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.

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.

Extraction of chitin from Litopenaeus vannamei shell and its subsequent characterization: an approach of waste valorization through microbial bioprocessing.

Chemical extraction of chitin is very hazardous and costly which can be overwhelmed by microbial bioprocessing. In this study, potent protease and lactic acid-producing bacteria were screened and identified as Alcaligens faecalis S3 and Bacillus coagulans L2, respectively. Productions of protease and lactic acid by the respective bacterial strains were optimized. The shell of Litopenaeus vannamei was sequentially treated with the partially purified protease and lactic acid and the treatment conditions were optimized for betterment of chitin yield. Spectral characterization by SEM-EDS, IR, XRD, NMR, XPS and thermal characterization by TG and DTG analysis of the extracted chitin was made and compared with commercial one. It was revealed that both the chitin have similar characteristics. Therefore, it can be articulated that chitin can be extracted from crustacean shells in pure form by microbial bioprocessing which will be a good catch for biorefinary industries for chitin extraction through greener route.

Co-cultures with integrated in situ product removal for lactate-based propionic acid production.

Propionic acid (PA) is a valuable organic acid for the food and feed industry, but no bioproduction at industrial scale exists so far. As product inhibition is a major burden for bioprocesses producing organic acids, in situ product removal (ISPR) is desirable. Here, we demonstrate a new strategy to produce PA with a co-culture coupled with ISPR using electrodialysis. Specifically, Bacillus coagulans first produces lactic acid (LA) from sugar(s) and LA is converted to PA using Veillonella criceti. Applying ISPR to the mentioned co-culture, the specific PA yield was increased from 0.35 to 0.39 g g-1 compared to no ISPR usage. Furthermore, the productivity was increased from 0.63 to 0.7 g L-1 h-1 by applying ISPR. Additionally, it was shown that co-consumption of xylose and glucose led to a higher PA productivity of 0.73 g L-1 h-1, although PA yield was only increased slightly up to 0.36 g g-1.

Survival of probiotic bacteria in the presence of food grade nanoparticles from chocolates: an in vitro and in vivo study.

The use of probiotics to treat gastrointestinal diseases such as diarrhea especially in children is becoming increasingly popular. Besides, the use of nanomaterials in food products is increasing rapidly especially in candies and chocolates. How these nanomaterials influence probiotic bacteria and their activity remains unexplored. Therefore, nanomaterials from commercial chocolate were purified and characterized by using SEM-EDS and XRD. The tested chocolate contained nano-TiO2 with an average size of ~ 40 nm. The influence of the extracted TiO2 on a commercial probiotic formulation usually used to treat diarrhea in children was studied. The probiotic formulation contained Bacillus coagulans, Enterococcus faecalis, and Enterococcus faecium as evident from 16S rRNA gene sequences and polyphasic characterization. Isolated bacteria exhibited known probiotic activities like biofilm formation, acid production, growth at 6% salt, and antibiotic resistance. TiO2 from chocolates inhibited the growth and activity of the probiotic formulation over a concentration range of 125-500µg/ml in vitro. Based on results, it is estimated that 20 g of such chocolate contains enough TiO2 to disturb the gut microbial community of children aged 2-8 years with a stomach capacity of ~ 0.5-0.9 l. The in vivo study on white albino mice shows the same response but with a higher dose. The results obtained by plate counts, MTT assay, live/dead staining, and qPCR suggest that TiO2 from chocolates inhibits the growth and viability of probiotic bacteria in mice gut even at a concentration of 50-100 µg/day/mice. Therefore, TiO2 in chocolate discourages survival of probiotic bacteria in the human gut.

The effects of probiotic Bacillus coagulans on the cytotoxicity and expression of alpha toxin gene of Clostridium perfringens type A.

Around the world, Clostridium perfringens type A is known to be a common foodborne pathogen. Therefore, the control and treatment of food poisoning caused by this pathogen are important. This study investigated, in vitro, the effects of Bacillus coagulans and its culture extracts on alpha toxin gene expression, growth inhibition, cytotoxicity, and apoptosis induced by C. perfringens spore, germinated spore and its enterotoxin. Flow cytometry was used to evaluate the apoptosis rate, and MTT test was used to evaluate cytotoxicity. Minimum inhibitory concentration was also used to measure the percentage of inhibition in the broth medium. Finally, RT-qPCR was used to evaluate alpha toxin gene expression. The results showed that the B. coagulans culture extract was able to inhibit the growth of the germinated spore of C. perfringens. Moreover, treating the extract with pepsin can reduce growth in the broth medium. MTT and flow cytometry showed that both B. coagulans and its extract can significantly reduce the cytotoxicity and apoptosis rate induced by C. perfringens type A. In addition, it was shown that the co-culture of B. coagulans and C. perfringens decreases alpha toxin gene expression. The findings of this study indicate that B. coagulans, with growth inhibition and reduced expression of alpha toxin in C. perfringens, can reduce the cytotoxicity and apoptosis rate induced on HT-29 cells.

Antagonism Against Fish Pathogens by Cellular Components/Preparations of Bacillus coagulans (MTCC-9872) and It's In Vitro Probiotic Characterisation.

