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Lactobacillus delbrueckii subsp. bulgaricus and Lactiplantibacillus plantarum are two lactic acid bacteria (LAB) widely used in the food industry. The objective of this work was to assess the resistance of these bacteria to freeze- and spray-drying and study the mechanisms involved in their loss of activity. The culturability and acidifying activity were measured to determine the specific acidifying activity, while membrane integrity was studied by flow cytometry. The glass transitions temperature and the water activity of the dried bacterial suspensions were also determined. Fourier transform infrared (FTIR) micro-spectroscopy was used to study the biochemical composition of cells in an aqueous environment. All experiments were performed after freezing, drying and storage at 4, 23 and 37 °C. The results showed that Lb. bulgaricus CFL1 was sensitive to osmotic, mechanical, and thermal stresses, while Lpb. plantarum WCFS1 tolerated better the first two types of stress but was more sensitive to thermal stress. Moreover, FTIR results suggested that the sensitivity of Lb. bulgaricus CFL1 to freeze-drying could be attributed to membrane and cell wall degradation, whereas changes in nucleic acids and proteins would be responsible of heat inactivation of both strains associated with spray-drying. According to the activation energy values (47–85 kJ/mol), the functionality loss during storage is a chemically limited reaction. Still, the physical properties of the glassy matrix played a fundamental role in the rates of loss of activity and showed that a glass transition temperature 40 °C above the storage temperature is needed to reach good preservation during storage. Key points • Specific FTIR bands are proposed as markers of osmotic, mechanic and thermal stress • Lb. bulgaricus CFL1 was sensitive to all three stresses, Lpb. plantarum WCFS1 to thermal stress only • Activation energy revealed chemically limited reactions ruled the activity loss in storage Graphical abstract

Lactobacillus delbrueckii subsp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ) are commonly used starters in milk fermentation. Fermentation experiments revealed that L. bulgaricus-S. thermophilus interactions ( LbSt I ) substantially impact dairy product quality and production. Traditional biological humidity experiments are time-consuming and labor-intensive in screening interaction combinations, an artificial intelligence-based method for screening interactive starter combinations is necessary. However, in the current research on artificial intelligence based interaction prediction in the field of bioinformatics, most successful models adopt supervised learning methods, and there is a lack of research on interaction prediction with only a small number of labeled samples. Hence, this study aimed to develop a semi-supervised learning framework for predicting LbSt I using genomic data from 362 isolates (181 per species). The framework consisted of a two-part model: a co-clustering prediction model (based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) dataset) and a Laplacian regularized least squares prediction model (based on K-mer analysis and gene composition of all isolates datasets). To enhance accuracy, we integrated the separate outcomes produced by each component of the two-part model to generate the ultimate LbSt I prediction results, which were verified through milk fermentation experiments. Validation through milk fermentation experiments confirmed a high precision rate of 85% (17/20; validated with 20 randomly selected combinations of expected interacting isolates). Our data suggest that the biosynthetic pathways of cysteine, riboflavin, teichoic acid, and exopolysaccharides, as well as the ATP-binding cassette transport systems, contribute to the mutualistic relationship between these starter bacteria during milk fermentation. However, this finding requires further experimental verification. The presented model and data are valuable resources for academics and industry professionals interested in screening dairy starter cultures and understanding their interactions.

Objective Probiotic bacteria can inhibit the growth of harmful bacterial species but may be limited in the bacteria they can counteract. We developed a novel co-culture assay in which candidate probiotic bacteria can be co-incubated with both Gram-positive and Gram-negative reporter pathogens that have been modified to emit light, so as to monitor pathogen growth simply by quantitation of emitted light from the culture. We used this assay to identify a novel probiotic bacterium with anti-microbial activity against both Gram-positive and Gram-negative pathogens. Results We isolated a novel bacterium from non-pasteurized milk and identified it as a strain of Lactobacillus delbrueckii . Testing in our assay confirmed that this bacterium was able to inhibit the growth of both Staphylococcus aureus and Escherichia coli , but in a delayed fashion, after allowing a period of early growth. This activity was not dependent on living cells but also a property of conditioned medium. We posited that this was because the pathogens stimulated the production of an inhibitory factor from the Lactobacillus , presumably a bacteriocin peptide. Pre-treatment of conditioned medium with a protease abolished this inhibitory activity, indicating that L. delbrueckii is stimulated to produce an antibacterial peptide in the presence of a bacterial pathogen.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-beta1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4(+)CD25(+) Tregs are involved in the suppressive effect induced by the lactobacilli mixture. Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.

