Comparative physiological and transcriptomic analysis of sono-biochemical control over post-acidification of Lactobacillus delbrueckii subsp. bulgaricus.

Thermosonication (UT) prestress treatments combining with varied fermentation patterns has been revealed as an effective method to regulate post-acidification as exerted by Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii), but sono-biochemical controlling mechanisms remain elusive. This study employed physiological and transcriptomic analysis to explore the response mechanism of L. delbrueckii to UT-induced microstress (600 W, 33 kHz, 10 min). UT stress-induced inhibition of acidification of L. delbrueckii during (post)-fermentation was first confirmed, relying on the UT process parameters such as stress exposure duration and UT power. The significantly enhanced membrane permeability in cells treated by 600 W for 10 min than the microbes stressed by 420 W for 20 min suggested the higher dependence of UT-derived stresses on the treatment durations, relative to the ultrasonic powers. In addition, ultrasonication treatment-induced changes in cell membrane integrity enhanced and/or disrupted permeability of L. delbrueckii, resulting in an imbalance in intracellular conditions associated with corresponding alterations in metabolic behaviors and fermentation efficiencies. UT-prestressed inoculum exhibited a 21.46% decrease in the membrane potential during the lag phase compared to untreated samples, with an intracellular pH of 5.68 ± 0.12, attributed to the lower activities of H+-ATPase and lactate dehydrogenase due to UT stress pretreatments. Comparative transcriptomic analysis revealed that UT prestress influenced the genes related to glycolysis, pyruvate metabolism, fatty acid synthesis, and ABC transport. The genes encoding 3-oxoacyl-[acyl-carrier-protein] reductases I, II, and III, CoA carboxylase, lactate dehydrogenase, pyruvate oxidase, glucose-6-phosphate isomerase, and glycerol-3-phosphate dehydrogenase were downregulated, thus identifying the relevance of the UT microstresses-downregulated absorption and utilization of carbohydrates with the attenuated fatty acid production and energy metabolisms. These findings could contribute to provide a better understanding of the inactivated effects on the post-acidification of L. delbrueckii by ultrasonic pretreatments, thus providing theoretical basis for the targeted optimization of acidification inhibition efficiencies for yogurt products during chilled preservation processes.

Multidrug resistance profile in Lactobacillus delbrueckii: a food industry species with probiotic properties.

Lactobacillus delbrueckii, a widely used lactic acid bacterium in the food industry, has been studied for its probiotic properties and reservoir of antibiotic-resistant genes, raising safety concerns for probiotic formulations and fermented products. This review consolidates findings from 60 articles published between 2012 and 2023, focusing on the global antibiotic resistance profile and associated genetic factors in L. delbrueckii strains. Resistance to aminoglycosides, particularly streptomycin, kanamycin, and gentamicin, as well as resistance to glycopeptides (vancomycin), fluoroquinolones (ciprofloxacin), and tetracyclines was predominant. Notably, although resistance genes have been identified, they have not been linked to mobile genetic elements, reducing the risk of dissemination. However, a significant limitation is the insufficient exploration of responsible genes or mobile elements in 80% of studies, hindering safety assessments. Additionally, most articles originated from Asian and Middle Eastern countries, with strains often isolated from fermented dairy foods. Therefore, these findings underscore the necessity for comprehensive analyses of new strains of L. delbrueckii for potential industrial and biotherapeutic applications and in combating the rise of antibiotic-resistant pathogens.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Functional genomic analysis of the isolated potential probiotic Lactobacillus delbrueckii subsp. indicus TY-11 and its comparison with other Lactobacillus delbrueckii strains.

