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In the study, in order to develop novel starter cultures, three types of yoghurt were produced with different combinations of autochthonous yoghurt bacteria and the samples were investigated in terms of fermentation, microbiological quality, physicochemical properties (pH, acidity, water-holding capacity, apparent viscosities, and rheological properties), organic and benzoic acid contents, and sensory properties during 28 days. Lactobacillus delbrueckii subsp. bulgaricus content of all samples was between 7.29 and 8.58, and Streptococcus thermophilus content was between 8.03 and 9.02 log CFU/g during storage. Yoghurt type and storage time statistically affected pH, acidity, water-holding capacity, apparent viscosity, rheological properties, organic and benzoic acid content, and some sensory parameters. The general acceptability scores of the probiotic, traditional, exopolysaccharide, and control samples during storage were 8.27, 7.82, 7.53, and 7.47 respectively. Moreover, the results revealed that the strain combinations for probiotic, traditional, and exopolysaccharide yoghurt samples can be used as novel starter cultures for yoghurt production. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are commonly used probiotics that improve human health in various aspects. We previously reported that yogurt starter strains, Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, potentially enhance the intestinal epithelial barrier function by inducing the expression of antimicrobial peptides in the small intestine. However, their effects on physical barrier functions remain unknown. In this study, we found that both strains ameliorated the decreased trans-epithelial resistance and the increased permeability of fluorescein isothiocyanate-dextran induced by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. We also demonstrated that LAB prevented a decrease in the expression and disassembly of tight junctions (TJs) induced by TNF-α and IFN-γ. To assess the repair activity of TJs, a calcium switch assay was performed. Both strains were found to promote the reassembly of TJs, and their activity was canceled by the inhibitor of AMP-activated protein kinase (AMPK). Moreover, these strains showed increased AMPK phosphorylation. These observations suggest that the strains ameliorated physical barrier dysfunction via the activation of AMPK. The activities preventing barrier destruction induced by TNF-α and IFN-γ were strain-dependent. Several strains containing L. bulgaricus 2038 and S. thermophilus 1131 significantly suppressed the barrier impairment, and L. bulgaricus 2038 showed the strongest activity among them. Our findings suggest that the intake of L. bulgaricus 2038 and S. thermophilus 1131 is a potential strategy for the prevention and repair of leaky gut. [ABSTRACT FROM AUTHOR]

Abstract \nHIGHLIGHTS Fermentation is one of the most effective methods of food preservation. Since ancient times, food has been fermented using lactic acid bacteria (LAB). Fermented milk is a very intricate fermentation ecosystem, and the microbial metabolism of fermented milk largely determines its metabolic properties. The two most frequently used dairy starter strains are Streptococcus thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). To enhance both the culture growth rate and the flavor and quality of the fermented milk, it has long been customary to combine S. thermophilus and L. bulgaricus in milk fermentation due to their mutually beneficial and symbiotic relationship. On the one hand, the symbiotic relationship is reflected by the nutrient co-dependence of the two microbes at the metabolic level. On the other hand, more complex interaction mechanisms, such as quorum sensing between cells, are involved. This review summarizes the application of LAB in fermented dairy products and discusses the symbiotic mechanisms and interactions of milk LAB starter strains from the perspective of nutrient supply and intra- and interspecific quorum sensing. This review provides updated information and knowledge on microbial interactions in a fermented milk ecosystem.The symbiotic relationship between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is reviewed.Their nutrient co-dependence is discussed.The role of quorum sensing in their interaction is discussed for the first time.This review is of interest to colleagues interested in exploiting LAB starter cultures. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

The present study investigated the suitability of combinations one Streptococcus thermophilus and five Lactobacillus delbrueckii subsp. bulgaricus isolates for industrial yogurt productions. The isolates were selected after a detailed pre-screening procedure based on acidification performances, production of volatile aroma compounds, texture developing properties and sensory evaluations of yogurts made from single cultures. The fermentation profiles of the combinations were investigated by monitoring the small oscillatory rheological parameters and pH reductions during fermentation. The variations in some physical, chemical and organoleptic characteristics of the yogurt samples were also monitored during cold storage at 4 °C for 21 days. Results indicated that all the combinations reduced the milk acidity to pH 4.6 within 4-5 hour at 43 °C. Gelation profiles of the samples were close to each other (close tan d values) with different elastic (G') and loss (G") moduli, indicating that similar chemical bondings took place in gel formation with different levels. All the samples showed a shear-thinning non-Newtonian flow behavior. Total number of starter bacteria was higher than 6 log cfu/mL in each sample after 21 days of storage. The post-acidification rates were also limited (between 4.21-4.37 after 21 days of cold storage). Untrained sensory evaluation panel found no marginal differences between the samples regarding appearance, body and texture, and aroma and flavor of the samples. To conclude, the combinations examined in this study were found to be suitable for commercial yogurt productions. [ABSTRACT FROM AUTHOR]

Aim This study investigates the individual and combined effects of fermentation parameters for improving cell biomass productivity and the resistance to freezing, freeze-drying, and freeze-dried storage of Lactobacillus delbrueckii subsp. bulgaricus CFL1. Methods and Results Cells were cultivated at different temperatures (42°C and 37°C) and pH values (5.8 and 4.8) and harvested at various growth phases (mid-exponential, deceleration, and stationary growth phases). Specific acidifying activity was determined after fermentation, freezing, freeze-drying, and freeze-dried storage. Multiple regression analyses were performed to identify the effects of fermentation parameters on the specific acidifying activity losses and to generate the corresponding 3D response surfaces. A multiobjective decision approach was applied to optimize biomass productivity and specific acidifying activity. The temperature positively influenced biomass productivity, whereas low pH during growth reduced the loss of specific acidifying activity after freezing and freeze-drying. Furthermore, freeze-drying resistance was favored by increased harvest time. Conclusions Productivity, and freezing and freeze-drying resistances of L. delbrueckii subsp. bulgaricus CFL1 were differentially affected by the fermentation parameters studied. There was no single fermentation condition that improved both productivity and resistance to freezing and freeze-drying. Thus, Pareto fronts were helpful to optimize productivity and resistance, when cells were grown at 42°C, pH 4.8, and harvested at the deceleration phase. [ABSTRACT FROM AUTHOR]

Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are commonly used together as a fermented milk starter. This study aimed to find the optimal ratio of two strains showing favorable fermentation characteristics, Lactobacillus delbrueckii subsp. bulgaricus T50 (T50) and Streptococcus thermophilus S10 (S10), in fermentation and explore their metabolic interaction. The optimal strain ratio was determined to be 1: 1000. The milk fermented with the optimal ratio had higher elasticity and viscosity, reached the gel point quickly, and had a strong gel structure compared to the commercial starters. T50+S10 exhibited favorable fermentation characteristics and storage stability, most similar to YF-L922, suggesting its potential to be developed as a commercial starter for fermented milk. Metabolomics analysis revealed that the T50+S10 combination enhanced the utilization of succinic acid, L-tyrosine, and L-tryptophan, promoting their mutualistic growth. T50 likely increased the availability of amino acid derivatives, small peptides, and calcium pantothenate to support S10 growth. These findings confirm the impact of L. bulgaricus and S. thermophilus ratio on fermented milk quality and provide insights into their metabolic-level interaction during fermentation. This study holds significance for the development and application of starter cultures in the dairy industry. [Display omitted] • Optimal starter ratio: 1:1000 of L. bulgaricus T50 and S. thermophilus S10. • Shortens gelation time, enhanced gel structure, and storage stability. • Enhances succinate, tyrosine, and tryptophan metabolism. • Provides amino acid derivatives and small peptides. • Facilitates pantothenate synthesis. [ABSTRACT FROM AUTHOR]

The multiple probiotic characteristics of strain TCI904 isolated in this study from natural fermented milk were investigated using a mouse model. TCI904 was identified as Lactobacillus delbrueckii subsp. bulgaricu (LDB), a well-known lactic acid starter bacterium found in yogurt. TCI904 exhibited an outstanding pancreatic lipase inhibition activity among several strains of lactic acid bacteria in vitro. Its in vivo effects were further studied. In a comparison of mice fed a high-fat diet (HFD) and those fed a HFD combined with TCI904 for 9 weeks, differences were observed in various aspects of health, and the adverse effects of a HFD were prevented in the latter group. TCI904 effectively prevented fat and body weight accumulation without reducing food intake; it also modulated innate immunity and increased the level of IgA in feces, reversing the increased blood sugar and insulin levels and attenuated the hyperlipidemia caused by a HFD. Based on biochemical test data, compared with the HFD group, a HFD combined with TCI904 induced significant lowering of insulin resistance indicator, homeostasis model assessment-insulin resistance (HOMA-IR) and atherogenic indices of plasma (AIP), the atherogenic coefficient (AC) and cardiac risk ratio (CRR) and increased the cardioprotective index (CPI). In addition, the administration of TCI904 alleviated mood disorders caused by a HFD. Taking the recommended human dose of TCI904 did not affect the liver or kidney function, indicating that TCI904 has sufficient in vivo safety. Taken together, the results of the present study contributed towards validation of the probiotic benefits of lactic acid starter microflora. Orally taken TCI904 exhibited positive immune- and metabolic-modulating, and anxiolytic properties, especially in HFD-induced obesity. [ABSTRACT FROM AUTHOR]

Simple Summary: Lactobacillus delbrueckii subsp. bulgaricus (LDB) is an important candidate for antibiotic replacement in pig production. In this study, LDB and antibiotic diets were fed to the LDB and antibiotic groups of female growing-finishing pigs, respectively. 16S rRNA sequencing was used to identify different microbiota. Liquid chromatography-mass spectrometry-based non-targeted metabolomics approaches were used to identify different metabolites. The co-occurrence network of the fecal microbiota and metabolite was analyzed. The results contain information on pig growth performance, microbiota data, metabolite data and co-occurrence networks, supporting the possibility of LDB as an antibiotics replacement in pig production. Lactobacillus delbrueckii subsp. bulgaricus (LDB) is an approved feed additive on the Chinese 'Approved Feed Additives' list. However, the possibility of LDB as an antibiotic replacement remains unclear. Particularly, the effect of LDB on microbiota and metabolites in the gastrointestinal tract (GIT) requires further explanation. This study aimed to identify the microbiota and metabolites present in fecal samples and investigate the relationship between the microbiota and metabolites to evaluate the potential of LDB as an antibiotic replacement in pig production. A total of 42 female growing-finishing pigs were randomly allocated into the antibiotic group (basal diet + 75 mg/kg aureomycin) and LDB (basal diet + 3.0 × 109 cfu/kg LDB) groups. Fecal samples were collected on days 0 and 30. Growth performance was recorded and assessed. 16S rRNA sequencing and liquid chromatography-mass spectrometry-based non-targeted metabolomics approaches were used to analyze the differences in microbiota and metabolites. Associations between the differences were calculated using Spearman correlations with the Benjamini–Hochberg adjustment. The LDB diet had no adverse effect on feed efficiency but slightly enhanced the average daily weight gain and average daily feed intake (p > 0.05). The diet supplemented with LDB increased Lactobacillus abundance and decreased that of Prevotellaceae_NK3B31_group spp. Dietary-supplemented LDB enhanced the concentrations of pyridoxine, tyramine, D-(+)-pyroglutamic acid, hypoxanthine, putrescine and 5-hydroxyindole-3-acetic acid and decreased the lithocholic acid concentration. The Lactobacillus networks (Lactobacillus, Peptococcus, Ruminococcaceae_UCG-004, Escherichia-Shigella, acetophenone, tyramine, putrescine, N-methylisopelletierine, N1-acetylspermine) and Prevotellaceae_NK3B31_group networks (Prevotellaceae_NK3B31_group, Treponema_2, monolaurin, penciclovir, N-(5-acetamidopentyl)acetamide, glycerol 3-phosphate) were the most important in the LDB effect on pig GIT health in our study. These findings indicate that LDB may regulate GIT function through the Lactobacillus and Prevotellaceae_NK3B31_group networks. However, our results were restrained to fecal samples of female growing-finishing pigs; gender, growth stages, breeds and other factors should be considered to comprehensively assess LDB as an antibiotic replacement in pig production. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate the effects of dietary non-encapsulated and microencapsulated Lactobacillus delbrueckii subsp. bulgaricus on growth performance, intestinal enzymatic activities, antioxidant capacity and hepato-biochemical parameters of rainbow trout before or after exposure to lead via diet. Fingerling fish (16 ± 4 g) were divided into four groups: negative control (NC), positive control (PC), probiotic (PR) and encapsulated probiotic (EN-PR). During the pre-exposure period (days 0–45), fish in the NC and PC groups received the basal diet, whereas fish in the PR and EN-PR groups were fed with basal diet containing 108 CFU g−1 feed of non-encapsulated and microencapsulated probiotic, respectively. During the exposure period (days 46–66), the fish in the probiotic and PC groups were co-treated with 500 μg g−1 feed of lead nitrate. Blood, liver and gut samples were taken at days 0, 45, 52, 59 and 66. The results revealed that growth performance and intestinal enzymatic activities were significantly (P<0.05) improved in the probiotic groups compared to the NC group (day 45). Dietary exposure to lead resulted in the highest levels of liver aspartate aminotransferase (AST), liver alkaline phosphatase (ALP) and serum malondialdehyde (MDA), and the lowest activities of serum superoxide dismutase (SOD) and catalase (CAT) in the PC group (day 66). The levels of liver ALP were significantly (P<0.05) lower in the probiotic groups compared to the NC and PC groups prior to and after exposure to dietary lead. Serum levels of total protein, albumin, SOD, CAT and glutathione (GSH) were significantly increased in fish fed with both non-encapsulated and microencapsulated probiotics (P<0.05). However, microencapsulated probiotic showed the greatest potential for alleviation of the disturbed activities of intestinal and hepatic enzymes, and improvement of serum biochemical and antioxidant parameters. Our findings suggest that L. delbrueckii subsp. bulgaricus, particularly in the microencapsulated form, can be used as a potential probiotic to protect rainbow trout from dietborne lead toxicity. [ABSTRACT FROM AUTHOR]

Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a microaerophylic anaerobe, which is widely used in the production of yogurt, cheese, and other fermented dairy products. L. bulgaricus and its partner Streptococcus thermophilus were used as starter cultures of yogurt in the world for thousands of years. In our previous study, L. bulgaricus LDB-C1 was obtained from traditional fermented milk, and possessed some characteristics like high exopolysaccharide yield and good fermentation performance. The analysis of its CRISPR–Cas system, antibiotic resistance, virulence factors, and mobile elements, was performed to reveal the stability of the strain LDB-C1. It was found that LDB-C1 contains a plenty of spacers in the CRISPR region, indicating it might have better performance against the infection of phages and plasmids. Furthermore, the acquired or transmittable antibiotic resistance/virulence factor genes were absent in the tested L. bulgaricus strain LDB-C1. [ABSTRACT FROM AUTHOR]

Gene feoA plays an important role in cell growth because of its function of transport Fe2+ which is a necessary element for cells. In this study, the recombinant plasmid pUC19-feoA-Tet was successfully constructed using the inserted gene inactivation method. Using the homologous recombination technique, the tet gene was used as a resistance screening marker to knock out the feoA gene of Lactobacillus delbrueckii subsp. bulgaricus 34.5 (strain 34.5). Comparative analysis of growth curves revealed the growth changes in the absence of feoA gene in strain 34.5. The results showed that the growth of the bacteria was prolonged by 2 h and could be restored in the stationary phase. To further study whether feoA is related to the cell division of strain 34.5, the qPCR experiment was carried out. The results showed that, compared with the wild-type strain, the expression of genes related to cell division in the mutant strain was up-regulated in the pre-log phase, down-regulated in the late-log phase, and returned to the original level in the stationary phase. These findings provide ideas for Lactobacillus delbrueckii subsp. bulgaricus to control division and cell cycle. [ABSTRACT FROM AUTHOR]

The probiotic efficacy and fermentative ability of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), a widely used probiotic, is majorly affected by its acid tolerance. Here, we conducted whole-genome sequencing of the high acid-tolerant L. bulgaricus LJJ stored in the laboratory. Compared with the whole genome of low acid-tolerant strain L. bulgaricus ATCC11842, the results show that 16 candidate acid-tolerant genes may be involved in the regulation of the acid tolerance of L. bulgaricus LJJ. Association analysis of candidate acid-tolerant genes and acid-tolerant traits of different L. bulgaricus strains revealed that the three genes dapA, dapH, and lysC are the main reasons for the strong acid tolerance of L. bulgaricus LJJ. The results of real-time quantitative PCR (RT-qPCR) supported this conclusion. KEGG pathway analysis showed that these three acid-tolerant genes are involved in the synthesis of lysine; the synthesis of lysine may confer L. bulgaricus LJJ strong acid tolerance. This study successfully revealed the acid tolerance mechanism of L. bulgaricus LJJ and provides a theoretical basis for the subsequent selection of strains with high acid tolerance for improved probiotic functions. Key points: • Three genes are identified as acid-tolerant genes, respectively, lysC, dapA, and dapH. • LysC and dapA are the major key genes in the synthesis of lysine. • The synthesis of lysine may confer L. bulgaricus LJJ strong acid tolerance. [ABSTRACT FROM AUTHOR]

The aim of this research was to determine the optimal medium for Exopolysaccharides (EPS) production by a Lactobacillus delbrueckii subsp. bulgaricus ropy strain isolated from a locally produced commercial fermented milk, in reconstituted skim milk (RSM) 10% (w/v), milk whey (MW), and soy milk whey (SMW), under optimal growth conditions for this strain. Milk whey was made by coagulating fresh milk using papaya latex 3% (v/v); soy milk whey was obtained from tofu household industry. The chemical composition of the substrate media was determined by proximate analysis, and sterilization was accomplished in an autoclave at 121°C for 15 min. Culture media were inoculated with 1% (v/v) of a starter culture of L. delbrueckii subsp. bulgaricus and then incubated at 30°C for 16 hr. EPS production, lactic acid content, cell counting, and pH were determined after the media were cooled at 5°C. Findings showed that on the basis of the growth characteristics of L. delbrueckii subsp. bulgaricus, the best medium for EPS production was RSM 10% (258.60 ± 26.86 mg/L) compared to the milk whey (69.60 ± 9.48 mg/L) and soy milk whey (49.80 ± 9.04 mg/L). [ABSTRACT FROM AUTHOR]

Gioddu is the sole variety of fermented milk originating in Italy. Despite the long history of consumption, Gioddu still represents an undisclosed source of microbial diversity. The present study was aimed to get an insight into the bacterial and fungal diversity of Gioddu samples collected from two artisan producers located in Sardinia. To this end 3 batches of Gioddu were collected from each producer and subjected to Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analyses. Gioddu was produced with sheep milk in accordance with the local tradition. Regarding the bacterial population, a low biodiversity emerged. In more detail, the sole species Lactobacillus delbrueckii subsp. bulgaricus was detected in all the samples, irrespective of the producer or the batch. A more ample microbial diversity was highlighted for the fungal population that included closest relatives to Pichia cactophila, Kluyveromyces marxianus and Galactomyces candidum. Based on the results, the detected bacterial and fungal species generally clustered in accordance with the producer, irrespective of the batch considered. It is noteworthy that, Gioddu revealed several microbiological similarities with kefir beverage, thus suggesting, by analogy, potential health benefits related to its consumption. More research is needed to better clarify the microbiota composition of Gioddu by using more powerful metagenomic techniques. [ABSTRACT FROM AUTHOR]

Background: Aerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective. Results: The differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus. Conclusions: Overall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators. [ABSTRACT FROM AUTHOR]

