Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid.

Agri-food residues offer significant potential as a raw material for the production of L-lactic acid through microbial fermentation. Weizmannia coagulans, previously known as Bacillus coagulans, is a spore-forming, lactic acid-producing, gram-positive, with known probiotic and prebiotic properties. This study aimed to evaluate the feasibility of utilizing untreated citrus waste as a sustainable feedstock for the production of L-lactic acid in a one-step process, by using the strain W. coagulans MA-13. By employing a thermophilic enzymatic cocktail (Cellic CTec2) in conjunction with the hydrolytic capabilities of MA-13, biomass degradation was enhanced by up to 62%. Moreover, batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into L-lactic acid, achieving a concentration of up to 44.8 g/L. These results point to MA-13 as a microbial cell factory for one-step production of L-lactic acid, by combining cost-effective saccharification with MA-13 fermentative performance, on agri-food wastes. Moreover, the potential of this approach for sustainable valorization of agricultural waste streams is successfully proven. KEY POINTS: • Valorization of citrus waste, an abundant residue in Mediterranean countries. • Sustainable production of the L-( +)-lactic acid in one-step process. • Enzymatic pretreatment is a valuable alternative to the use of chemical.

Extracellular vesicles of Weizmannia coagulans lilac-01 reduced cell death of primary microglia and increased mitochondrial content in dermal fibroblasts in vitro.

We investigated the properties of extracellular vesicles from the probiotic Weizmannia coagulans lilac-01 (Lilac-01EVs). The phospholipids in the Lilac-01EV membrane were phosphatidylglycerol and mitochondria-specific cardiolipin. We found that applying Lilac-01EVs to primary rat microglia in vitro resulted in a reduction in primary microglial cell death (P < .05). Lilac-01EVs, which contain cardiolipin and phosphatidylglycerol, may have the potential to inhibit cell death in primary microglia. The addition of Lilac-01EVs to senescent human dermal fibroblasts suggested that Lilac-01 EVs increase the mitochondrial content without affecting their membrane potential in these cells.

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.

Microalgal upgrading of the fermentative biohydrogen produced from Bacillus coagulans via non-pretreated plant biomass.

BACKGROUND: Hydrogen is a promising source of alternative energy. Fermentative production is more feasible because of its high hydrogen generation rate, simple operating conditions, and utilization of various organic wastes as substrates. The most significant constraint for biohydrogen production is supplying it at a low cost with fewer impurities. RESULTS: Leaf biomass of Calotropis procera was used as a feedstock for a dark fermentative production of hydrogen by Bacillus coagulans AH1 (MN923076). The optimum operation conditions for biohydrogen production were 5.0% substrate concentrationand pH 9.0, at 35 °C. In which the biohydrogen yield was 3.231 mmol H2/g dry biomass without any pretreatments of the biomass. A freshwater microalga Oscillatroia sp was used for upgrading of the produced biohydrogen. It sequestrated 97 and 99% % of CO2 from the gas mixture when it was cultivated in BG11 and BG11-N media, respectively After upgrading process, the residual microalgal cells exhibited 0.21mg/mL of biomass yield,high content of chlorophyll-a (4.8 µg/mL) and carotenoid (11.1 µg/mL). In addition to Oscillatroia sp residual biomass showed a lipid yield (7.5-8.7%) on the tested media. CONCLUSION: Bacillus coagulans AH1 is a promising tool for biohydrogen production avoiding the drawbacks of biomass pretreatment. Oscillatroia sp is encouraged as a potent tool for upgrading and purification of biohydrogen. These findings led to the development of a multiproduct biorefinery with zero waste that is more economically sustainable.

Bacillus coagulans GBI-30, 6086 (BC30) improves lactose digestion in rats exposed to a high-lactose meal.