Probiotics refer to living microorganisms that exert a variety of beneficial effects on human health. On the contrary, they also can cause infection, produce toxins within the body, and transfer antibiotic-resistant genes to the other microorganisms in the digestive tract necessitating a comprehensive safety assessment. This study aimed to conduct functional genomic analysis and some relevant biochemical tests to uncover the probiotic potentials of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed transmission electron microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. The strain TY-11 was identified as Lactobacillus delbrueckii subsp. indicus, whose genome (1,916,674 bp) contained 1911 CDS, and no gene was identified for either antibiotic resistance or toxic metabolites. It carried genes for the degradation of toxic metabolites, treatment of lactose intolerance, toll-like receptor 2-dependent innate immune response, heat and cold shock, bile salts tolerance, and acidic pH tolerance. Genes were annotated for inhibiting pathogenic bacteria by inhibitory substances [bacteriocin: Helveticin-J (331 bp) and Enterolysin-A (275 bp), hydrogen peroxide, and acid]; blockage of adhesion sites; and competition for nutrients. The genes involved in its metabolic pathway were detected as suitable for digesting indigestible nutrients in the human gut. The TY-11 genome possessed an additional 37 core genes of subspecies indicus which were deficient in the core genome of the most popular subsp. bulgaricus. During the phenotypic testing, the isolate TY-11 demonstrated high antagonistic activity (inhibition zone of 21.33 ± 1.53 mm) against Escherichia coli ATCC 8739 and was not sensitive to any of the 10 tested antibiotics. This study was the first study to explore the molecular insights into probiotic roles, including antimicrobial activities and antibiotic sensitivity, of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus. IMPORTANCE: This study aimed to conduct functional genomic analysis to uncover the probiotic potential of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed transmission electron microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. In our current investigation, we revealed a number of common and unique excellences of the probiotic Lactobacillus delbrueckii subsp. indicus TY-11 that are likely to be important to illustrate its intestinal residence and probiotic roles. This is the first study to explore the molecular insights into intestinal residence and probiotic roles, including antimicrobial activities and antibiotic sensitivity, of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Effect of probiotic administration during pregnancy on the functional diversity of the gut microbiota in healthy pregnant women.

Our study aims to investigate the impact of probiotic consumption during pregnancy on gut microbiota functional diversity in healthy pregnant women. Thirty-two pregnant women were randomly assigned to two groups. The probiotic group (PG) consisted of pregnant women who consumed triple viable Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, and Streptococcus thermophilus tablets from the 32nd week of pregnancy until delivery. The functional profiles of the gut microbiota were predicted through high-throughput 16S rRNA sequencing results using PICRUSt software and referencing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In the gut microbiota of the PG, the genera Blautia and Ruminococcus, as well as the species Subdoligranulum, showed significantly higher relative abundances compared to the control group (CG) (P < 0.05). At Level 1 of the KEGG signaling pathways, there was a significant reduction in the functional genes of the gut microbiota involved in Organismal Systems in the PG (P < 0.05). In Level 2 of the KEGG signaling pathways, there was a significant reduction in the functional genes of the gut microbiota involved in Infectious Disease in the PG (P < 0.05). In Level 3 of the KEGG signaling pathways, the PG exhibited a significant increase in the functional genes of the gut microbiota involved in ABC transporters, Oxidative phosphorylation, Folate biosynthesis, and Biotin metabolism (P < 0.05). The CG showed a significant increase in the functional genes related to Cysteine and methionine metabolism, Vitamin B6 metabolism, Tuberculosis, and Vibrio cholerae pathogenic cycle (P < 0.05). In conclusion, our findings suggest that probiotic supplementation during pregnancy has a significant impact on functional metabolism in healthy pregnant women. IMPORTANCE: Probiotics are considered beneficial to human health. There is limited understanding of how probiotic consumption during pregnancy affects the functional diversity of the gut microbiota. The aim of our study is to investigate the impact of probiotic consumption during pregnancy on the functional diversity of the gut microbiota. Our findings suggest that probiotic supplementation during pregnancy has a significant impact on functional metabolism. This could potentially open up new avenues for preventing various pregnancy-related complications. This also provides new insights into the effects of probiotic consumption during pregnancy on the gut microbiota and offers a convenient method for exploring the potential mechanisms underlying the impact of probiotics on the gut microbiota of pregnant women.

In vitro investigation of the effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 exopolysaccharides on tight junction damage caused by influenza virus infection.

It is a problem that influenza virus infection increases susceptibility to secondary bacterial infection in lungs leading to lethal pneumonia. We previously reported that exopolysaccharides (EPS) derived from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) could prevent against influenza virus infection followed by secondary bacterial infection in vitro. Therefore, the present study assessed whether EPS derived OLL1073R-1 protects the alveolar epithelial barrier disfunction caused by influenza virus infection. After A549 cells treated with EPS or without EPS were infected influenza virus A/Puerto Rico/8/34 (IFV) for 12 h, the levels of tight junction genes expression and inflammatory genes expression were measured by reverse transcription polymerase chain reaction. As results, EPS treatment could protect against low-titer IFV infection, but not high-titer IFV infection, followed by suppression of the increased expression of inflammatory cytokine gene levels and recovery of the decrease in the expression level of ZO-1 gene that was caused by low-titer IFV infection, leading to an improvement trend in the barrier function. Our findings showed that EPS derived from OLL1073R-1 could inhibit low-titer IFV infection leading to maintenance of the epithelial barrier function through the suppression of inflammatory cytokine genes expression.

