Deciphering the pancreatic cancer microbiome in Mainland China: Impact of Exiguobacterium/Bacillus ratio on tumor progression and prognostic significance.

The existing body of research underscores the critical impact of intratumoral microbiomes on the progression of pancreatic ductal adenocarcinoma (PDAC), particularly in reshaping the tumor microenvironment and influencing gemcitabine resistance. However, peritumoral tissues' microbiome, distinct from PDAC tumors, remain understudied, and Western-centric analyses overlooking potential variations in dietary-influenced microbiomes. Our study addresses this gap by 16 S rRNA sequencing of PDAC tumors and matched peritumoral tissues from Chinese Mainland patients. Our research has uncovered that the microbiome composition within tumors and paired peritumoral tissues exhibits a high degree of similarity, albeit with certain discrepancies. Notably, Exiguobacterium is found to be more abundant within the tumor tissues. Further investigations have revealed that a lower Exiguobacterium/Bacillus ratio in both the tumor and peritumoral tissues of PDAC patients is indicative of a more favorable prognosis. Further exploration utilizing an orthotopic tumor model demonstrates that the probiotic Bacillus Coagulans impedes PDAC progression, accompanied by an increased infiltration of inflammatory neutrophils in tumors. Additionally, in the subgroup with a low Exiguobacterium/Bacillus ratio, whole-exome sequencing reveals elevated missense mutations in ABL2 and MSH2. The elevated expression of ABL2 and MSH2 has been correlated with poorer prognostic outcomes in PDAC patients. Together, these insights shed light on risk factors influencing PDAC progression and unveil potential therapeutic targets, alongside probiotic intervention strategies.

Photosynthesizing carbonate/nitrate into Chlorococcum humicola biomass for biodiesel and Bacillus coagulans-based biohydrogen production.

Biofuel can be generated by different organisms using various substrates. The green alga Chlorococcum humicola OQ934050 exhibited the capability to photosynthesize carbonate carbon, maybe via the activity of carbonic anhydrase enzymes. The optimum treatment is C:N ratio of 1:1 (0.2 mmoles sodium carbonate and 0.2 mmoles sodium nitrate) as it induced the highest dry mass (more than 0.5 mg.mL-1). At this combination, biomass were about 0.2 mg/mL-1 carbohydrates, 0.085 mg/mL-1 proteins, and 0.16 mg/mL-1 oil of this dry weight. The C/N ratios of 1:1 or 10:1 induced up to 30% of the Chlorococcum humicola dry mass as oils. Growth and dry matter content were hindered at 50:1 C/N and oil content was reduced as a result. The fatty acid profile was strongly altered by the applied C.N ratios. The defatted leftovers of the grown alga, after oil extraction, were fermented by a newly isolated heterotrophic bacterium, identified as Bacillus coagulans OQ053202, to evolve hydrogen content as gas. The highest cumulative hydrogen production and reducing sugar (70 ml H2/g biomass and 0.128 mg/ml; respectively) were found at the C/N ratio of 10:1 with the highest hydrogen evolution efficiency (HEE) of 22.8 ml H2/ mg reducing sugar. The optimum treatment applied to the Chlorococcum humicola is C:N ratio of 1:1 for the highest dry mass, up to 30% dry mass as oils. Some fatty acids were induced while others disappeared, depending on the C/N ratios. The highest cumulative hydrogen production and reducing sugar were found at the C/N ratio of 10:1.

Investigate the relationship between Bacillus coagulans and its inhibition of chemotherapy-induced lung cancer resistance.

Lung cancer is a leading cause of death globally, with lung adenocarcinoma being the most common subtype. Despite advancements in targeted therapy, drug resistance remains a major challenge. This study investigated the impact of Bacillus coagulans on drug resistance in lung adenocarcinoma cells. The cells were pretreated with B. coagulans culture filtrate (BCCF), and functional assays were performed, including cell proliferation, cell cycle, apoptosis, and immunofluorescence staining. Results showed that BCCF induced cell cycle arrest at the S phase, reducing cell proliferation and suppressing drug resistance marker P-glycoprotein expression in BCCF-treated resistant cells rather than BCCF-treated control cells. Moreover, drug-resistant cells exhibited the ability for epithelial-mesenchymal transition, which could contribute to their necrosis through the iron-mediated cell death pathway upon BCCF treatment. Proteomic analysis identified downregulation of DNA mismatch repair protein PMS2 after BCCF treatment. These findings suggest that B. coagulans may modulate the DNA repair pathway, influencing drug resistance in lung adenocarcinoma cells. In conclusion, this study highlights the potential impact of B. coagulans on drug-resistant lung adenocarcinoma cells. Further investigation and understanding of the regulatory mechanisms by which B. coagulans modulates drug resistance in lung adenocarcinoma can aid in the development of more effective treatment strategies to improve the prognosis of lung cancer patients.