Summary: Lactobacillus delbrueckii subsp. bulgaricus CH9‐3 proteinase is an enzyme that plays an important role in yogurt fermentation, by which its use is linked to gel properties of yogurt. In this work, a modified glucono‐δ‐lactone (GDL) acidification model was used to simulate the fermentation of yogurt and study the effect of CH9‐3 proteinase on GDL‐acidified milk gel. The results showed that the proteinase significantly improved the quality of milk gel by increasing its firmness, consistency, and viscosity index, while decreasing its syneresis. Furthermore, the proteinase could accelerate the formation of milk gel as indicated by the decrease in surface charge of milk gel. The findings demonstrate that CH9‐3 proteinase has a positive effect on the properties of milk gel, and thus may have promising applications in the yogurt making industry. [ABSTRACT FROM AUTHOR]

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography–mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621–7633, 2017) showed that β-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the proliferation effect of fractionated lotus seed resistant starch (LRS3-20%, LRS3-30%) and the structural changes following fermentation. Fractionated lotus seed resistant starch, especially LRS3-20%, promoted the proliferation of these probiotics, and Bifidobacterium longum produced large quantities of acetic acid, while Lactobacillus delbrueckii subsp. bulgaricus produced lactic acid. Additionally, the probiotics with lotus seed resistant starch (LRS3) and LRS3-20% survived more successfully in simulated gastrointestinal solutions compared to glucose, LRS3-30% and high amylose maize starch, but LRS3 and LRS3-20% differed in their abilities to enhance the tolerance to acidic buffer, bile acid and simulated gastric juice. Furthermore, after fermentation, fractionated lotus seed resistant starch displayed a loose, rough surface. The crystalline and double helical structure increased as the amount of amorphous and sub-crystalline structure reduced following fermentation. Interestingly, structural changes were correlated with the original structures and utilisation by the probiotics. [ABSTRACT FROM AUTHOR]

Lactobacillus delbrueckii subsp. bulgaricus is currently one of the most valuable lactic acid bacteria (LAB) and widely used in global dairy industry. The acid tolerance and adaptation ability of LAB is the key point of their survival and proliferation during fermentation process and in gastrointestinal tract of human body. Two component system (TCS) is one of the most important mechanisms to allow bacteria to sense and respond to changes of environmental conditions. TCS typically consists of a histidine protein kinase (HPK) and a corresponding response regulator (RR). Our previous study indicated a TCS (JN675228/JN675229) was involved in acid adaptation in L. bulgaricus. To reveal the role of JN675228 (HPK1)/JN675229 (RR1) in acid adaptation, the target genes of JN675228 (HPK1)/JN675229 (RR1) were identified by means of a proteomic approach complemented with transcription data in the present study. The results indicated that HPK1/RR1 regulated the acid adaptation ability of bacteria by means of many pathways, including the proton pump related protein, classical stress shock proteins, carbohydrate metabolism, nucleotide biosynthesis, DNA repair, transcription and translation, peptide transport and degradation, and cell wall biosynthesis, etc. To our knowledge, this is the first report with the effect of acid adaptation-related TCS HPK1/RR1 on its target genes. This study will offer experimental basis for clarifying the acid adaptation regulation mechanism of L. bulgaricus, and provide a theoretical basis for this bacterium in industry application. [ABSTRACT FROM AUTHOR]

• Cell envelope proteinase (CEP) extracted from Lactobacillus delbrueckii subsp. bulgaricus. • CEP mainly degraded soybean protein near 35 kDa. • Hydrolysis time affects structural properties and antioxidant activity of hydrolysates. • Hydrolysates after 4 h have the highest antioxidant activity. In this work, we investigated the structural and biological properties of soybean protein isolate (SPI) after 0–8 h hydrolyzation with cell envelope proteinase (CEP) extracted from Lactobacillus delbrueckii subsp. bulgaricus. CEP hydrolysis increased the β-sheet and red-shifted the fluorescence peak, while decreasing the α-helix, indicating the unfolding of soybean proteins. Increased surface hydrophobicity and fluorescence of the soybean protein hydrolysates were correlated with the increased hydrophobic amino acid (from 209.67 to 217.6 mg/100 g). CEP tended to hydrolyze the N- and C-terminal regions of sequences dominated by Gly and Leu, which enhanced the antioxidant activity of the SPHs (lowest IC 50s value of ABTS•+ and hydroxyl radical scavenging activity were 0.324 ± 0.006 mg/mL and 0.365 ± 0.001 mg/mL after 4 h hydrolysis). Comparison with the database of bioactive peptides suggested various potential biological activities, including antioxidant activity, angiotensin-converting enzyme inhibitory activity and dipeptidyl peptidase-IV inhibitory activity. The study findings have theoretical significance for the development of CEP hydrolysis and novel bioactive soybean peptides. [ABSTRACT FROM AUTHOR]

Background. The efficacy of Lactobacillus delbrueckii subsp. bulgaricus as starter cultures for the dairy industry depends largely on the number of viable and active cells. Freeze-drying is the most convenient and successful method to preserve the bacterial cells. However, not all strains survived during freeze-drying. Methods. The effects of six substances including NaCl, sorbitol, mannitol, mannose, sodium glutamate, betaine added to the MRS medium on the growth and freeze-drying survival rate and viable counts of Lb. delbrueckii subsp. bulgaricus were studied through a single-factor test and Plackett-Burman design. Subsequently, the optimum freeze-drying conditions of Lb. delbrueckii subsp. bulgaricus were determined. Results. Lb. delbrueckii subsp. bulgaricus survival rates were up to the maximum of 42.7%, 45.4%, 23.6%, while the concentrations of NaCl, sorbitol, sodium glutamate were 0.6%, 0.15%, 0.09%, respectively. In the optimum concentration, the viable counts in broth is 6.1, 6.9, 5.13 (×108 CFU/mL), respectively; the viable counts in freeze-drying power are 3.09, 5.2, 2.7 (×1010 CFU/g), respectively. Conclusion. Three antifreeze factors including NaCl, sorbitol, sodium glutamate have a positive effect on the growth and freeze-drying of Lb. delbrueckii subsp. bulgaricus. The results are beneficial for developing Lb. delbrueckii subsp. bulgaricus. [ABSTRACT FROM AUTHOR]

The rheological and physical characteristics of non-fat set yogurt produced using co-cultures of ropy EPS-producingStreptococcus thermophilusS3.3 andLactobacillus delbrueckiisubsp.bulgaricusLTM and ofStreptococcus thermophilusS3.3 andLactobacillus delbrueckiisubsp.bulgaricusH+-ATPase-defective mutant L6 under different conditions were studied. Yogurts produced with mutant L6 had a higher pH and water-holding capacity and better textural properties compared to those produced with the LTM strain. The highest storage modulus (G′) and thixotropic loops were found for yogurt made with L6 at 42°C on day 21 of cold storage. This yogurt contained a network of thick, continuous protein aggregates and large void spaces. [ABSTRACT FROM AUTHOR]