Deciphering the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice fermentation using phenotypic and transcriptional analysis.

In the context of sustainable diet, the development of soy-based yogurt fermented with lactic acid bacteria is an attractive alternative to dairy yogurts. To decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice (SJ) fermentation, the whole genome of the strain CIRM-BIA865 (Ld865) was sequenced and annotated. Then Ld865 was used to ferment SJ. Samples were analyzed throughout fermentation for their cell number, carbohydrate, organic acid, free amino acid, and volatile compound contents. Despite acidification, the number of Ld865 cells did not rise, and microscopic observations revealed the elongation of cells from 3.6 µm (inoculation) to 36.9 µm (end of fermentation). This elongation was observed in SJ but not in laboratory-rich medium MRS. Using transcriptomic analysis, we showed that the biosynthesis genes of peptidoglycan and membrane lipids were stably expressed, in line with the cell elongation observed, whereas no genes implicated in cell division were upregulated. Among the main sugars available in SJ (sucrose, raffinose, and stachyose), Ld865 only used sucrose. The transcriptomic analysis showed that Ld865 implemented the two transport systems that it contains to import sucrose: a PTS system and an ABC transporter. To fulfill its nitrogen needs, Ld865 probably first consumed the free amino acids of the SJ and then implemented different oligopeptide transporters and proteolytic/peptidase enzymes. In conclusion, this study showed that Ld865 enables fast acidification of SJ, despite the absence of cell division, leads to a product rich in free amino acids, and also leads to the production of aromatic compounds of interest. IMPORTANCE: To reduce the environmental and health concerns related to food, an alternative diet is recommended, containing 50% of plant-based proteins. Soy juice, which is protein rich, is a relevant alternative to animal milk, for the production of yogurt-like products. However, soy "beany" and "green" off-flavors limit the consumption of such products. The lactic acid bacteria (LAB) used for fermentation can help to improve the organoleptic properties of soy products. But metabolic data concerning LAB adapted to soy juice are lacking. The aim of this study was, thus, to decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during fermentation of a soy juice, based on a multidisciplinary approach. This result will contribute to give tracks for a relevant selection of starter. Indeed, the improvement of the organoleptic properties of these types of products could help to promote plant-based proteins in our diet.

Development of a single nucleotide polymorphism-based strain-identified method for Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 using pan-genomics analysis.

The health benefits conferred by probiotics is specific to individual probiotic strains, highlighting the importance of identifying specific strains for research and production purposes. Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 are exceedingly valuable for commercial use with an excellent mixed-culture fermentation. To differentiate these 2 strains from other S. thermophilus and L. delbrueckii ssp. bulgaricus, a specific, sensitive, accurate, rapid, convenient, and cost-effective method is required. In this study, we conducted a pan-genome analysis of S. thermophilus and L. delbrueckii ssp. bulgaricus to identify species-specific core genes, along with strain-specific SNPs. These genes were used to develop suitable PCR primers, and the conformity of sequence length and unique SNPs was confirmed by sequencing for qualitative identification at the strain level. The results demonstrated that SNPs analysis of PCR products derived from these primers could distinguish CICC 6038 and CICC 6047 accurately and reproducibly from the other strains of S. thermophilus and L. delbrueckii ssp. bulgaricus, respectively. The strain-specific PCR method based on SNPs herein is universally applicable for probiotics identification. It offers valuable insights into identifying probiotics at the strain level that is fit-for-purpose in quality control and compliance assessment of commercial dairy products.

Intraspecific microdiversity and ecological drivers of lactic acid bacteria in naturally fermented milk ecosystem.

Traditional fermented milks are produced by inoculating technique, which selects well-adapted microorganisms that have been passed on through generations. Few reports have used naturally fermented milks as model ecosystems to investigate the mechanism of formation of intra-species microbial diversity. Here, we isolated and whole-genome-sequenced a total of 717 lactic acid bacterial isolates obtained from 12 independent naturally fermented milks collect from 12 regions across five countries. We further analyzed the within-sample intra-species phylogenies of 214 Lactobacillus helveticus isolates, 97 Lactococcus lactis subsp. lactis isolates, and 325 Lactobacillus delbrueckii subsp. bulgaricus isolates. We observed a high degree of intra-species genomic and functional gene diversity within-/between-sample(s). Single nucleotide polymorphism-based phylogenetic reconstruction revealed great within-sample intra-species heterogeneity, evolving from multiple lineages. Further phylogenetic reconstruction (presence-absence gene profile) revealed within-sample inter-clade functional diversity (based on carbohydrate-active enzyme- and peptidase-encoding genes) in all three investigated species/subspecies. By identifying and mapping clade-specific genes of intra-sample clades of the three species/subspecies to the respective fermented milk metagenome, we found extensive potential inter-/intra-species horizontal gene transfer events. Finally, the microbial composition of the samples is closely linked to the nucleotide diversity of the respective species/subspecies. Overall, our results contribute to the conservation of lactic acid bacteria resources, providing ecological insights into the microbial ecosystem of naturally fermented dairy products.