Synbiotic as an ameliorating factor in the health-related quality of life in women with polycystic ovary syndrome. A randomized, triple-blind, placebo-controlled trial.

BACKGROUND: There are complicated mechanisms that link the disruption of the gut microbiome to the symptoms and complications of polycystic ovary syndrome (PCOS). In this study, an attempt was made to assess the effects of synbiotics on the health-related quality of life (HRQoL) in women with PCOS . METHODS: Fifty-six women with PCOS were enrolled in a triple-blind controlled trial for 12 weeks. They were randomly assigned to receive a daily 2-gram synbiotic sachets (containing Bacillus coagulans (GBI-30), Lactobacillus rhamnosus, Lactobacillus helveticus, and fructooligosaccharide) (n = 28) or placebo (n = 28). To evaluate the impact on the HRQoL, participants were required to fill 26-Item Polycystic Ovary Syndrome Health-Related Quality of Life Questionnaire (PCOSQ-26), 12-Item Short-Form Health Survey (SF-12) and Perceived Stress Scale (PSS-10) pre and post the intervention. RESULTS: Finally, statistical analyses were performed on 52 participants who finished the trial. Synbiotic supplementation improved the scores of emotional (P = 0.044), body hair (P = 0.016), weight (P = 0.033) and infertility domains (P = 0.027) of PCOSQ-26 compared to placebo group. The physical score within SF-12 also had a significant enhancement (P = 0.035). No significant improvement was seen in the PSS-10 score at the end of the trial. CONCLUSION: This study illustrated the advantageous effects of synbiotics on the health-related quality of life in women with PCOS. Further studies are required to confirm our findings. TRIAL REGISTRATION: http://www.irct.ir : IRCT20211108053007N1; date of registration: 14/02/2023.

In-silico analysis of probiotic attributes and safety assessment of probiotic strain Bacillus coagulans BCP92 for human application.

The whole genome sequence (WGS) of Bacillus coagulans BCP92 is reported along with its genomic analysis of probiotics and safety features. The identification of bacterial strain was carried out using the 16S rDNA sequencing method. Furthermore, gene-related probiotic features, safety assessment (by in vitro and in silico), and genome stability were also studied using the WGS analysis for the possible use of the bacterial strain as a probiotic. From the BLAST analysis, bacterial strain was identified as Bacillus (Heyndrickxia) coagulans. WGS analysis indicated that the genome consists of a 3 475 658 bp and a GC-content of 46.35%. Genome mining of BCP92 revealed that the strain is consist of coding sequences for d-lactate dehydrogenase and l-lactate dehydrogenases, 36 genes involved in fermentation activities, 29 stress-responsive as well as many adhesions related genes. The genome, also possessing genes, is encoded for the synthesis of novel circular bacteriocin. Using an in-silico approach for the bacterial genome study, it was possible to determine that the Bacillus (Heyndrickxia) coagulans strain BCP92 contains genes that are encoded for the probiotic abilities and did not harbour genes that are risk associated, thus confirming the strain's safety and suitability as a probiotic to be used for human application.

Novel microbial synthesis of titania nanoparticles using probiotic Bacillus coagulans and its role in enhancing the microhardness of glass ionomer restorative materials.