Lactic acid production from renewable resources such as pods of carob alternate to derived products from oil present a growing interest, in particular for the production of biodegradable polylactic acid polymer. The carob pods syrup is a by-product composed of sugars and its use as substrate of low cost in various industries is considered however, few microorganisms consume quickly and effectively sugars to give a single product. Lactic acid bacteria were screened for sugars fermentation and Lactobacillus delbrueckii subsp. bulgaricus showed the highest levels of lactic acid production. The physicochemical analysis of carob pods syrup showed that it presents a suitable quality. The micro-organism implemented is kind Lactobacillus delbrueckii subsp. bulgaricus, cultivated in bathe fermentation. Three culture mediums were tested, the carob syrup enriched with sweet cheese whey (CS+SCW), carob syrup enriched with Tween80 (CS+T80) and carob syrup enriched with sweet cheese whey+Tween80 (CS+SCW+T80), the addition of the growth factors improve the culture, that is why the production of lactate is very important improved 52.76 g/l. [ABSTRACT FROM AUTHOR]

This study was carried out to determine the properties of set type yoghurts made using microwave heating after yoghurt fermentation during storage period of 28 d at 4±1°C. In this way, it will be possible to increase the yoghurt shelf-life, by preventing post-acidification. Yoghurts samples were subjected to three different microwave heating (10,20 and 30 sec) at 720 Watt power level. Viability of yoghurt bacteria ( Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) was assessed during 28 d of storage. Samples were subjected also physicochemical analysis (pH and titratable acidity).The results showed that microwave applications significantly affected viable counts of L. delbrueckii ssp. bulgaricus and S. thermophilus (P<0.05). The counts of L. bulgaricus and S. thermophilus increased in heated yoghurts but a delay was observed during of storage period. Also the results indicated that the microwave heating had significant effect on the titratable acidity values (P<0.05).The titratable acidity and pH showed similar patterns of increase or decrease during storage period. During refrigerated storage, decline in pH was greater in control group yoghurts than heated yoghurts. The titratable acidity increased in all yoghurt samples. However, the rate of increase was affected by the microwave heating time. The results of the study showed that the microwave heating had significant effect on the acceptability of yoghurt during shelf-life [ABSTRACT FROM AUTHOR]

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear double-stranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry. [ABSTRACT FROM AUTHOR]

Lactobacillus delbrueckii subsp. bulgaricus is an acid-tolerant species used in yoghurt production and considered important in postacidification reductions in palatability of yoghurt during storage. In this study, a variant ( ND06-2) of L. delbrueckii subsp. bulgaricus ND06 was selected using neomycin sulphate. ND06-2 exhibited sensitivity to acids and lower H+- ATPase activity compared with ND06. High-pressure liquid chromatography ( HPLC) was used to demonstrate that the capacity of ND06-2 to produce lactic acid was significantly lower than for ND06 during fermentation or storage. These results suggest that the L. delbrueckii subsp. bulgaricus variant could be used in yoghurt starters and have a lower tendency to cause postacidification. [ABSTRACT FROM AUTHOR]

Red beans exhibit many biofunctions, including the stimulation of intestinal motility, improvement of anemia and elimination of edema. This study was conducted to evaluate the functional properties of microbially fermented red beans produced under different fermentation conditions and to establish the optimum fermentation conditions for the production of fermented red beans with multiple biofunctions. The optimum fermentation conditions were the fermentation of red beans by a co-culture of Bacillus subtilis and Lactobacillus bulgaricus in the presence of 1% glucose, incubated at 30 °C for 120 h and stirred every 24 h. Compared with unfermented red beans, red beans fermented under the optimum conditions contained a higher concentration of antioxidant substances, including total phenolics, anthocyanin, flavonoids and vitamins C and E. The results of tests for DPPH-radical scavenging, ferrous ion chelation and reducing power implied a high antioxidant content. Fermented red beans exhibited nattokinase activity and contained a significant amount of potential probiotics. [ABSTRACT FROM AUTHOR]

Yogurt is a fermented milk product produced by two homofermentative lactic acid bacteria,Lactobacillus delbrueckiisubsp.bulgaricusandStreptococcus thermophilus. It is consumed throughout the world as a healthy, nutritional food. In this study, the strains ofLactobacillusandStreptococcuswere isolated from raw milk samples collected from different local geographical regions of Turkey. One hundred and thirty isolates were characterized to genus level by classical morphological, physiological and biochemical tests. Twenty six of all isolates were identified asL. delbrueckiisubsp.bulgaricusand 47 isolates were identified asS. thermophilususing API 50 CHL system. RAPD-PCR was used to explain molecular characterization and genetic differentiation of a total of 73 strains. The whole cell protein profiles of these strains were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this study, all strains were identified by the RAPD-PCR technique to species or strain level. SDS-PAGE was also shown to be an effective method for molecular differentiation all of the strains. Phenotypic identification, RAPD-PCR and SDS-PAGE proved to be informative methods suitable to explain molecular characterization and genetic differentiation ofL. delbrueckiisubsp.bulgaricusandS. thermophilusstrains isolated from raw milk. In the present study, we described an identification system by using RAPD-PCR and SDS-PAGE techniques and used this system for genetic differentiation ofL. delbrueckiisubsp. bulgaricus andS. thermophilusstrains isolated from raw milk. It is believed that these two molecular techniques allow researchers to fill the significant gaps present in our understanding of these organisms by providing a comprehensive view potentially involved in industrially relevant phenotypes from different geographical origin. [ABSTRACT FROM AUTHOR]

Glutamate decarboxylase enzyme produces γ-aminobutyric acid (GABA) in a non-reversible decarboxylation reaction of glutamate. GABA is a major inhibitory neurotransmitter of the brain and it is also present at high concentration in other organs such as pancreatic islets. GABA has effects on blood pressure, diabetes, inflammation, sleeplessness and depression. Some bacteria such as Lactobacillus strains are capable of GABA production. Identification of these bacteria is important both for researchers and industry. The aim of this study was molecular gene cloning and sequencing of glutamate decarboxylase (gad) from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272. These strains were cultured in MRS medium at 37°C for 24 hours. For cloning gad gene from these strains, polymerase chain reaction (PCR) was performed using specific primers designed by Oligo7 software. PCR production was extracted from agarose gel and was inserted into PGEM-T vector using T4 DNA ligase enzyme and then it was transformed to E. coli XL1Blue. In the final step, white colonies were selected and after plasmid extraction, the existence of gad gene in recombinants was confirmed by PCR. Gad gene was cloned from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272. It is for the first time that the gad gene sequences from these bacteria were registered on NCBI with accession numbers KF751355 and KF751352 respectively. The result of this research indicates that the two aforementioned bacteria contain glutamate decarboxylase gene and therefore they possibly can be used for industrial γ-aminobutyric acid production. [ABSTRACT FROM AUTHOR]

