Genetic diversity and phylogenetic relationships of Streptococcus thermophilus isolates from traditional Turkish yogurt: multilocus sequence typing (MLST).

Yogurt, a globally consumed fermented dairy product, is recognized for its taste and potential health benefits attributed to probiotic bacteria, particularly Streptococcus thermophilus. In this study, we employed Multilocus Sequence Typing (MLST) to investigate the genetic diversity and phylogenetic relationships of 13 S. thermophilus isolates from traditional Turkish yogurt samples. We also assessed potential correlations between genetic traits and geographic origins. The isolates were identified as S. thermophilus using VITEK® MALDI-TOF MS, ribotyping, and 16S rRNA analysis methods. MLST analysis revealed 13 different sequence types (STs), with seven new STs for Turkey. The most prevalent STs were ST/83 (n = 3), ST/135 (n = 2), and ST/134 (n = 2). eBURST analysis showed that these isolates mainly were singletons (n = 7) defined as sequence types (STs) that cannot be assigned to any group and differ at two or more alleles from every other ST in the sample. This information suggests that the isolates under study were genetically distinct from the other isolates in the dataset, highlighting their unique genetic profiles within the population. Genetic diversity analysis of ten housekeeping genes revealed polymorphism, with some genes showing higher allelic variation than others. Tajima's D values suggested that selection pressures differed among these genes, with some being more conserved, likely due to their vital functions. Phylogenetic analysis revealed distinct genetic diversity between Turkish isolates and European and Asian counterparts. These findings demonstrate the genetic diversity of S. thermophilus isolates in Turkish yogurt and highlight their unique evolutionary patterns. This research contributes to our understanding of local microbial diversity associated with yogurt production in Turkey and holds the potential for identifyic strains with enhanced functional attributes.

Optimization of cryoprotectant formulation to enhance the viability of Lactiplantibacillus plantarum NBC99 isolated from human origin.

Freeze drying has been well applied in the preparation of high-efficiency viability probiotic powders. However, the process is generally accompanied by probiotic viability deficiency, which poses a problem for further application. In this study, various kinds of cryoprotectant formulations (skim milk, maltodextrin, and sucrose) were tested to enhance the survival of Lactiplantibacillus plantarum NBC99 after freezing and freeze-drying. An I-optimal experimental design-oriented optimization model was presented to optimize the cryoprotective medium, and the highest cell survival was observed with 25% skim milk, 8.71% maltodextrin, and 1.13% sucrose cryoprotectant as the optimum condition. L. plantarum NBC99 has been a good potential strain for the manufacture of an industrial probiotic, and this research has aimed to investigate the long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria. The results showed the potential value of freeze-dried probiotic L. plantarum NBC99 culture for commercialization.

Optimization of asymmetric bioreduction conditions of 1-indanone by Leuconostoc mesenteroides N6 using a face-centered design-based multi-objective optimization model.

There has been an increasing interest in biocatalysts over the past few decades in order to obtain high efficiency, high yield, and environmentally benign procedures aiming at the manufacture of pharmacologically relevant chemicals. Lactic Acid Bacteria (LAB), a microbial group, can be employed as biocatalysts while performing asymmetric reduction of prochiral ketones. In this study, Leuconostoc mesenteroides N6 was used for the asymmetric bioreduction 1-indanone. And then, a novel and innovative face-centered design-based multi-objective optimization model was used to optimize experimental conditions. Also, the experimental design factors were defined as agitation speed, incubation period, pH, and temperature for optimization to acquire the maximum enantiomeric excess (ee) and conversion rate (cr) values. When using the face-centered design-based multi-objective optimization model, the optimum culture conditions corresponded to 96.34 and 99.42%, ee and cr responses, respectively, were pH = 5.87, incubation temperature = 35 °C, incubation period = 50.88 h, and agitation speed = 152.60 rpm. Notably, the validation experiment under the optimum model conditions confirmed the model results. This study demonstrated the importance of the optimization and the efficiency of the face-centered design-based multi-objective model.

Optimization of asymmetric bioreduction conditions of 1-(thiophen-2-yl)ethanone by Weissella cibaria N9 using a desirability function-embedded face-centered optimization model.