In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii.

Lactobacillus delbrueckii, the type species of the genus Lactobacillus, is widely recognized as the primary starter culture in the dairy industry due to its proteolytic activity, which enables it to growth in milk. In this study, a comprehensive genomic analysis of the proteolytic system was conducted on L. delbrueckii strains. The analysis included 27 genomes of L. delbrueckii, with a specific focus on the key enzyme involved in this system, the cell envelope-associated proteinase (CEP). The amino acid sequences, as well as the protein-structure prediction of the CEPs, were compared. Additionally, syntenic analysis of the genomic locus related to the CEPs revealed high conservation in L. delbrueckii subsp. bulgaricus strains, while L. delbrueckii subsp. lactis strains exhibited greater variability, including the presence of insertion sequences, deletions, and rearrangements. Finally, the CEP promoter region and putative regulatory elements responsible for controlling the expression of the proteolytic system in lactobacilli were investigated. Our genomic analysis and in silico characterization of the CEPs contribute to our understanding of proteolytic activity and the potential applications of these lactic acid bacteria in the dairy industry. Further research in this area will expand our knowledge and potential practical uses of these findings.

Dynamic metagenome-scale metabolic modeling of a yogurt bacterial community.

Genome-scale metabolic models and flux balance analysis (FBA) have been extensively used for modeling and designing bacterial fermentation. However, FBA-based metabolic models that accurately simulate the dynamics of coculture are still rare, especially for lactic acid bacteria used in yogurt fermentation. To investigate metabolic interactions in yogurt starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, this study built a dynamic metagenome-scale metabolic model which integrated constrained proteome allocation. The accuracy of the model was evaluated by comparing predicted bacterial growth, consumption of lactose and production of lactic acid with reference experimental data. The model was then used to predict the impact of different initial bacterial inoculation ratios on acidification. The dynamic simulation demonstrated the mutual dependence of S. thermophilus and L. d. bulgaricus during the yogurt fermentation process. As the first dynamic metabolic model of the yogurt bacterial community, it provided a foundation for the computer-aided process design and control of the production of fermented dairy products.

Inulin increases the EPS biosynthesis of Lactobacillus delbrueckii ssp. bulgaricus LDB-C1.

OBJECTIVE: Its eps gene cluster, the antioxidant activity and monosaccharide composition of exopolysaccharides, the expression levels of related genes at different fermentations were analyzed for clarifying the exopolysaccharide biosynthesis mechanism of Lactobacillus delbrueckii subsp. bulgaricus LDB-C1. RESULTS: The comparison analysis of eps gene clusters indicated that the gene clusters present diversity and strain specificity. The crude exopolysaccharides from LDB-C1 exhibited a good antioxidant activity. Compared with glucose, fructose, galactose, and fructooligosaccharide, inulin significantly improved the exopolysaccharide biosynthesis. The structures of EPSs were significantly different under different carbohydrate fermentation conditions. Inulin obviously increased the expressions of most EPS biosynthesis related genes at fermentation 4 h. CONCLUSION: Inulin accelerated the beginning of the exopolysaccharide production in LDB-C1, and the enzymes promoted by inulin was beneficial for the accumulation of exopolysaccharide at the whole fermentation process.

Subspecies-level genome comparison of Lactobacillus delbrueckii.

Lactobacillus delbrueckii comprises six subspecies, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. jakobsenii, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. sunkii, and L. delbrueckii subsp. indicus. We investigated the evolution of the six subspecies of L. delbrueckii using comparative genomics. While the defining feature of the species was the gene number increment driven by mobile elements and gene fragmentation, the repertoire of subspecies-specific gene gains and losses differed among the six subspecies. The horizontal gene transfer analyses indicated that frequent gene transfers between different subspecies had occurred when the six subspecies first diverged from the common ancestor, but recent gene exchange was confined to a subspecies implying independent evolution of the six subspecies. The subspecies bulgaricus is a homogeneous group that diverged from the other subspecies a long time ago and underwent convergent evolution. The subspecies lactis, jakobsenii, delbrueckii, and sunkii were more closely related to each other than to other subspecies. The four subspecies commonly show increasing genetic variability with increasing genome size. However, the four subspecies were distinguished by specific gene contents. The subspecies indicus forms a branch distant from the other subspecies and shows an independent evolutionary trend. These results could explain the differences in the habitat and nutritional requirements of the subspecies of L. delbrueckii.