In order to purify proteinase fromLactobacillus delbrueckiisubsp.bulgaricus, chromatography methods including ion-exchange and hydrophobic interaction were combined. Chromatographic conditions were structured and optimized. The final sample volume, flow rate, and length of gradient for anion exchange chromatography were 20 mL, 5 mL/min and 10 column volume length of gradient, in that order. Besides, the flow rate and length of gradient for hydrophobic interaction chromatography were 1 mL/min and 10 column volume. Proteinase was purified up to 43-fold with a specific activity of 54.4 U/mg protein, and the recovery of 46.6%. The molecular weight of the purified proteinase was about 40 kDa. [ABSTRACT FROM PUBLISHER]

Yogurt is a fermented milk product produced by two homofermentative lactic acid bacteria, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. It is consumed throughout the world as a healthy, nutritional food. In this study, the strains of Lactobacillus and Streptococcus were isolated from raw milk samples collected from different local geographical regions of Turkey. One hundred and thirty isolates were characterized to genus level by classical morphological, physiological and biochemical tests. Twenty six of all isolates were identified as L. delbrueckii subsp. bulgaricus and 47 isolates were identified as S. thermophilus using API 50 CHL system. RAPD-PCR was used to explain molecular characterization and genetic differentiation of a total of 73 strains. The whole cell protein profiles of these strains were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this study, all strains were identified by the RAPD-PCR technique to species or strain level. SDS-PAGE was also shown to be an effective method for molecular differentiation all of the strains. Phenotypic identification, RAPD-PCR and SDS-PAGE proved to be informative methods suitable to explain molecular characterization and genetic differentiation of L. delbrueckii subsp. bulgaricus and S. thermophilus strains isolated from raw milk. In the present study, we described an identification system by using RAPD-PCR and SDS-PAGE techniques and used this system for genetic differentiation ofL. delbrueckii subsp. bulgaricus and S. thermophilus strains isolated from raw milk. It is believed that these two molecular techniques allow researchers to fill the significant gaps present in our understanding of these organisms by providing a comprehensive view potentially involved in industrially relevant phenotypes from different geographical origin. [ABSTRACT FROM AUTHOR]

[Display omitted] • L. bulgaricus L7 with antioxidant capacity was isolated from Xinjiang cheese. • Both fermented milk and glutathione could relieve alcohol-induced oxidative stress and inflammatory responses. • Fermented milk in restoring alcohol-induced gut dysbiosis stronger than glutathione. An isolate of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus L7) with robust antioxidant capacity was screened from traditional fermented Xinjiang cheese. Here, we focused on evaluating the effect of L. bulgaricus fermented milk on acute alcoholic liver disease prevention using a mice model, with glutathione treated mice as positive control. The results showed that both fermented milk and glutathione feeding could relieve alcohol-induced oxidative stress and inflammatory responses significantly. However, fermented milk feeding seemed to hold more promise in restoring gut dysbiosis relative to glutathione. Enhanced abundance of potential pathogens, Porphyromonas sp. and Enterococcus sp., induced by alcohol was considerably attenuated by additional feeding of fermented milk. Different scenario was firstly observed when additionally fed with glutathione in this study, showing remarkably increased abundance of Bacteroides sp. and Enterococcus sp. Our results suggested the more robust capability of L. bulgaricus fermented milk in restoring alcohol-induced gut dysbiosis relative to glutathione, indicating its potential protective effects on alcohol-induced hepatic injury. [ABSTRACT FROM AUTHOR]

Abstract: The ability of two types of galacto-oligosaccharides (GOS) and lactulose to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze-drying was evaluated on the basis of their capacity to form glassy structures, using near infrared (NIR) spectroscopy. After freeze-drying in the presence of protective agents, microorganisms were stored at 4°C at different relative humidities (RH) (from 11 to 80%) up to 21days. The loss of viability was determined by plate count and referred to viability before freeze-drying. For each storage condition, glass transition temperatures (Tg) were determined by differential scanning calorimetry (DSC). In parallel, near infrared spectra registered in the 900–1700nm region provided information about viability and RHs that could be correlated with the results obtained by plate counts and DSC. The comparison of NIR spectra of freeze-dried samples stored at the same RH but differing in the time of storage allowed the identification of spectral features related with viability. The spectral differences observed on samples stored in the same conditions (same time of storage and same protective agent) but differing in RHs were ascribed to the formation of amorphous and rubbery states. Based on the spectral information, a multivariate based classification model (PLS-DA) was defined to determine whether samples are in a rubbery or an amorphous state. As the physical state of starters is related with their shelf-life, this model provided support for the use of NIR spectroscopy as a quick method to assess the proper storage of starters. [Copyright &y& Elsevier]

In this work, five ultrafiltered fractions ( UFs) with molecular mass less than 3 kDa (kilo-daltons) from casein hydrolysates treated with alcalase, flavourzyme, neutrase, papain and trypsin, respectively, were obtained. The effect of five UFs on the fermentation for lactic acid ( LA) production by mixed cultures of Streptococcus thermophilus ( St) and Lactobacillus delbrueckii subsp. bulgaricus ( Lb) during 72 h of cultivation was investigated. Compared with the control, LA production was, respectively, enhanced by 23.66%, 39.01%, 29.74%, 49.64% and 47.40% with the supplement of UF- A, UF- F, UF- N, UF- P and UF- T at 24 h. The possible mechanism of LA production enhanced was elucidated by the time course analysis of the specific activity of glucokinase, phosphoglucose isomerase, 6-phosphofructokinase, pyruvate kinase and lactate dehydrogenase during fermentation process. In addition, the results obtained showed the diverse influence of five UFs on the bacterial fermentation was attributed to their different amino acid distribution. [ABSTRACT FROM AUTHOR]