Dental caries is a commonly occurring non-communicable disease throughout the world that might compromise the quality of any individual's life. Glass ionomer cements (GIC) are the most acceptable restorative materials due to their ease of manipulation, minimal tooth loss and least invasive strategy; however, they lack mechanical stability that has become a point of concern. Nanoparticles (NPs) are an outstanding option for modifying and enhancing the properties of dental materials. The focus of this study was to prepare novel, biocompatible titania dioxide (TiO2) NPs as a dental-restorative material using an efficient probiotic Bacillus coagulans. The prepared NPs were incorporated into glass ionomer restorative material at varying concentrations and investigated for cell viability percentage, microhardness and surface morphology. Results indicated that pure 100% anatase phase TiO2 NPs with particle size of 21.84 nm arranged in smooth, spherical agglomerates and clusters forms. These NPs depicted cell viability > 90%, thus confirming their non-cytotoxic behavior. GIC restorative materials reinforced by 5% titania (TiO2) NPs demonstrated the highest microhardness in comparison to the control group and other experimental groups of the study. Surface morphology analysis revealed a reduction in cracks in this novel dental-restorative material supporting its compatible biological nature with better hardness strength and negligible crack propagation. Overall, these results indicated that TiO2 NPs produced using a biological approach could be easily used as restorative materials in dental applications.

Effects of Partial Replacement of Sugar with Fig Syrup on the Survival of Bacillus coagulans and the Physicochemical Properties of Probiotic Ice Cream.

Research background: Various sectors of the food industry demand the enrichment of food with functional compounds. Probiotic products with valuable nutritional and therapeutic properties have attracted great attention in the fields of industry, nutrition and medicine. The aim of the present study is to investigate the sensory and physicochemical properties of probiotic ice cream containing fig syrup and to evaluate the survival of Bacillus coagulans after 90 days of storage at -18 °C. Experimental approach: In this study, four experimental groups of ice cream were produced as follows: plain dairy ice cream (without additives), ice cream containing 109 CFU/g B. coagulans, ice cream containing 25 % fig syrup as sugar substitute and ice cream containing 25 % fig syrup as sugar substitute and 109 CFU/g B. coagulans. They were stored at -18 °C for 3 months. Texture, pH, acidity and viscosity were analysed and microbial counts were determined after 1, 30, 60 and 90 days of storage. The organoleptic evaluation was carried out on days 1 and 90. Results and conclusions: The results showed that during the initial freezing process and the transformation of the mixture into ice cream, the number of B. coagulans decreased from 109 to 107 CFU/g, without significant changes observed over the 90-day period. No significant changes were found in the sensory and textural properties of the samples either. Replacement of 25 % sugar with fig syrup reduced the pH, increased the acidity of the ice cream and improved their viscosity. In conclusion, the production of functional ice cream using fig syrup and B. coagulans is recommended for their health benefits. Novelty and scientific contribution: The results of this study can be used to prepare functional and healthy foods. Our results suggest that fig syrup has the potential to be used as a natural sweetener or sugar substitute in various products.

Influence of dietary Bacillus coagulans and/or Bacillus licheniformis-based probiotics on performance, gut health, gene expression, and litter quality of broiler chickens.

Probiotics are non-pathogenic microorganisms that are potentially important non-antibiotic alternatives. This study aimed to compare novel multi-strain and single-strain Bacillus probiotics and their respective influences on broiler chickens' performance, gut health, litter quality, immune response, and NBN and TLR gene expression. A total of 1200 Arbor-Acres 1-day-old broiler chicks were randomly allocated into three treatments (T1 was a control, T2 was supplemented with a combined Bacillus coagulans (2 × 109 cfu/g) and Bacillus licheniformis (8 × 109 cfu/g) probiotic strains (0.2 kg/ton of feed), and T3 was supplemented with Bacillus licheniformis (3.2 × 109 cfu/g) probiotic (0.5 kg/ton of feed) with eight replicas of each. Supplementing the broiler diet with either the single-strain (T3) or the multi-strain (T2) Bacillus-based probiotic raised the overall birds' body weight, body weight gain, feed conversion ratio, and European production efficiency factor compared to the control (T1), with a significant enhancement achieved by the multi-strain Bacillus product (P = 0.005). T2 and T3 exhibited significantly improved cholesterol, Alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase levels than the control (P ≤ 0.05). The transcript levels of both NBN and TLR genes were upregulated in the liver in the T2 and T3 groups. The T2 group experienced significant reductions in gut bacterial counts, especially for Clostridia, and recorded the lowest litter moisture and nitrogen. In conclusion, supplementing broiler diets with probiotics of multiple Bacillus strains increased production profitability by promoting bird growth, improving feed intake, enhancing gut mucosa and immune organs, and upregulating genes responsible for immunity. All these inhibit the overgrowth of enteric pathogens and sustain litter quality.