PURPOSE: Bacillus coagulans GBI-30, 6086 (BC30) was previously shown to improve nutrient digestibility and amino acid absorption from milk protein in vitro. However, the effect of supplementation with this probiotic on lactose digestibility has not yet been evaluated in vivo. METHODS: Wistar female rats were exposed to an acute high-lactose diet (LD; 35% lactose) meal challenge after 7 days of administration of BC30 (LD-BC; n = 10) or vehicle (LD-C; n = 10). Rats treated with vehicle and exposed to control diet (CD; 35% corn starch) meal were used as controls (CD-C; n = 10). Carbohydrate oxidation (CH_OX) and lipid oxidation (L_OX) were monitored by indirect calorimetry before and after lactose challenge. After the challenge, rats were treated daily with vehicle or probiotic for an additional week and were fed with CD or LD ad libitum to determine the effects of BC30 administration in a lactose-induced diarrhoea and malnutrition model. RESULTS: LD-C rats showed lower CH_OX levels than CD rats, while LD-BC rats showed similar CH_OX levels compared to CD rats during the lactose challenge, suggesting a better digestion of lactose in the rats supplemented with BC30. BC30 completely reversed the increase in the small intestine length of LD-C animals. LD-BC rats displayed increased intestinal mRNA Muc2 expression. No significant changes were observed due to BC30 administration in other parameters, such as serum calprotectin, intestinal MPO activity, intestinal A1AT and SGLT1 levels or intestinal mRNA levels of Claudin2 and Occludin. CONCLUSION: Treatment with BC30 improved the digestibility of lactose in an acute lactose challenge and ameliorated some of the parameters associated with lactose-induced malnutrition.

Inhibitory Effects of Bacillus Coagulans TL3 on the Ileal Oxidative Stress and Inflammation Induced by Lipopolysaccharide in Rats.

This study aimed to explore the effect and mechanism of Bacillus coagulans TL3 (B. coagulans TL3) on ileal inflammatory injury induced by lipopolysaccharide (LPS). Animal models were established wherein male Wistar rats were randomly divided into four groups: a control group, an LPS group, a high-concentration B. coagulans (HBC) group, and a low-concentration B. coagulans (LBC) group. The results showed that the biochemical indices changed, significant pathological changes were found, the number of apoptotic cells increased in the ileal tissue of the LPS group rats; the protein expressions of NFκB, MYD88, TLR4, TNF-α, Il-6, IL-1ß, Claudin-1, Occludin, and ZO-1 in the LPS group were significantly decreased. The biochemical indices, pathological changes, and protein expressions in rats subjected to intragastric administration with high or low concentrations of B. coagulans TL3, were significantly reversed compared with the LPS group. These results indicated that TL3 strain could protect rats against ileal oxidative stress and inflammation induced by LPS and the protective mechanism was related to inhibition of the toll-like receptor 4 (TLR4) / myeloid differentiation factor-88 (MyD88) signaling pathway.

Nutritional Interventions with Bacillus coagulans Improved Glucose Metabolism and Hyperinsulinemia in Mice with Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) is a metabolic disorder caused by mutations in the porphobilinogen deaminase (PBGD) gene, encoding the third enzyme of the heme synthesis pathway. Although AIP is characterized by low clinical penetrance (~1% of PBGD mutation carriers), patients with clinically stable disease report chronic symptoms and frequently show insulin resistance. This study aimed to evaluate the beneficial impact of nutritional interventions on correct carbohydrate dysfunctions in a mouse model of AIP that reproduces insulin resistance and altered glucose metabolism. The addition of spores of Bacillus coagulans in drinking water for 12 weeks modified the gut microbiome composition in AIP mice, ameliorated glucose tolerance and hyperinsulinemia, and stimulated fat disposal in adipose tissue. Lipid breakdown may be mediated by muscles burning energy and heat dissipation by brown adipose tissue, resulting in a loss of fatty tissue and improved lean/fat tissue ratio. Probiotic supplementation also improved muscle glucose uptake, as measured using Positron Emission Tomography (PET) analysis. In conclusion, these data provide a proof of concept that probiotics, as a dietary intervention in AIP, induce relevant changes in intestinal bacteria composition and improve glucose uptake and muscular energy utilization. Probiotics may offer a safe, efficient, and cost-effective option to manage people with insulin resistance associated with AIP.