Objectives: Genetic diversity and resistance of Lactobacillus bulgaricus sbsp. delbrueckii collection with 100 isolates from different home-made yogurt in rural Bulgarian areas were determined. Methods: The strain K98 was the most resistant to bile salts and low pH. Survival and effects on short chain fatty acids production were tested in 20 healthy volunteers. High genetic diversity was observed in the L. bulgaricus collection by RAPD, whereas the ability of tolerate high deoxycholic acid concentrations, and different acid pHs was variable. The strain K98 was selected and used to prepare a homemade yogurt which was administered to 20 healthy volunteers (500 ml/day during 15d). A basal faecal sample and another after yogurt intake were recovered. Results: DGGE experiments, using both universal and Lactic Acid Bacteria (LAB) primers, demonstrated no significant changes in the qualitative composition of gut microbiota. A band corresponding to L. bulgaricus was observed in all 20 samples. Viable L. bulgaricus K98 strain was only recovered in one volunteer. After yogurt intake we found an increase of LAB and Clostridium perfringens, and a decrease of Bacteroides- Prevotella-Porphyromonas. In addition, increases of acetic, butyric and 2-hydroxy-butyric acids in faeces were detected. Conclusions: Genetic diversity of L delbrueckii subsp. bulgaricus especie is high We have isolated a probiotic resistant strain to bile and high acidity, L delbrueckii subsp. bulgaricus-K98. Qualitative and quantitative changes in the intestinal microbiota are found after ingestion of a homemade yogurt containing this strain, with a concomitant increase in faecal SCFA. Our findings support the interest in developing further studies providing different amounts of L delbrueckii subsp. bulgaricus-K98, and should evaluate its clinical effects in human disease. [ABSTRACT FROM AUTHOR]

Continued acid production by Lactobacillus delbrueckii subsp . bulgaricus during the chilled storage of yogurt is the major cause of postacidification, resulting in a short shelf life. Two H+-ATPase defective variants of L. delbrueckii subsp . bulgaricus were successfully isolated and their H+-ATPase activities were reduced by 51.3% and 34.3%, respectively. It was shown that growth and acid production of variants were remarkably inhibited. The variants were more sensitive to acidic condition and had a significant rate for inactivation of H+-ATPase by N, N-dicyclohexylcarbodiimide (DCCD), along with a low H+-extrusion, suggesting that H+-ATPase is direct response for H+-extrusion. In addition, the variants were also more sensitive to NaCl, while H+-ATPase activities of variants and parent strain were significantly enhanced by NaCl stress. Obviously, H+-ATPase might be involved in Na+ transportation. Furthermore, variants were inoculated in fermented milk to ferment yogurt. There was no significant difference in flavor, whereas the postacidification of yogurt during chilled storage was remarkably inhibited. It is suggested that application of L. delbrueckii subsp . bulgaricus with reduced H+-ATPase activity in yogurt fermentation is one of effect, economic and simple avenues of inhibiting postacidification of yogurt during refrigerated storage, giving a longer shelf life. Practical Application: During yogurt fermentation, continued acid production by Lactobacillus delbrueckii subsp . bulgaricus during the chilled storage of yogurt leads to milk fermentation with high postacidification, resulting in a short shelf life. In this work, 2 acid-sensitive variant strains of L. delbrueckii subsp . bulgaricus were isolated. The characteristics related to H+-ATPase were compared and it was observed that milk fermented by the variants had lower postacidification, giving a longer shelf life. Application of L. delbrueckii subsp . bulgaricus with reduced H+-ATPase activity in yogurt fermentation might be one of effect, economic and simple avenues of inhibiting yogurt postacidification during chilled storage, giving a longer shelf life. [ABSTRACT FROM AUTHOR]

Abstract: The Gram-positive bacterium Lactobacillus delbrueckii subsp. bulgaricus is of vital importance to the food industry, especially to the dairy industry. Two component systems (TCSs) are one of the most important mechanisms for environmental sensing and signal transduction in the majority of Gram-positive and Gram-negative bacteria. A typical TCS consists of a histidine protein kinase (HPK) and a cytoplasmic response regulator (RR). To investigate the functions of TCSs during acid adaptation in L. bulgaricus, we used quantitative PCR to reveal how TCSs expression changes during acid adaptation. Two TCSs (JN675228/JN675229 and JN675230/JN675231) and two HPKs (JN675236 and JN675240) were induced during acid adaptation. These TCSs were speculated to be related with the acid adaptation ability of L. bulgaricus. The mutants of JN675228/JN675229 were constructed in order to investigate the functions of JN675228/JN675229. The mutants showed reduced acid adaptation compared to that of wild type, and the complemented strains were similar to the wild-type strain. These observations suggested that JN675228 and JN675229 were involved in acid adaptation in L. bulgaricus. The interaction between JN675228 and JN675229 was identified by means of yeast two-hybrid system. The results indicated there is interaction between JN675228 and JN675229. [Copyright &y& Elsevier]

Abstract: Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥106 viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45°C for 72h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO2 atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37°C for 72h. [Copyright &y& Elsevier]

The primary purpose of this research was to monitor the influence of sunflower honey addition (2%, 4% and 6% w/v) to yoghurt milk on survival of the microbial flora of yoghurt and the physicochemical and sensory characteristics during refrigerated storage for 4 weeks. The water activity decreased according to the addition of honey with higher concentrations in the storage period (4°C). At the end of fermentation, pH values of yoghurt samples ranged between 4.33 (without honey) and 4.52 (addition of honey with 6%). The highest water holding capacity, consistency and the lowest brightness values were determined in the groups produced with 6% honey addition. The water holding capacity and a* (redness) values of the honey incorporated yoghurt samples increased during storage. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus values of the yoghurt with honey increased compared with the control group samples (P < 0.01). Addition of honey to yoghurt milk has affected the vitality of the characteristics starters in the incubation and storage time of the yoghurt samples (P < 0.01). Optimum sweetness was obtained with the samples containing 4% honey level. [ABSTRACT FROM AUTHOR]

Superior artisanal isolates of thermophilic lactic acid bacteria producing high lactic acid and β-galactosidase were isolated from traditional Turkish yogurt samples from the Toros mountain region from a highly bio-diverse environment. A full factorial statistical design, with the factors of types of strains and medium formulations under static and agitation conditions, were applied to investigate the effects on β-galactosidase and lactic acid production. Streptococcus thermophilus 95/2 and Lactobacillus delbrueckii subsp. thermophilus 77 exhibited remarkable potential as promising starter culture candidates valuable to various applications in the dairy industry. The efficiency of cell disruption methods was investigated on the extraction of intracellular β-galactosidase enzyme. Lysozyme enzyme treatment was determined as the most effective method, which resulted in approximately 1.5 and 10 times higher enzyme activity than glass bead and homogenization treatment, respectively. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to study the effect of different gaseous conditions on the physico-chemical properties of yoghurt. Four conditions of oxidoreduction potential (Eh7), +433mV (milk gassed with air), +405mV (ungassed milk), +283mV (milk gassed with N2) and −349mV (milk gassed with N2–H2) were applied to milk. The rheological properties and microstructure of these yoghurts were determined by measuring apparent viscosity, whey separation (WS) and using confocal laser scanning microscopy. Exopolysaccharide (EPS) production by lactic acid bacteria (LAB) was also studied, and production was increased for extreme Eh7 values: the highest values were obtained for air (oxidizing condition) and for N2–H2 (reducing condition). Yoghurts made under bubbled N2–H2 clearly led to low WS with higher gel aggregation and a lower proportion of pores. [Copyright &y& Elsevier]