Alleviative effects of a novel strain Bacillus coagulans XY2 on copper-induced toxicity in zebrafish larvae.

Copper (Cu) is a kind of micronutrient element that is essential for human metabolism. However, it is also considered as an environmental pollutant which is toxic to organisms at a high concentration level. Probiotics, regarded as beneficial microorganisms for promoting human health, have functions of antioxidant capacity, immune-enhancing properties, intestinal barrier protection and regulation. Several studies have reported that probiotics show positive effects on alleviating and intervening heavy metals toxicity. However, evidence for relieving copper-induced toxicity by probiotics is still limited. In this study, we firstly conducted a zebrafish larvae model to screen out microorganisms which are helpful for CuSO4 toxicity resistance and one novel strain named as Bacillus coagulans XY2 was discovered with the best protective activity. B. coagulans XY2 significantly reduced the mortality of zebrafish larvae exposed to 10 µmol/L CuSO4 for 96 hr, as well as alleviated the neutrophils infiltration in the larvae lateral line under a 2 hr exposure. B. coagulans XY2 exhibited a high in vitro antioxidant activity and against CuSO4-induced oxidative stress in zebrafish larvae by up-regulating sod1, gstp1 and cat gene transcriptional levels and relevant enzymatic activities. CuSO4 stimulated the inflammation process resulting in obvious increases of gene il-1ß and il-10 transcription, which were suppressed by B. coagulans XY2 intervention. Overall, our results underline the bio-function of B. coagulans XY2 on protecting zebrafish larvae from copper toxicity, suggesting the potential application values of probiotics in copper toxicity alleviation on human and the environment.

Identification of a major facilitator superfamily protein that is beneficial to L-lactic acid production by Bacillus coagulans at low pH.

BACKGROUND: Product inhibition is one of the major problems in lactic acid (LA) fermentation. Our previous study revealed that Bacillus coagulans 2-6 was an efficient producer of high-optical-purity L-LA. Its mutant strain B. coagulans Na-2 has better resistance to sodium lactate stress but the resistance mechanism has not been understood. RESULTS: In this study, the whole-genome sequencing of B. coagulans Na-2 was performed and one mutant gene mfs coding for the major facilitator superfamily (MFS) protein was revealed by comparative genome analysis. Ten mutation sites were identified between the wild (MFS-2-6) and mutant (MFS-Na-2) proteins, among which T127A and N154T were predicted locating in the center of the transmembrane transport channel. The MFS-2-6 and MFS-Na-2 were expressed separately in a genetically operable strain, B. coagulans DSM1, using the genes' native promoter. The expression of the two MFS proteins had no effect and a negative effect on L-LA production when the pH was controlled at 6.0 and 7.0 by sodium hydroxide, respectively. However, 4.2 and 4.6-fold of L-LA concentrations were obtained at pH 5.0 by the strains expressing MFS-2-6 and MFS-Na-2 than that by the control strain, respectively. The intracellular pH values of the strains expressing MFS-2-6 and MFS-Na-2 were approximately 0.69 and 0.45 higher than that of the control strain during pH-controlled fermentation at 5.0. Results suggest that the expression of MFS-2-6 and MFS-Na-2 were both conducive to L-LA production at low pH, while the better performance of the latter was probably due to the more appropriate intracellular pH during the whole fermentation process. CONCLUSIONS: The MFS protein identified here can improve the ability of B. coagulans to resist acidic environments and produce more L-LA at low pH. The MFS protein has an application potential in environment-friendly L-LA production.

Effective gossypol removal from cottonseed meal through optimized solid-state fermentation by Bacillus coagulans.