Lactobacilli isolated from various sources were identified on the basis of 16S–23S rRNA gene intergenic region amplification and subsequent sequencing of the smaller intergenic region. An in vitro analysis of probiotic properties including binding, ability to tolerate different concentrations of bile, survival in acidic buffer and antimicrobial activity of four different isolates and two standard strains (Lactobacillus plantarum American Type Culture Collection (ATCC) 8014 and L. rhamnosus GG (LGG)) was carried out. The ability of each isolate to stimulate Caco-2 cells, human peripheral blood mononuclear cells (PBMC) and THP-1 cells resulting in immunomodulation of these cells was analysed. Isolates L. rhamnosus CS25 and L. delbrueckii M and standard strain ATCC 8014 showed broad antimicrobial activity, and isolates CS25 (percentage of survival 6·9 % at pH 2·5, 5·1 % at pH 2·0) and L. plantarum CS23 (5·7 % at pH 2·5, 4·9 % at pH 2·0) have shown good tolerance to acidic pH. Isolate CS23 showed a good survival (14 %) after 2 h incubation in de Man, Rogosa and Sharpe (MRS) medium containing 3 % bile salts. Isolates CS23, CS25 and L. fermentum ASt1 could stimulate Caco-2 cells, human PBMC and THP-1 cells for a strong and varied immunomodulatory response in these cells. Though LGG showed poor antimicrobial activity as well as bile and acid tolerance, it was found to be the best binding strain tested. Child faecal isolate CS23 from the present study showed high binding ability (seventeen bacteria/Caco-2), high tolerance to acidic pH and bile salts and significant immunomodulation; therefore it is a good potential probiotic candidate.

Microbiome-derived metabolites have emerged as key mediators of the gut-brain axis, influencing cognitive function and neuroprotection. This study investigated whether subsp. CKDB001 alleviates scopolamine-induced memory impairment through metabolic modulation, and how its effects compare with those of donepezil. ICR mice were administered CKDB001 or donepezil for 4-5 weeks and evaluated through behavioral, microbiome, metabolomic, and molecular analyses. CKDB001 significantly improved spatial working memory in a dose-dependent manner, with the high-dose group showing improvements comparable to those of the donepezil-treated group, while passive avoidance showed a non-significant but positive trend. Both CKDB001 and donepezil modulated gut microbial composition, leading to a partial divergence from the scopolamine-disrupted community structure, with CKDB001 inducing dose-dependent intestinal colonization. Metabolomic profiling revealed that both treatments increased tryptophan-derived indole metabolites and altered lipid and short-chain fatty acid metabolite profiles, although these effects were more pronounced in CKDB001-treated mice. At the molecular level, both CKDB001 and donepezil reduced hippocampal tau phosphorylation, downregulated glycogen synthase kinase-3 (GSK-3) signaling, enhanced intestinal tight-junction proteins, and partially normalized acetylcholinesterase activity, with CKDB001 restoring AChE levels more closely toward the normal control. Together, these findings suggest that CKDB001 mitigates cognitive deficits through coordinated modulation of microbial, metabolic, and neuronal pathways, offering a microbiome-based therapeutic approach that may provide benefits comparable to donepezil with potentially fewer limitations.