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.

The effect of Bacillus coagulans Unique IS-2 supplementation on plasma amino acid levels and muscle strength in resistance trained males consuming whey protein: a double-blind, placebo-controlled study.

PURPOSE: The aim of the present study was to assess the effect of Bacillus coagulans Unique IS-2 supplementation on absorption and utilization of protein in resistance-trained males. METHODS: In this double blind, placebo-control trial, resistance-trained males (21.08 ± 2.84 years) were randomized to consume, either 20 g of whey protein powder {80% whey protein concentrate (WPC80), amounting to 15.4 g protein} with 2 billion CFU Bacillus coagulans Unique IS-2 (supplemental group) or 20 g of whey protein powder and lactose instead of Bacillus coagulans (placebo group) once daily for 60 days with a controlled resistance exercise protocol. The whey protein concentrate (WPC-80) given to both groups had a lactose content of 6.8%. Plasma-free amino acids (PFAAs) were determined at baseline, at 30 and 60 days of supplementation. Muscle strength, hypertrophy, VO2 max, and body composition, and other biochemical parameters were assessed at baseline and end line. RESULTS: A positive effect of probiotic Bacillus coagulans Unique IS-2 supplementation was observed on protein absorption as evidenced by an increase in total PFAA by + 16.1% (p = 0.004). Branched chain amino acids (BCAA) comprising isoleucine (p = 0.016), leucine (p = 0.001), and valine (p = 0.002) were increased by + 33.1% in ITT analysis as compared to placebo after 60 days. At 30 days an increase in isoleucine by + 35% (p = 0.113), leucine by + 43% (p = 0.032), and valine by + 32% (p = 0.017) was observed in ITT analysis. Probiotic effect was shown on exercise performance as evidenced by an increase in one RM of leg press and vertical jump power by + 16.61% (p = 0.024) and + 7.86% (p = 0.007), respectively. CONCLUSION: Significantly increased absorption of BCAA with supplementation of B. coagulans Unique IS-2 along with whey protein and improvement in leg press and vertical jump power was noted indicating the positive effect of the probiotic on muscle power in the lower body. TRIAL REGISTRATION NUMBER: CTRI/2017/03/008117; Date:16.03.2017.

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.

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.

Cytotoxicity, apoptosis, and IL-8 gene expression induced by some foodborne pathogens in presence of Bacillus coagulans in HT-29 cells.

Food poisoning caused by bacteria is one of the most important concerns in food hygiene. The use of probiotics in prevention, control, and treatment of these infections has been considerably increased in recent years. This study evaluated the effect of B. coagulans cell free supernatant (CFS) on growth of Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, non-pathogenic Escherichia coli, and Escherichia coli 0157:H7 by the broth dilution method. The cytotoxicity, and apoptosis induced by pathogens alone and in co-culture with B. coagulans or its CFS were measured by trypan blue, and fluorescence staining methods. The expression level of interleukin-8 (IL-8) cytokine-encoding genes was also investigated by a qRT-PCR assay in all pathogens and co-cultured groups in HT-29 cells. Our results showed that 4% B. coagulans CFS reduced pathogen growth. The highest rate of growth inhibition was observed in L. monocytogenes. We also found that B. coagulans, and its 4% CFS reduced the cytotoxic effects of pathogens, with the exception of S. aureus. Non-pathogenic E. coli also had no significant cytotoxic effect on the cells. Examination of the treated cells with acridine orange/ethidium bromide staining showed reductions in the rate of cell damage (including early apoptosis, late apoptosis, and necrosis) in pathogen-probiotic co-cultures. Furthermore, we showed that co-culture of pathogens with B. coagulans significantly down-regulated IL-8 gene expression (P < 0.05). The greatest down-regulation compared with pathogen alone was observed in S. aureus. Hence, B. coagulans can be considered as an appropriate probiotic to diminish cytotoxicity, and inflammatory response of enteropathogenic bacteria.

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).