BACKGROUND: Cottonseed meal (CSM) is the main by-product of the cottonseed oil extraction process with high protein content, which is an important protein source for feed industry. However, CSM contains free gossypol (FG), a toxic substance that is detrimental to animal health and greatly limits its application. Microbial fermentation is currently considered to be one of the most effective methods to reduce FG and other anti-nutritional factors in CSM. Previously, yeast and bacteria species are used for degradation of FG in CSM, but showing less detoxification efficiency. Bacillus coagulans combines the properties of both lactic acid bacteria and Bacillus, producing both lactic acid and spores, and is considered a potential probiotic. In this study, we aimed to evaluate and optimize the effect of the solid-state fermentation process using a Bacillus coagulans to gossypol removal contained cottonseed meal. RESULTS: 36 B. coagulans strains were isolated and found to have the ability to remove free gossypol. Through the evaluation of strains and optimization of fermentation conditions including fermentation temperature, ratio of material to water, inoculation amount, fermentation time and pH, we have established a solid-state fermentation process using a Bacillus coagulans strain S17 on CSM substrate with 1:1 of the material-to-water ratio, 15% (v/w) seed inoculation, 2% expanded corn flour, 1% bran, and 0.3%-0.8% metal irons at 40 °C for 52 h. After fermentation, the FG content in CSM was reduced from 923.80 to 167.90 mg/kg with 81.83% detoxification efficiency. Meanwhile, the crude protein content in CSM increased from 47.98 to 52.82%, and importantly, the spore concentration of strain S17 reached 1.68 × 1010 CFU/g dry material. CONCLUSION: The study showed that B. coagulans have the potential strong ability to degrade free gossypol through cottonseed meal fermentation. This study presents a feasible process for improving the resource utilization rate and nutritional value of CSM via solid-state fermentation through B. coagulans S17.

Theaflavin and its derivatives exert antibacterial action against Bacillus coagulans through adsorption to cell surface phospholipids.

AIMS: To investigate the antibacterial effects of tea theaflavins and catechins against Bacillus coagulans and the underlying mechanism of antibacterial action. METHODS AND RESULTS: Bactericidal activities of theaflavin and its analogues were evaluated and compared with that of epigallocatechin gallate. Theaflavin derivatives exhibited high bactericidal activity at 50 µmol L-1 , whereas epigallocatechin gallate did not, even at 500 µmol L-1 . Furthermore, we investigated the adsorption of theaflavins to model phospholipid membranes and corresponding effects on membrane fluidity to reveal their effects on the B. coagulans cell surface. Cell membrane fluidity was decreased after treatment with theaflavin derivatives with one or more galloyl moieties. Quartz-crystal microbalance analysis showed a strong affinity of the membrane phosphatidyl glycerol (PG) bilayers for theaflavin derivatives, correlating their bactericidal activity. CONCLUSION: These findings suggest that theaflavins could effectively inhibit B. coagulans by decreasing cell membrane fluidity. SIGNIFICANCE AND IMPACT: Bacillus coagulans is a spore-forming heat-resistant bacterium responsible for spoilage in low-acidic beverages. Natural antimicrobial components in tea-based beverages are central to reducing microbial contamination and product quality deterioration, although mechanisms underlying their antimicrobial action remain obscure. This study highlights the inhibitory action of theaflavins on B. coagulans and their potential application in food and beverage industries.

A novel probiotic strain exerts therapeutic effects on mouse model of multiple sclerosis by altering the expression of inflammasome and IDO genes and modulation of T helper cytokine profile.

Multiple sclerosis is an inflammatory demyelinating disease that commences to neuronal cell destruction. Recently, a promising evidence of synergic effects of combined supplementation with vitamin D and probiotics in modulating the gut microbiota and metabolome is emerging. Bacillus Coagulans IBRC-M10791 as a novel strain was chosen, prevention and treatment impacts of regular administered were studied in Cuprizone-induced C57bl/6 mouse of demyelination. The mice were divided into six groups and received a daily dose of cuprizone or probiotics. To investigate the effect of probiotic, the IDO-1, CYP27B1, NLRP1, NLRP3, and AIM2 expression were estimated by Real-Time PCR, and IL-4, IL-17, IFN-gamma, and TGF-beta cytokines were measured by ELISA. The results showed that there was significant decrease in IL-17 and IFN-γ and modulatory effects on IL-4 and TGF-ß. On the other hand, we demonstrated that there are significant decrease for expression of IDO-1, CYP27b1, NLRP1, NLRP3 and AIM2 genes in prevention and treatment groups compared to cuprizone group. Also, a significant enhancement in rate of remyelination and alternations proved by LFB staining and Y-Maze test. In conclusion, our study provides insight into how the therapeutic effect of the chosen strain of probiotic was correlated with the modulation of the level of inflammatory and anti-inflammatory cytokines. Further, we demonstrated that the expression of genes related to Tryptophan, Vitamin D and Inflammasome pathways could be affected by B.coagulans. Our study could be beneficial to provide a novel Co-therapeutic strategy for Multiple sclerosis.