Lactobacillus delbrueckii reduces anxiety-like behavior in zebrafish through a gut microbiome - brain crosstalk.

Certain bacteria possess the ability to reduce anxiety- and stress-related behaviors through the gut microbiome-brain axis. Such bacteria are called psychobiotics, and can be used to improve mood and cognition. However, only a few bacteria have been characterized as psychobiotics, and their exact mechanism of action remains unclear. Hence, in this study we analyzed three different species under the Lactobacillacea family, namely, Lactobacillus delbrueckii, Lacticaseibacillus casei, and Lacticaseibacillus paracasei for their potential psychobiotic activities. L. delbrueckii treatment reduced anxiety-like behavior and increased brain and gut glutamic acid decarboxylase (gad) gene expression in zebrafish. It also altered zebrafish gut microbial community as determined by PCR-DGGE and 16S rRNA-based metagenomics analysis. Overall, this paper showed that L. delbrueckii but not L. paracasei and L. casei, induced a consistent improvement in anxiety-like behavior in zebrafish, implicating its potential role as a psychobiotic to reduce anxiety. This article is part of the Special Issue on 'Microbiome & the Brain: Mechanisms & Maladies'.

Short-length Homologous Region exhaustive Search algorithm (SHRS): A primer design algorithm for differentiating bacteria at the species, subspecies, or strain level based on a whole genome sequence.

In fields such as the food industry, it is very important to identify target bacteria at the species level or lower for optimal product quality control. Bacteria identification at the subspecies or lower level requires time-consuming and high-cost analyses such as multi-locus sequence typing and amplified fragment length polymorphism analyses. Herein, we developed a primer design algorithm for precisely identifying bacteria based on a whole genome DNA sequence that is easy to apply. The algorithm designs primer sets that produce fragments from all input sequences and maximizes the differences in the amplicon size or amplicon sequence among input sequences. We demonstrate that the primer sets designed by the algorithm clearly classified six subspecies of Lactobacillus delbrueckii, and we observed that the resolution of the method is equal to that of a multi-locus sequence analysis. The algorithm allows the easy but precise identification of bacteria within a short time. (SHRS is available freely from PyPI under the MIT license.).

In Vitro Production of Galactooligosaccharides by a Novel ß-Galactosidase of Lactobacillus bulgaricus.

ß-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of ß-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the ß-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other ß-galactosidases of this species. The molecular model revealed that the new ß-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The ß-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme's temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, ß-Gal produced a large amount of GOS with DP3 containing ß-(1→3) and ß-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of ß-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

Genome-Scale Metabolic Modeling Combined with Transcriptome Profiling Provides Mechanistic Understanding of Streptococcus thermophilus CH8 Metabolism.

Streptococcus thermophilus is a lactic acid bacterium adapted toward growth in milk and is a vital component of starter cultures for milk fermentation. Here, we combine genome-scale metabolic modeling and transcriptome profiling to obtain novel metabolic insights into this bacterium. Notably, a refined genome-scale metabolic model (GEM) accurately representing S. thermophilus CH8 metabolism was developed. Modeling the utilization of casein as a nitrogen source revealed an imbalance in amino acid supply and demand, resulting in growth limitation due to the scarcity of specific amino acids, in particular sulfur amino acids. Growth experiments in milk corroborated this finding. A subtle interdependency of the redox balance and the secretion levels of the key metabolites lactate, formate, acetoin, and acetaldehyde was furthermore identified with the modeling approach, providing a mechanistic understanding of the factors governing the secretion product profile. As a potential effect of high expression of arginine biosynthesis genes, a moderate secretion of ornithine was observed experimentally, augmenting the proposed hypothesis of ornithine/putrescine exchange as part of the protocooperative interaction between S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yogurt. This study provides a foundation for future community modeling of food fermentations and rational development of starter strains with improved functionality. IMPORTANCE Streptococcus thermophilus is one the main organisms involved in the fermentation of milk and, increasingly, also in the fermentation of plant-based foods. The construction of a functional high-quality genome-scale metabolic model, in conjunction with in-depth transcriptome profiling with a focus on metabolism, provides a valuable resource for the improved understanding of S. thermophilus physiology. An example is the model-based prediction of the most significant route of synthesis for the characteristic yogurt flavor compound acetaldehyde and identification of metabolic principles governing the synthesis of other flavor compounds. Moreover, the systematic assessment of amino acid supply and demand during growth in milk provides insights into the key challenges related to nitrogen metabolism that is imposed on S. thermophilus and any other organism associated with the milk niche.

