probiotic administration (baseline = control < probiotics). Additionally, levels of IL-1ß, IL-2, IL-12, and IFN-γ significantly increased among the three groups (baseline < control < probiotics). This result demonstrated that probiotics helped to shift the anti-inflammatory state to a pro-inflammatory state. The correlation analysis outcome suggested that the relationship between the microbiota and the cytokines was not strain-dependent. The gut microbiota varied during the third trimester. The probiotics demonstrated immunomodulation effects that helped to switch over to a pro-inflammatory immune state in the third trimester, which was important for labor.

Gut microbiota of preterm infants supplemented with probiotics: sub-study of the ProPrems trial.

BACKGROUND: The ProPrems trial, a multi-center, double-blind, placebo-controlled randomized trial, previously reported a 54% reduction in necrotizing enterocolitis (NEC) of Bell stage 2 or more from 4.4 to 2.0% in 1099 infants born before 32 completed weeks' gestation and weighing < 1500 g, receiving probiotic supplementation (with Bifidobacterium longum subsp. infantis BB-02, Streptococcus thermophilus TH-4 and Bifidobacterium animalis subsp. lactis BB-12). This sub-study investigated the effect of probiotic supplementation on the gut microbiota in a cohort of very preterm infants in ProPrems. RESULTS: Bifidobacterium was found in higher abundance in infants who received the probiotics (AOR 17.22; 95% CI, 3.49-84.99, p < 0.001) as compared to the placebo group, and Enterococcus was reduced in infants receiving the probiotic during the supplementation period (AOR 0.27; 95% CI, 0.09-0.82, p = 0.02). CONCLUSION: Probiotic supplementation with BB-02, TH-4 and BB-12 from soon after birth increased the abundance of Bifidobacterium in the gut microbiota of very preterm infants. Increased abundance of Bifidobacterium soon after birth may be associated with reducing the risk of NEC in very preterm infants.

Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase.

Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.

Evaluation of the biosafety of recombinant lactic acid bacteria designed to prevent and treat colitis.

Inflammatory bowel diseases (IBDs) affect the gastrointestinal tract and are characterized by recurrent inflammation that requires lifelong therapies. Probiotics such as lactic acid bacteria (LAB) have been proposed to complement current treatment protocols for these patients; however, their characteristics are strain dependent. In this regard, certain novel characteristics are only possible through the genetic modification of these beneficial micro-organisms. Different delivery systems, such as protein delivery of anti-oxidant enzymes and anti-inflammatory cytokines, have been shown to be effective in preventing and treating IBD in animal models. In this study, the safety of the recombinant LAB (recLAB) Streptococcus thermophilus CRL807 : CAT, S. thermophilus CRL807 : SOD, Lactococcus lactis NCDO2118 pXILCYT : IL-10, L. lactis MG1363 pValac : IL-10 and L. lactis MG1363 pGroESL : IL-10 with proven beneficial effects was compared to their progenitor strains S. thermophilus CRL807, L. lactis NCDO2118 or L. lactis MG1363. The prolonged administration of these genetically modified strains showed that they were just as safe as the native strains from which they derive, as demonstrated by normal animal growth and relative organ weights, absence of microbial translocation from the gastrointestinal tract, normal blood parameters and intestinal histology. The results show the potential use of these recLAB in future therapeutic formulations; however, the use of modern bio-containment systems is required for the future acceptance of these recLAB by the medical community and patients with IBD.

Development of a simple IgE-independent anaphylactic model using buckwheat antigen and B-type CpG oligodeoxynucleotide from Streptococcus thermophilus.

We developed a severe anaphylactic model in mice using buckwheat antigen and B-type CpG-oligodeoxynucleotides (CpG-ODNs) from Streptococcus thermophilus genome. In typical systemic anaphylaxis models, animals are challenged with large quantity of antigens via an intravenous (i.v.) route. Here, we showed a simple anaphylactic shock after challenge via intraperitoneal (i.p.) route. The i.p. method is simpler than i.v. administration and has a lower risk for failure. To generate this anaphylactic model, 5-week-old female BALB/c mice were first i.p. sensitized with buckwheat antigen mixed with B-type CpG-ODN. After 2 weeks, mice were challenged with antigen to induce anaphylactic shock, which was evaluated by scoring the severity symptoms and measuring serum levels of various proteins and splenic cell producing cytokines. Immunoglobulin (Ig)G2a production and interferon-γ positive cells were markedly increased in mice immunized with antigen mixed with B-type CpG-ODN, whereas serum IgE levels were decreased by B-type CpG-ODN. We also examined the effects of various ODNs (A, B and C-type CpG-ODNs) and antigens (buckwheat, α-casein, ß-lactoglobulin and ovalbumin) on anaphylactic severity, and found that the combination of buckwheat and B-type CpG-ODN induced the most intense anaphylactic shock. This model is expected to contribute to the study of the prevention of anaphylactic shock.