In vitro and in vivo evaluation of probiotic potential and safety assessment of Bacillus coagulans SKB LAB-19 (MCC 0554) in humans and animal healthcare.

Bacillus coagulans is Gram positive, spore forming and high lactic acid producing bacteria; however, probiotic and safety assessment of the isolated strain need to be investigated for commercial applications. Current study aimed to screen SKB LAB-19 for potential probiotic characteristics viz. enzyme production, antimicrobial properties, pH/bile salt tolerance, temperature stability, antidiarrheal activity in Swiss albino mice and Wistar rats; and acute oral toxicity in mice. The results showed that, SKB LAB-19 produces eight potential enzymes, effective against E. coli and C. perfringensis, tolerant to bile salt (0.3%w/v)/gastric pH (2.5), stable at 40-90 °C and nontoxic to cells. SKB LAB-19 was found to be safe and displayed promising results to reverse E. coli and castor oil induced diarrhoea. Histopathological studies showed repair to damaged mucosal epithelium cells and improves integrity of the goblet cells of colon. SKB LAB-19 showed immunomodulatory effects with increased immunoglobulins in blood and augmented weight of spleen and thymus. In addition, SKB LAB-19 showed significant in-vitro antioxidant activity (82.93%), reducing capacity and ascorbate auto-oxidation inhibition effect (94.62%). These preliminary results suggested that, SKB LAB-19 was found to be safe and has the potential to be used as effective probiotic and anti-diarrhoeal agent in humans and animal healthcare.

Phylogeny of the HO family in cyprinus carpio and the response of the HO-1 gene to adding Bacillus coagulans in feed under Cd2+ stress.

The heavy metal cadmium (Cd2+) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd2+, the increased accumulation of Cd2+ in fish has become a food safety and public health concern. Heme oxygenase (HO) is an important antioxidant enzyme that plays a key role in defending the body against oxidative damage, but little research has been done in common carp. In this study, 6 HO genes were identified in the common carp genome database. Comparative genomics analysis showed considerable expansion of the HO genes and verified the four-round whole genome duplication (WGD) event in common carp. Phylogenetic analysis revealed that all HO genes of common carp were clustered into orthologous groups, indicating high conservation during evolution. In addition, the tissue distribution results showed that most HO genes had extensive tissue distribution and showed tissue-specific expression patterns. Exposure to 0.5 mg/L Cd2+ significantly reduced the expression of TGF-ß and IL-10 in common carp, which may indicate that Cd2+ exposure can destroy the physical barrier function of the intestine, inhibit intestinal immune defense and induce intestinal inflammation. To find a suitable concentration of Bacillus coagulans that could activate HO-1 genes and the immunity of the organism, we investigated the changes in HO-1 gene expression levels in the intestinal tract of common carp under Cd2+ stress at 30 days and 60 days by adding different concentrations of B. coagulans to the feed. Compared with the Cd2+ stress group without supplementation, the expression levels of the HO-1 gene in the gut of three different concentrations of B. coagulans were almost increased. And B. coagulans with L2 concentrations had better activation effect on the HO-1 gene. Similarly, compared to the Cd2+ stressed group, adding B. coagulans to the diet can almost cause the early upregulation of IL-10 and TGF-ß genes. Therefore, the addition of appropriate concentrations of B. coagulans may be a good way to activate HO-1, IL-10, and TGF-ß genes, reduce oxidative damage, and encourage the immune.

Integration of metabolic pathway manipulation and promoter engineering for the fine-tuned biosynthesis of malic acid in Bacillus coagulans.

Bacillus coagulans, a thermophilic facultative anaerobe, is a favorable chassis strain for the biosynthesis of desired products. In this study, B. coagulans was converted into an efficient malic acid producer by metabolic engineering and promoter engineering. Promoter mapping revealed that the endogenous promoter Pldh was a tandem promoter. Accordingly, a promoter library was developed, covering a wide range of relative transcription efficiencies with small increments. A reductive tricarboxylic acid pathway was established in B. coagulans by introducing the genes encoding pyruvate carboxylase (pyc), malate dehydrogenase (mdh), and phosphoenolpyruvate carboxykinase (pckA). Five promoters of various strengths within the library were screened to fine-tune the expression of pyc to improve the biosynthesis of malic acid. In addition, genes involved in the competitive metabolic pathways were deleted to focus the substrate and energy flux toward malic acid. Dual-phase fed-batch fermentation was performed to increase the biomass of the strain, further improving the titer of malic acid to 25.5 g/L, with a conversion rate of 0.3 g/g glucose. Our study is a pioneer research using promoter engineering and genetically modified B. coagulans for the biosynthesis of malic acid, providing an effective approach for the industrialized production of desired products using B. coagulans.