Background/Objectives: Lactobacillus delbrueckii subsp. lactis CKDB001 (LL) has demonstrated anti-inflammatory, antioxidant, and lipid-regulatory effects in vitro and in vivo, including attenuation of hepatic steatosis and modulation of lipid metabolism. Given the known interactions between host metabolism and gut microbiota, these findings suggest a potential role for LL in modulating microbial composition under conditions of diet-induced obesity. This study aimed to investigate the microbiome-related effects of LL using an established murine model. To evaluate the effect of LL supplementation on gut microbial composition and predict microbial metabolic functions in mice with high-fat diet-induced obesity. Methods: Male C57BL/6J mice were fed a high-fat diet and administered LL orally for 12 weeks. Fecal samples were collected and analyzed using 16S rRNA gene sequencing. Microbial taxonomic profiles were assessed using linear discriminant analysis effect size, and functional predictions were performed using PICRUSt2. Results: LL supplementation significantly altered the gut microbiota by increasing the relative abundance of Lactobacillus and other commensal taxa while reducing the prevalence of pro-inflammatory genera such as Alistipes and Bilophila. Functional prediction analysis revealed a downregulation of lipopolysaccharide and ADP-L-glycero-β-D-manno-heptose biosynthesis pathways. Microbial functions associated with carbohydrate metabolism and short-chain fatty acid production were enriched in the LL-treated group. Conclusions: LL modulated gut microbial composition and suppressed pro-inflammatory microbial pathways while enhancing beneficial metabolic functions in high-fat diet-fed mice. These findings support the potential of LL as a safe and effective microbiota-targeted probiotic for managing obesity-related metabolic disorders.

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

Lactic acid bacteria (LAB) have been widely used as probiotics which contribute to our health. We previously reported that subsp. 2038 and 1131, two yogurt starter strains, ameliorate the intestinal barrier dysfunction caused by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. However, Caco-2 cells differ from living organisms in various ways. We have developed a human induced pluripotent stem cell-derived crypt-villus structural small intestine (hiPSC-SI) was established with a villus-like structure containing constituent cells of the small intestine. A hiPSC-SI and LAB co-culture model was established to assess the impact of LAB on barrier function and elucidate the underlying mechanisms. The medium on the luminal side for co-culturing cells and bacteria was examined and determined to use Hanks' balanced salt solution without glucose in terms of bacterial survival rate. LAB were found to ameliorate permeability and decrease the gene expression of tight junction associated proteins induced by TNF-α and IFN-γ. Regarding cell differentiation, LAB suppressed the downregulation of , , and by cytokines. Moreover, they ameliorated reduced mucin 2 protein production and decreased the number of mucin 2-positive cells. Finally, transcriptome analysis suggested that they ameliorated the aberration in cytokine-induced cell differentiation via an anti-inflammatory effect on intestinal stem cells. The results indicate that LAB ameliorate the cytokine-induced dysfunction of intestinal barrier integrity and homeostasis disrupted by cytokines in a co-culture model of hiPSC-SI and LAB.

The homeostatic systems, such as the nervous and immune systems, show deterioration in aging as a consequence of the age-related oxidative-inflammatory stress establishment. The supplementation with fermented milk containing probiotic bacteria could be a good nutritional strategy to improve homeostatic system functions in aged individuals through the modulation of their redox state. The aim of the present study was to evaluate the effect of 2-week supplementation with a commercial fermented milk containing yogurt species (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), and the probiotic Lactobacillus casei DN-114001 on behavior, redox state, and immune cell functions of aged mice as well as on their life span. Aged female ICR-CD1 mice were supplemented with fermented milk containing these probiotics for 2 weeks. After this period, a variety of behavioral tests were performed and several parameters of redox state and function of peritoneal leukocytes were analyzed. The results showed that the 2-week supplementation of fermented milk containing probiotics improved behavior (such as muscular vigor, exploratory activity, and anxiety-like behavior) as well as the redox state and functions of peritoneal immune cells in aged mice. In conclusion, the present study shows that the supplementation with fermented milk containing probiotics for a short period of time could be a good nutritional strategy to promote healthy aging.