Prochiral ketones can be effectively bio-reduced to chiral secondary alcohols by whole-cell biocatalysts, which are possible useful precursors to synthesize physiologically active chemicals and natural products. When whole-cell biocatalysts strains are used, bioreduction process can be influenced by various cultural factors, and it is vital to optimize these factors that affect selectivity, conversion rate, and yield. In this study, Weissella cibaria N9 was used as whole-cell biocatalyst for bioreduction of 1-(thiophen-2-yl)ethanone, and cultural design factors were optimized using a desirability function-embedded face-centered optimization model. For this, effects of pH (4.5-5.5-6.5, x1), (2) temperature (25-30-35 °C, x2), (3) incubation period (24-48-72 h, x3), and (4) agitation speed (100-150-200 rpm, x4) on two response variables; (1) ee (%) and (2) cr (%) were tested. Next, desirability function-embedded face-centered optimization model revealed that a pH of 6.43, a temperature of 26.04 °C, an incubation period of 52.41 h, and an agitation speed of 150 rpm were the optimum levels and the estimated ee and cr responses were 99.31% and 98.16%, respectively. Importantly, the actual experimental ee and cr responses were similar to the estimated values indicating the capability of the offered desirability function-embedded face-centered optimization model when using the optimum cultural conditions.

Isolation and characterization of yogurt starter cultures from traditional yogurts and growth kinetics of selected cultures under lab-scale fermentation.

The development of new starter cultures is a crucial task for the food industry to meet technological requirements and traditional products are important reservoirs for new starter cultures. In this respect, this study aimed to isolate, identify, and determine the technological characteristics of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains originated from traditional yogurt samples. Genotypic discrimination of 200 isolates revealed the presence of distinct 19 S. thermophilus and 11 Lb. delbrueckii subsp. bulgaricus strains as potential starter cultures. Strain-specific properties determined the acidification capacity of the yogurt starter cultures and a higher acidification capacity was observed for S. thermophilus strains compared to Lb. delbrueckii subsp. bulgaricus strains. Proteolytic activity was found between 0.012-0.172 and 0.078-0.406 for S. thermophilus and Lb. delbrueckii subsp. bulgaricus strains, respectively. 4 of S. thermophilus and 3 of Lb. delbrueckii subsp. bulgaricus strains were found resistant to all tested bacteriophages. The antibiotic susceptibility tests of the isolates revealed that a very low antibiotic resistance was observed for the yogurt starter cultures. Finally, the growth kinetics of selected strains were determined and the maximum specific growth rate of selected S. thermophilus and Lb. delbrueckii subsp. bulgaricus was calculated as 0.527 h-1 and 0.589 h-1, respectively.

Optimization of asymmetric reduction conditions of 1-(benzo [d] [1,3] dioxol-5-yl) ethanone by Lactobacillus fermentum P1 using D-optimal experimental design-based model.

The biocatalytic asymmetric reduction of prochiral ketones is a significant transformation in organic chemistry as chiral carbinols are biologically active molecules and may be used as precursors of many drugs. In this study, the bioreduction of 1-(benzo [d] [1,3] dioxol-5-yl) ethanone for the production of enantiomerically pure (S)-1-(1,3-benzodioxal-5-yl) ethanol was investigated using freeze-dried whole-cell of Lactobacillus fermentum P1 and the reduction conditions was optimized with a D-optimal experimental design-based optimization methodology. This is the first study using this optimization methodology in a biocatalytic asymmetric reduction. Using D-optimal experimental design-based optimization, optimum reaction conditions were predicted as pH 6.20, temperature 30 °C, incubation time 30 h, and agitation speed 193 rpm. For these operating conditions, it was estimated that the product could be obtained with 94% enantiomeric excess (ee) and 95% conversion rate (cr). Besides, the actual ee and cr were found to be 99% tested under optimized reaction conditions. These findings demonstrated that L. fermentum P1 as an effective biocatalyst to obtain (S)-1-(1,3-benzodioxal-5-yl) ethanol and with the D-optimal experimental design-based optimization, this product could be obtained with the 99% ee and 99% cr. Finally, the proposed mathematical optimization technique showed the applicability of the obtained results for asymmetric reduction reactions.