Inhibitory Effect of Lactobacillus delbrueckii subsp. bulgaricus KSFY07 on Kappa-Carrageenan-Induced Thrombosis in Mice and the Regulation of Oxidative Damage.

A mouse thrombosis model was established by kappa-carrageenan to observe the inhibitory effect of Lactobacillus delbrueckii subsp. bulgaricus KSFY07 (LDSB-KSFY07) on thrombosis and the oxidative stress response. Mouse serum, liver tissue-related indicators, and intestinal microbial composition were measured by examining the expression of microbes in mouse faeces using a biochemical kit, slice observations, and quantitative polymerase chain reaction (qPCR) experiments. The results showed that LDSB-KSFY07 effectively reduced the degree of black tail in thrombotic mice, increased activated partial thromboplastin time (APTT), and decreased thrombin time (TT), fibrinogen (FIB), and prothrombin time (PT) in thrombotic mice. LDSB-KSFY07 was also able to reduce malondialdehyde (MDA) levels and increase superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) levels in the serum and liver tissues of thrombotic mice. Pathological observations showed that LDSB-KSFY07 reduced liver tissue lesions and tail vein thrombosis. Further, experimental results showed that LDSB-KSFY07 was able to upregulate the mRNA expression of copper/zinc-SOD (Cu/Zn-SOD), manganese-SOD, and GSH-Px in the liver tissue of thrombotic mice. Moreover, LDSB-KSFY07 was also able to downregulate the mRNA expression of NF-κB p65, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in tail vein vascular tissue. Meanwhile, LDSB-KSFY07 could raise plasminogen activator inhibitor-1 (PAI-1) mRNA expression and reduce tissue plasminogen activator (t-PA) expression in heart and tail vein vascular tissues of thrombotic mice. A mouse faeces examination revealed that LDSB-KSFY07 could also upregulate Bacteroides, Lactobacterium, and Bifidobacterium microbial expression and downregulate Firmicutes expression in the gut. These results indicate that LDSB-KSFY07 was able to inhibit mouse thrombosis and reduce liver oxidative stress damage in thrombus mice and show that high concentrations of LDSB-KSFY07 provided a better response similar to that of the drug heparin.

Effects of probiotics on Zebrafish model infected with Aeromonas hydrophila: spatial distribution, antimicrobial, and histopathological investigation.

Usage of "probiotics" for treatment of food-borne pathogens associated diseases, makes a significant reduction in transmission of resistant bacteria, and antimicrobial resistance genes from aquaculture environments to humans. In this research, the authors aim to evaluate the immunomodulatory, and histological effects of two probiotic strains on the Zebrafish model. Fish models were treated with Lactobacillus delbrueckii (G2), Lactobacillus acidophilus (G3) and both probiotics (G4) and compared with the control group (G1) (only infected by pathogen and receiving no probiotic). Biometric tests, height, weight, and mortality rate of the fishes were assessed. Afterward, RT-PCR was conducted for bacterial existence of probiotic strains, and quantitative assessment of alterations in targeted immune genes. Subsequently, histological sampling was done for investigation of spatial distribution, and villus length in proximal, middle, and distal sections of intestinal tissues. Based on the results, G4 showed the highest gene expression for Lactobacillus acidophilus after 28 days (P < 0.05). G4 also showed an increase in the number of goblet cells and villus length in the middle and distal sections of intestinal tissue after 56 days. Furthermore, after 56 days, the highest number of intraepithelial cells was observed in the proximal sections of intestinal tissue in G4. G2 and G3 showed significant differences in comparison with G1 (P < 0.05). After 60 days, the highest gene expression for Lactobacillus bulgaricus was found in group treated with only this probiotic bacteria. The highest expression level of IL-1ß and TNF-α were found in G1. The highest survival rate was in the case of groups only treated with Lactobacillus bulgaricus (G2). To sum up, it seems that usage of probiotics for the improvement of public health and fisheries industries can be helpful.