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In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201, and Lactobacillus delbrueckii ssp. bulgaricus 92059, showed the highest activities following growth in milk. All three strains degraded α- and β-casein, but did not hydrolyse κ-casein. HPLC analysis of skim milk fermentation revealed increasing amounts of peptides after 5 and 10 h with Lb. d. ssp. bulgaricus 92059. Hydrolysates obtained with Lb. d. ssp. lactis 92202 and Lb. d. ssp. bulgaricus 92059 revealed the highest angiotensin-converting enzyme-inhibitory effect. The effect was dose dependent. Almost no effect (<10%) was seen for Lb. helveticus 92201. For Lb. d. ssp. bulgaricus 92059, maximal inhibition of approx. 65% was reached after 25 h of fermentation. In an in vitro assay measuring potential immunomodulation, hydrolysates of the three strains yielded anti-inflammatory activities in the presence of TNF-α. However, the effects were more pronounced at lower hydrolysate concentrations. In the absence of TNF-α, slight pro-inflammatory effects were observed. The hydrolysate of Lb. d. ssp. bulgaricus 92059, when purified by means of solid-phase extraction, exhibited pro-inflammatory activity. Sour whey containing Lb. d. ssp. bulgaricus 92059 cells showed pro-inflammatory activity while cell-free sour whey was clearly anti-inflammatory. In the purified hydrolysate, 20 different α- and β-casein (CN)-derived peptides could be identified by LC-MS. Most peptides originated from the central and C-terminal regions of β-casein. Peptide length was between 9 (β-CN(f 59–67)) and 22 amino acids (β-CN(f 117–138)).

In this study, the effect of several organic nitrogen sources (namely peptone, meat extract—ME, yeast extract—YE, and corn steep liquor—CSL) on d-lactic acid production by Lactobacillus delbrueckii ssp. delbrueckii has been studied. While lactic acid bacteria (LAB) are well-known for their complex nutritional requirements, organic nitrogen source-related cost can be as high as 38% of total operational costs (OPEX), being its nature and concentration critical factors in the growth and productivity of the selected strain. Corn steep liquor (CSL) has been chosen for its adequacy, on the grounds of the d-lactic acid yield, productivity, and its cost per kilogram of product. Finally, orange peel waste hydrolysate supplemented with 37 g/l CSL has been employed for d-lactic acid production, reaching a final yield of 88% and a productivity of 2.35 g/l h. CSL cost has been estimated at 90.78$/ton of d-lactate.

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

Background CodY (Control of dppY) is a global transcriptional regulator of Streptococcus thermophilus (ST). In this study, we utilized a S. thermophilus codY gene deletion mutant strain to explore metabolites that may affect the oxidative-stress resistance of Lactobacillus delbrueckii subsp. bulgaricus . The context of this research is to understand the role of CodY in the interaction between S. thermophilus and L. delbrueckii , particularly focusing on the impact of CodY on the oxidative-stress resistance of L. delbrueckii . Results Firstly, the ST-1Δ codY mutant strain was constructed by the knockout technique. We found that codY gene deletion significantly reduced the growth rate and resistance to oxidative stress of L. delbrueckii in co-culture, with a decrease in viable bacterial count to 5.26 ± 0.04 log(cfu/mL), and a decrease in survival by 15% and 25% under 5 mM and 10 mM H 2 O 2 stress, respectively. Metabolites were comprehensively analyzed under three different culture conditions, and we identified 35 differentiated metabolites associated with CodY regulation. These metabolites were screened by their significant differential metabolite expression folds (up- or down-regulation) under different culture conditions, and Variable Importance in Projection (VIP) values assessed their significance, fold changes, and P values. Further, we identified six compounds, including lysine, 4-hydroxyphenylacetic acid, cycloleucine, glycine-L-lysine, 3-hydroxyphenylacetic acid, and N 6 -acetyl-L-lysine, which significantly enhanced the oxidative-stress resistance of L. delbrueckii . The viable counts of L. delbrueckii were increased by 7.73%, 6.49%, 4.27%, 3.62%, 2.12%, and 1.38%, respectively, in the medium supplemented with these substances under the stress of 10 mM H 2 O 2 . Conclusions The study results are important for understanding the synergistic effect of the two bacteria during fermentation and their response to environmental stresses. The findings provide insights into the role of CodY in modulating the oxidative-stress resistance of L. delbrueckii and highlight the potential of identified metabolites to enhance the performance of this bacterium under stress conditions. This could have implications for the development of strategies to improve the robustness of industrial fermentation processes involving these bacteria.

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.

Probiotic fermentation with a defined consortium ( Saccharomyces cerevisiae , Lactobacillus helveticus , and Lactobacillus delbrueckii subsp. bulgaricus ) successfully transformed pumpkin juice into a functional beverage with enhanced bioactivity and appealing aroma. The fermented product achieved a high viable count of 7.37 log CFU mL −1 , along with significant increases in vitamin C, total phenolics, flavonoids, and carotenoids compared to unfermented juice. Fermentation also imparted a distinct volatile profile enriched with esters and alcohols—such as phenylethyl alcohol, phenethyl acetate, and ethyl caprylate—which contributed intense fruity and floral notes, as confirmed by OPLS-DA and sensory evaluation. The resulting juice retained its original color (ΔE < 2) and developed desirable rheological behavior. Thus, accordingly the pumpkin juice fermented with the composite probiotics received the highest sensory evaluation. This study demonstrates that composite-culture fermentation effectively enhances the nutritional value, aroma, and sensory quality of pumpkin juice, highlighting its potential as a novel probiotic beverage.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

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.

Today, probiotics are considered to be living microorganisms whose consumption has a certain number of beneficial effects on the consumer. The present study aimed to investigate the effect of a new probiotic extract ( Lactobacillus delbrueckii subsp. lactis KUMS Y33) on the differentiation process of human adipose-derived stem cells (hADSCs) into adipocytes and osteocytes and, as a result, clarify its role in the prevention and treatment of bone age disease. Several bacteria were isolated from traditional yogurt. They were evaluated to characterize the probiotic’s activity. Then, the isolated hADSCs were treated with the probiotic extract, and then osteogenesis and adipogenesis were induced. To evaluate the differentiation process, oil red O and alizarin red staining, a triglyceride content assay, an alkaline phosphatase (ALP) activity assay, as well as real-time PCR and western blot analysis of osteocyte- and adipocyte-specific genes, were performed. Ultimately, the new strain was sequenced and registered on NBCI. In the probiotic-treated group, the triglyceride content and the gene expression and protein levels of C/EBP-α and PPAR-γ2 (adipocyte-specific markers) were significantly decreased compared to the control group ( P  <  0.05 ), indicating an inhibited adipogenesis process. Furthermore, the probiotic extract caused a significant increase in the ALP activity, the expression levels of RUNX2 and osteocalcin, and the protein levels of collagen I and FGF-23 (osteocyte-specific markers) in comparison to the control group ( P  <  0.05 ), indicating an enhanced osteogenesis process. According to the results of the present study, the probiotic extract inhibits adipogenesis and significantly increases osteogenesis, suggesting a positive role in the prevention and treatment of osteoporosis and opening a new aspect for future in-vivo study.

Lactobacillus delbrueckii JCM 1002 T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF . When freshly cultured cells of the recombinant B. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to “monopolize” inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.