Production and characterization of yeast extracts produced by Saccharomyces cerevisiae, Saccharomyces boulardii and Kluyveromyces marxianus.

In recent years, prejudice in society against monosodium glutamate (MSG) has directed food manufacturers to alternative sources. Yeast extracts are considered as "natural" due to the production process and stand out due to their nutritional properties as well as giving a flavor similar to MSG. In this study, chemical, functional and flavor properties of yeast extract powders produced from Saccharomyces cerevisiae TGM10, Saccharomyces boulardii S11 and Kluyveromyces marxianus TGM66 were evaluated. Results revealed that the most protein-rich sample was S. cerevisiae TGM10 extract (69.17%), followed by S. boulardii S11 (66.16%) and K. marxianus TGM66 (62.42%) extracts, respectively and S. cerevisiae TGM10 extract was also the richest yeast extract for essential amino acids. Additionally, flavor-enhancing amino acids such as glutamic acid, aspartic acid, alanine and glycine were dominant in S. cerevisiae TGM10 extract (47.41 g/100 g protein). Sensorial evaluation of yeast extracts demonstrated that salty taste, umami taste and meaty flavor scores of yeast extracts were lower than MSG whereas for fruity flavor, yeast extracts had the highest scores. These findings revealed the potential of three yeast strains to produce yeast extracts in order to increase the nutritional value and flavor of foods.

Effects of GSM 1800 band radiation on composition, structure and bioactivity of exopolysaccharides produced by yoghurt starter cultures.

In this study, the effects of GSM 1800 band radiation on composition, structure and bioactivity of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were determined. For this, GSM 1800 band radiation was applied to both cultures and characteristics of EPSs extracted from the control groups (K) and the radiation stressed groups (R) were determined. An alteration in the chemical composition of the EPSs was observed and EPS production levels and molecular weights of the EPSs increased following the GSM 1800 band radiation application. Alterations in the functional groups, thermal and morphological characteristics of EPSs following the GSM 1800 band radiation application were confirmed by FTIR, TGA and SEM analysis, respectively. Importantly no alterations in the antioxidant and antibacterial activity of the EPSs were observed following the radiation application. These results suggested the effects of the GSM radiation on final characteristics of EPSs from yogurt starter cultures.

Comparison of culture-dependent and culture-independent techniques in the detection of lactic acid bacteria biodiversity and dynamics throughout the ripening process: The case of Turkish artisanal Tulum cheese produced in the Anamur region.

Our objective was to analyze the diversity of the microbiota over 180 d of ripening of eight batches of artisanal goatskin Tulum cheeses by culture-dependent and culture-independent (PCR-DGGE) methods. V3 region of the bacterial 16S rRNA gene was amplified with the PCR after direct DNA isolation from the cheese samples. Nine different species and five genera were determined by culturing, while 11 species were identified in the PCR-DGGE technique. This diversity revealed the uniqueness of artisanal cheese varieties. The dominant genera in all the cheese samples were composed of Enterococcus species. The culture-dependent method revealed five genera (Enterococcus,Bacillus,Lactococcus,Lactobacillus, Sphingomonas) while three genera (Enterococcus, Streptococcus, Lactococcus) were detected in the culture-independent method. It was concluded that combining the two methods is important for characterizing the whole microbiota of the Tulum cheese varieties produced in the Anamur region.

Synthesis of silver nanoparticles prepared with a dextran-type exopolysaccharide from Weissella cibaria MED17 with antimicrobial functions.

Microbial polysaccharides can find distinct applications as stabilizer agents including synthesis of nanoparticles. In this study, a dextran-type exopolysaccharide (EPS) was used as the stabilizer agent for the green synthesis of silver nanoparticles (AgNPs-Dex) with antimicrobial characteristics. UV-vis spectroscopy analysis was used to test the formation of AgNPs-Dex. The uniform morphology at around 10 nm size was observed for AgNPs-Dex by TEM analysis and importantly EDX analysis demonstrated the embedment of Ag+ ions within dextran as the stabilizer agent. XRD analysis confirmed the crystalline nature of AgNPs-Dex and FTIR analysis demonstrated the interactions of dextran functional groups with silver. DSC and TGA analysis showed the alteration in the thermal stability of AgNPs-Dex compared to the stabilizer dextran. The antibacterial and antifungal activities of AgNPs-Dex were determined against food originated pathogenic bacteria and fungi and important inhibition levels were observed at 1 mg ml-1 concentration of AgNPs-Dex and this activity was observed to be concentration dependent.