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 ß-galactosidase.

BACKGROUND: The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and ß-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract. RESULTS: In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on ß-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as ß-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant ß-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated. CONCLUSIONS: Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and ß-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases.

Revisiting the production of L( +)-lactic acid from vine shoots: bioconversion improvements by employing thermotolerant bacteria.

Vine shoots (Vitis vinifera L.) constitute an abundant lignocellulosic source which is frequently underutilised. Alkaline and acidic pretreatments (with and without washing steps) were compared and optimised to release fermentable sugars from vine shoots. An acidic pretreatment using 1.72% H2SO4 at 134 °C for 17 min (with 10% w/w solid biomass), followed by an enzymatic hydrolysis, offered the most cost-effective results, releasing 40.21 g/L sugars. Three thermotolerant strains, namely, Bacillus coagulans DSM 2314, Geobacillus stearothermophilus DSM 2313, and G. stearothermophilus DSM 494, were assessed to produce lactic acid from vine-shoot hydrolysates under aerobic and non-sterile conditions, without the need of detoxification steps. In addition, wine lees were satisfactorily employed as nitrogen sources for the fermentation, providing similar results to yeast extract and being the only nutrient added to vine-shoot hydrolysates. Under optimal conditions, B. coagulans DSM 2314 produced 29.21 ± 0.23 g/L lactic acid in 24 h, with a sugar consumption of 98.74 ± 0.07% and a yield of 96.38 ± 0.76%, when supplemented with red wine lees. The purity of the isomer L( +) reached 97.59 ± 1.35% of the total lactic acid produced. Although G. stearothermophilus was able to transform the hexoses from vine-shoot hydrolysates into lactic acid, it proved to be inefficient for metabolising pentoses, thus obtaining lower lactic acid values (16-18 g/L).

Comparative evaluation for thermostability and gastrointestinal survival of probiotic Bacillus coagulans MTCC 5856.

Thermal stability (D-value and pasteurization) and gastric acid resistance of spore forming and nonspore forming probiotic strains were evaluated in this study. Bacillus coagulans MTCC 5856 spores showed highest thermal resistance (D-value 35.71 at 90 °C) when compared with other Bacillus strains and Lactobacillus species. B. coagulans strains exhibited significantly higher resistance to simulated gastric juice (pH 1.3, 1.5, and 2.0) compared to Lactobacillus strains. It also showed high resistance to cooking conditions of chapati (whole wheat flour-based flatbread) (88.94% viability) and wheat noodles (and 94.56% viability), suggesting remarkable thermal resistance during food processing. Furthermore, B. coagulans MTCC 5856 retained 73% viability after microwave cooking conditions (300 s, at 260 °C) and 98.52% in milk and juice at pasteurization temperature (420 min, at 72 °C). Thus, B. coagulans MTCC 5856 clearly demonstrated excellent resistance to gastric acid and high temperature (90 °C), thereby suggesting its extended application in functional foods (milk, fruit juices, chapati, and wheat noodles) wherein high temperature processing is involved.

Improving probiotic spore yield using rice straw hydrolysate.

Spore-forming Bacillus sp. has been extensively studied for their probiotic properties. In this study, an acid-treated rice straw hydrolysate was used as carbon source to produce the spores of Bacillus coagulans. The results showed that this hydrolysate significantly improved the spore yield compared with other carbon sources such as glucose. Three significant medium components including rice straw hydrolysate, MnSO4 and yeast extract were screened by Plackett-Burman design. These significant variables were further optimized by response surface methodology (RSM). The optimal values of the medium components were rice straw hydolysate of 27% (v/v), MnSO4 of 0·78 g l-1 and yeast extract of 1·2 g l-1 . The optimized medium and RSM model for spore production were validated in a 5 l bioreactor. Overall, this sporulation medium containing acid-treated rice straw hydrolysate has a potential to be used in the production of B. coagulans spores.