Enhanced Heavy Metal Tolerance and Accumulation by Transgenic Sugar Beets Expressing Streptococcus thermophilus StGCS-GS in the Presence of Cd, Zn and Cu Alone or in Combination.

Phytoremediation is a promising means of ameliorating heavy metal pollution through the use of transgenic plants as artificial hyperaccumulators. A novel Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) that synthesizes glutathione (GSH) with limited feedback inhibition was overexpressed in sugar beet (Beta vulgaris L.), yielding three transgenic lines (s2, s4 and s5) with enhanced tolerance to different concentrations of cadmium, zinc and copper, as indicated by their increased biomass, root length and relative growth compared with wild-type plants. Transgenic sugar beets accumulated more Cd, Zn and Cu ions in shoots than wild-type, as well as higher GSH and phytochelatin (PC) levels under different heavy metal stresses. This enhanced heavy metal tolerance and increased accumulation were likely due to the increased expression of StGCS-GS and consequent overproduction of both GSH and PC. Furthermore, when multiple heavy metal ions were present at the same time, transgenic sugar beets overexpressing StGCS-GS resisted two or three of the metal combinations (50 µM Cd-Zn, Cd-Cu, Zn-Cu and Cd-Zn-Cu), with greater absorption in shoots. Additionally, there was no obvious competition between metals. Overall, the results demonstrate the explicit role of StGCS-GS in enhancing Cd, Zn and Cu tolerance and accumulation in transgenic sugar beet, which may represent a highly promising new tool for phytoremediation.

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress.

Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H2O2 and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn(2+)-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.

Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk.

AIM: The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant. METHODS AND RESULTS: The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617(T) and its derivative, carbamoylphosphate synthetase-negative mutant A17(DeltacarB), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO(2) depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l-arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine. CONCLUSIONS: Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.

Folate: a functional food constituent.

Folate, a water-soluble vitamin, includes naturally occurring food folate and synthetic folic acid in supplements and fortified foods. Mammalian cells cannot synthesize folate and its deficiency has been implicated in a wide variety of disorders. A number of reviews have dwelt up on the health benefits associated with increased folate intakes and many countries possess mandatory folate enrichment programs. Lately, a number of studies have shown that high intakes of folic acid, the chemically synthesized form, but not natural folates, can cause adverse effects in some individuals such as the masking of the hematological manifestations of vitamin B(12) deficiency, leukemia, arthritis, bowel cancer, and ectopic pregnancies. As fermented milk products are reported to contain even higher amounts of folate produced by the food-grade bacteria, primarily lactic acid bacteria (LAB), the focus has primarily shifted toward the natural folate, that is, folate produced by LAB and levels of folate present in foods fermented by/or containing these valuable microorganisms. The proper selection and use of folate-producing microorganisms is an interesting strategy to increase "natural" folate levels in foods. An attempt has been made through this review to share information available in the literature on wide ranging aspects of folate, namely, bioavailability, analysis, deficiency, dietary requirements, and health effects of synthetic and natural folate, dairy and nondairy products as a potential source of folate, microorganisms with special reference to Streptococcus thermophilus as prolific folate producer, and recent insight on modulation of folate production levels in LAB by metabolic engineering.

Glutamine synthesis is essential for growth of Streptococcus thermophilus in milk and is linked to urea catabolism.

Growth of a glutamine synthetase-deficient mutant of Streptococcus thermophilus was compared to that of the parent strain in milk that was not supplemented or was supplemented with ammonium chloride, glutamine, or the urease inhibitor flurofamide. It was concluded that one of the functions of urease is to supply ammonia for the synthesis of glutamine.