Synthesis of alternan-stabilized zinc nanoparticles: morphological, thermal, antioxidant and antimicrobial characterization.

This study aimed to synthesize zinc nanoparticles with antimicrobial and antioxidant activities using alternan (ZnNPs-Alt) produced by Lactobacillus reuteri E81 as the stabilizing agent. For the characterization of the ZnNPs-Alt, UV-vis spectroscopy, SEM, TEM and EDX analysis, XRD, FTIR and DSC analysis were applied. The functional role of ZnNPs-Alt was tested by determination of their antioxidant activities by DPPH and CUPRAC methodologies and their antibacterial and antifungal activities. Results of this study demonstrated that alternan was utilized as a successful stabilizer to produce crystalline, thermally stabile ZnNPs-Alt with a particle size of ∼ 100 nm. Importantly, strong antioxidant and antimicrobial activities were observed for ZnNPs-Alt in a concentration dependent manner. These findings confirmed the role of alternan as a stabilizing agent for the production of ZnNPs-Alt with functional roles.

Determining the optimum model parameters for oligosaccharide production efficiency using response surface integrated particle swarm optimization method: an experimental validation study.

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45 U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.

Whole cell application of Lactobacillus paracasei BD101 to produce enantiomerically pure (S)-cyclohexyl(phenyl)methanol.

In this study, a total of 10 bacterial strains were screened for their ability to reduce cyclohexyl(phenyl)methanone 1 to its corresponding alcohol. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful biocatalyst to reduce the ketones to the corresponding alcohols. The reaction conditions were systematically optimized for the reducing agent L paracasei BD101, which showed high enantioselectivity and conversion for the bioreduction. The preparative scale asymmetric reduction of cyclohexyl(phenyl)methanone (1) by L paracasei BD101 gave (S)-cyclohexyl(phenyl)methanol (2) with 92% yield and >99% enantiomeric excess. The preparative scale study was carried out, and a total of 5.602 g of (S)-cyclohexyl(phenyl)methanol in high enantiomerically pure form (>99% enantiomeric excess) was produced. L paracasei BD101 has been shown to be an important biocatalyst in asymmetric reduction of bulky substrates. This study demonstrates the first example of the effective synthesis of (S)-cyclohexyl(phenyl)methanol by the L paracasei BD101 as a biocatalyst in preparative scale.

Response surface methodology as optimization strategy for asymmetric bioreduction of acetophenone using whole cell of Lactobacillus senmaizukei.

Whole cell applications are one of the main methodologies for the bioreduction of prochiral ketones to obtain enantiomerically rich chiral secondary alcohols which are mainly affected by the culture parameters of the whole cell. In this study, whole cell of Lactobacillus senmaizukei as a safe Lactic Acid Bacteria (LAB) was used for the reduction of acetophenone and Response Surface Methodology (RSM) application was used to optimize the culture parameters in terms of temperature, pH, incubation time, and agitation level to obtain the highest enantiomeric excess (ee) and conversion rate. The predicted optimum conditions for the bioreduction with whole cell Lactobacillus senmaizukei were found to be pH of 5.25, temperature of 25 °C, incubation time of 72 hr, and agitation level of 100 rpm. Importantly, the efficiency of the reduction of the acetophenone was significantly affected by the linear and quadratic effects of culture parameters. These findings are important to show the role of culture parameters for the bioreduction reactions and also the efficiency of the RSM technique to optimize these parameters.

Synthesis of Enantiomerically Enriched Drug Precursors by Lactobacillus paracasei BD87E6 as a Biocatalyst.