Aim: The aim of the study was to evaluate the adhesion of Lactobacillus delbrueckii subsp . bulgaricus and Lactobacillus rhamnosus strain GG to saliva-coated surfaces in vitro. Methods: Fifteen radiolabeled dairy L. delbrueckii subsp . bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite beads and polystyrene microtiter plates and the radioactivity was measured by liquid scintillation counter. The effects of lysozyme on the adhesion of lactobacilli and of pretreatment with lactobacilli on the adhesion of Streptococcus sanguinis were also assessed. Results: All strains tested adhered to saliva-coated surfaces but with significantly different binding frequencies. The adhesion of the L. delbrueckii subsp . bulgaricus strains remained lower in comparison to L. rhamnosus strain GG. One L. delbrueckii subsp . bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of the samples significantly increased lactobacillus adhesion to saliva-coated surfaces. Conclusion: The present results showed significant variations in the adhesion capacity of the Lactobacillus strains studied. Adhesion to oral surfaces is of primary importance for bacterial colonization in the mouth. Only one of the L. delbrueckii subsp . bulgaricus dairy starter culture strains investigated had a high adhesion percentage. This strain might then be considered for further investigations in the oral environment. [ABSTRACT FROM AUTHOR]

Background. Some researchers have indicated that Lactobacillus delbrueckii subsp. bul garicus may provide additional health benefits, reduce serum cholesterol level, for example. The aim of this study was to determine cholesterol uptake by Lb. delbrueckii subsp. bulgaricus commercial yoghurt starter isolates in artificial GIT fluids. Material and methods. Lb. delbrueckii subsp. bulgaricus isolates were cultured in MRS broth and in artificial GIT fluids contained cholesterol at initial concentration ca. 560 μg/mL, as well as in MRS broth with cholesterol addition. Results. All Lb. delbrueckii subsp. bulgaricus showed ability to uptake of cholesterol from MRS broth and artificial GIT fluids. The isolates incubated in artificial gastric fluid removed the minimal amounts of cholesterol in comparison to the same isolates incubated in MRS broth. Only two isolates removed significantly more cholesterol from MRS broth that from duodenal fluid. The amount of removed cholesterol from artificial duodenal fluid ranged from 20 μg/mL to 78 μg/mL. All Lb. delbrueckii subsp. bulgaricus isolates survived worse in artificial GIT fluids than in MRS broth. The viability of Lb. delbrueckii subsp. bulgaricus in GIT fluids depended on isolate. Conclusions. These results proved that Lb. delbrueckii subsp. bulgaricus shows ability to uptake cholesterol from MRS broth and artificial GIT fluids, and the degree of cholesterol uptake depends on isolate and incubation environment. The ability of Lb. delbrueckii subsp. bulgaricus to survive in GIT varies according to the isolates and incubation environment. [ABSTRACT FROM AUTHOR]

Abstract: Enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus is a priority due to their importance in yogurt production. Capillary electrophoresis (CE) of both bacteria could be achieved in 7.2min with a resolution of 3.2 in the background electrolyte (BGE) containing 4.5mM Tris(hydroxymethyl) amminomethane (TRIS)–4.5mM boric acid–0.1mM ethylenediamine tetraacetate (EDTA) (TBE) buffer (pH 8.4) and 0.05% (v/v) polyethylene oxide (PEO), using a capillary of 47.5cm (effective length)×100μm i.d., injection of 50mbar×3s followed by −5kV×120s, a voltage and temperature of 20kV and 25°C, respectively. Appropriate amounts of PEO in the BGE, sample preparation (i.e. vortex) and introduction were key factors for their separation. A short hydrodynamic injection followed by applying reversed polarity voltage could compress the bacteria into narrow zones, which were detected as separated single peaks. Method linearity (r 2 >0.99), precision (%RSDs<9.3%), recovery (%R =91.7–106.7%) and limit of quantitation (1.0×106 colony forming unit per mL (CFU/mL)) were satisfactory. Results from the CE analysis of both bacteria in yogurt were not statistically different from those of the plate count method (P >0.05). The CE method can be used as an alternative for quantitation of L. delbrueckii subsp. bulgaricus and S. thermophilus in yogurt since it was reliable, simple, cost and labor effective and rapid, allowing the analysis of 3 samples/h (comparing to 2d/sample by plate count method). [Copyright &y& Elsevier]

Abstract: Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+-ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+-ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P ≤0.05) reduced H+-ATPase activity were successfully isolated. Yoghurt fermented using L. delbrueckii subsp. bulgaricus SBT0164 No. 55-1 (mutant) starter culture had markedly reduced post-acidification and maintained viability (≥108 CFU/ml) of both Bifidobacteruim breve JCM 1192T and Bifidobacteruim breve JCM 7017 during storage at 10 °C for 21 days. These results clearly showed that yoghurt fermented by mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+-ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during refrigerated storage. [Copyright &y& Elsevier]

Abstract: Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria. There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production. [Copyright &y& Elsevier]

Abstract: Responses of Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus to a rapid change in extracellular pH (pHex) from pHex 6 to a range of concentrations down to pHex 3.0 were examined, using HCl and lactic acid (LA) as acidulants. A new fluorescent probe 5-(and-6)-carboxy-2′, 7′-dichlorofluorescein diacetate succinimidyl ester (CDCFDA-SE) was employed that enabled reliable measurements of intracellular pH (pHi) to a minimum pHi of 4.0. Changes in pHi and H+ fluxes from immobilised bacteria were measured using fluorescence ratio imaging microscopy (FRIM) and a non-invasive ion flux measuring technique (MIFE), respectively. L. innocua maintained a relatively constant pHi of 5.5–6.1 at pHex 4 and 5 via H+ extrusion. In contrast, L. delbrueckii subsp. bulgaricus progressively lowered pHi towards pHex over the entire pHex range examined. The type of acidulant used influenced pH regulation with both pHi and H+-fluxes being more severely affected by LA compared to HCl. Overall, our data demonstrated different adaptive strategies in these two bacteria. While L. innocua expels protons to maintain a constant pHi, L. delbrueckii subsp. bulgaricus allows proton entry after acidic treatment so that pHi follows pHex. [Copyright &y& Elsevier]

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions ( D=0.10 h−1), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h−1, and further to 0.35 h−1, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved. [ABSTRACT FROM AUTHOR]