Global sales of single enantiomeric drug products are growing at an alarming rate every year. A total of 7 bacterial strains were screened for their ability to reduce acetophenones to its corresponding alcohol. Among these strains Lactobacillus paracasei BD87E6 was found to be the most successful biocatalyst to reduce the ketones to the corresponding alcohols. The reaction conditions were systematically optimized for the reducing agent Lactobacillus paracasei BD87E6, which showed high enantioselectivity and conversion for the bioreduction. The preparative scale asymmetric reduction of 3-methoxyacetophenone (1h) by Lactobacillus paracasei BD87E6 gave (R)-1-(3-methoxyphenyl)ethanol (2h) with 92% yield and 99% enantiomeric excess. Compound 2h could be used for the synthesis of (S)-rivastigmine which has a great potential for the treatment of Alzheimer's disease. This study demonstrates that Lactobacillus paracasei BD87E6 can be used as a biocatalyst to obtain chiral carbinol with excellent yield and selectivity. The whole cell catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that Lactobacillus paracasei BD87E6 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

Glucan type exopolysaccharide (EPS) shows prebiotic effect and reduces syneresis in chocolate pudding.

Exopolysaccharides (EPS) of lactic acid bacteria are important biopolymers that can improve the physicochemical properties of food products and act as prebiotics. In this study the physicochemical role and the prebiotic effects of a glucan type EPS with (α1-3) and (α1-6) linkages were assessed in chocolate pudding containing Lactobacillus rhamnosus GG as a probiotic strain. The functions of EPS were determined by developing three different formulations: control, probiotic (Lactobacillus GG) and symbiotic pudding (Lactobacillus GG + EPS) samples. The pH and acidity of the symbiotic pudding sample were higher than the probiotic and the control samples during the 28-day of storage period. Similarly, an important level of increment in Lactobacillus GG levels in symbiotic sample was observed compared to the probiotic sample suggesting the prebiotic role of the α-glucan. Importantly, the syneresis in symbiotic pudding sample reduced significantly compared to other pudding samples which is related with the physicochemical role of glucan type EPS. This study reveals the prebiotic and physicochemical roles of α-glucan type EPS in a chocolate pudding model.

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst.

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1-phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4-nitroacetophenone (1m) by C. zeylanoides P1 gave (S)-1-(4-nitrophenyl)ethanol (2m) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.

Highly Enantioselective Production of Chiral Secondary Alcohols Using Lactobacillus paracasei BD101 as a New Whole Cell Biocatalyst and Evaluation of Their Antimicrobial Effects.

Chiral secondary alcohols are valuable intermediates for many important enantiopure pharmaceuticals and biologically active molecules. In this work, we studied asymmetric reduction of aromatic ketones to produce the corresponding chiral secondary alcohols using lactic acid bacteria (LAB) as new biocatalysts. Seven LAB strains were screened for their ability to reduce acetophenones to their corresponding alcohols. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful at reducing the ketones to the corresponding alcohols. The reaction conditions were further systematically optimized for this strain and high enantioselectivity (99%) and very good yields were obtained. These secondary alcohols were further tested for their antimicrobial activities against important pathogens and significant levels of antimicrobial activities were observed although these activities were altered depending on the secondary alcohols as well as their enantiomeric properties. The current methodology demonstrates a promising and alternative green approach for the synthesis of chiral secondary alcohols of biological importance in a cheap, mild, and environmentally useful process.

Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785.

BACKGROUND: The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens. RESULTS: Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses. CONCLUSIONS: This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.

Impact of exopolysaccharide production on functional properties of some Lactobacillus salivarius strains.

The aim of this work was to characterize functional properties of Lactobacillus salivarius strains isolated from chicken feces. Detection of genes responsible for exopolysaccharide (EPS) production revealed that all strains harbored a dextransucrase gene, but p-gtf gene was only detected in strain E4. Analysis of EPS production levels showed significant alterations among strains tested. Biofilm formation was found to be medium composition dependant, and there was a negative correlation with biofilm formation and EPS production. Autoaggregation properties and coaggregation of L. salivarius strains with chicken pathogens were appeared to be specific at strain level. An increment in bacterial adhesion to chicken gut explants was observed in L. salivarius strains with the reduction in EPS production levels. This study showed that strain-specific properties can determine the functional properties of L. salivarius strains, and the interference of these properties might be crucial for final selection of these strains for technological purposes.