Bacterial fish pathogens are pervasive in aquaculture. Control of bacterial fish pathogen is a difficult task among aquaculture practitioners. A large number of antibiotics are used for the control of prevalent bacterial pathogens in aquaculture. This may lead to drug resistance among pathogens and further treatment will be ineffective. Here, we can use probiotic bacteria as a biocontrol agent in fish disease and it is a novel field. In this study, antimicrobial potential of the bacterium Bacillus coagulans (MTCC-9872) has been evaluated through in vitro antagonistic activity of cellular preparations/components against potent pathogens. The cellular preparations/components such as Ethyl acetate extract, whole-cell product, heat-killed whole-cell product, and filtered broth were exhibited bactericidal activity against the tested pathogens. Bactericidal activity varied among different cellular preparation/components. The tested bacterium effectively produced biofilm as significant as tested positive control in a microtitre plate and effectively adhered on to the glass slide. In addition, the bacterium was capable of producing extracellular enzymes necessary for the digestion of food materials and was capable to grow in fish mucus from Oreochromis niloticus. The bacterium tolerated bile juice secreted by the host. Moreover, intraperitoneal injection of the bacterium did not induce any pathological signs, symptoms or mortalities in Oreochromis niloticus and revealed the safety of this bacterium in the fish.

Efficient in situ separation and production of L-lactic acid by Bacillus coagulans using weak basic anion-exchange resin.

To get rid of the dependence on lactic acid neutralizer, a simple and economical approach for efficient in situ separation and production of L-lactic acid was established by Bacillus coagulans using weak basic anion-exchange resin. During ten tested resins, the 335 weak basic anion-exchange resins demonstrated the highest adsorption capacity and selectivity for lactic acid recovery. The adsorption study of the 335 resins for lactic acid confirmed that it is an efficient adsorbent under fermentation condition. Langmuir models gave a good fit to the equilibrium data at 50 °C and the maximum adsorption capacity for lactic acid by 335 resins was about 402 mg/g. Adsorption kinetic experiments showed that pseudo-second-order kinetics model gave a good fit to the adsorption rate. When it was used for in situ fermentation, the yield of L-lactic acid by B. coagulans CC17 was close to traditional fermentation and still maintained at about 82% even after reuse by ten times. These results indicated that in situ separation and production of L-lactic acid using the 335 resins were efficient and feasible. This process could greatly reduce the dosage of neutralizing agent and potentially be used in industry.

Systematic development and optimization of chemically defined medium supporting high cell density growth of Bacillus coagulans.

With determined components and experimental reducibility, the chemically defined medium (CDM) and the minimal chemically defined medium (MCDM) are used in many metabolism and regulation studies. This research aimed to develop the chemically defined medium supporting high cell density growth of Bacillus coagulans, which is a promising producer of lactic acid and other bio-chemicals. In this study, a systematic methodology combining the experimental technique with flux balance analysis (FBA) was proposed to design and simplify a CDM. The single omission technique and single addition technique were employed to determine the essential and stimulatory compounds, before the optimization of their concentrations by the statistical method. In addition, to improve the growth rationally, in silico omission and addition were performed by FBA based on the construction of a medium-size metabolic model of B. coagulans 36D1. Thus, CDMs were developed to obtain considerable biomass production of at least five B. coagulans strains, in which two model strains B. coagulans 36D1 and ATCC 7050 were involved.

High-throughput system for screening of high L-lactic acid-productivity strains in deep-well microtiter plates.

For strain improvement, robust and scalable high-throughput cultivation systems as well as simple and rapid high-throughput detection methods are crucial. However, most of the screening methods for lactic acid bacteria (LAB) strains were conducted in shake flasks and detected by high-performance liquid chromatography (HPLC), making the screening program laborious, time-consuming and costly. In this study, an integrated strategy for high-throughput screening of high L-lactic acid-productivity strains by Bacillus coagulans in deep-well microtiter plates (MTPs) was developed. The good agreement of fermentation results obtained in the MTPs platform with shake flasks confirmed that 24-well U-bottom MTPs could well alternate shake flasks for cell cultivation as a scale-down tool. The high-throughput pH indicator (bromocresol green) and L-lactate oxidase (LOD) assays were subsequently developed to qualitatively and quantitatively analyze L-lactic acid concentration. Together with the color halos method, the pH indicator assay and LOD assay, the newly developed three-step screening strategy has greatly accelerated the screening process for LAB strains with low cost. As a result, two high L-lactic acid-productivity mutants, IH6 and IIIB5, were successfully screened out, which presented, respectively, 42.75 and 46.10 % higher productivities than that of the parent strain in a 5-L bioreactor.

Development of a semi-dynamic in vitro model and its testing using probiotic Bacillus coagulans GBI-30, 6086 in orange juice and yogurt.

A dynamic system mimicking the gastrointestinal tract (GIT) conditions (fluids, pH, temperature, and residence time) was used to evaluate the behavior of Bacillus coagulans GBI-30, 6086 (BC) incorporated in yogurt and orange juice. BC counts were monitored in samples collected before the in vitro digestion, after initial contact with gastric fluids (30 min), static (1 h 15 min) and dynamic (2 h) stages in the gastric compartment, static (3 h) and dynamic (4 h) stages in the duodenal compartment, static (5 h) and dynamic (6 h) stages in the jejunal compartment, and after digestion. BC presented high survival in juice and yogurt over the digestion stages. The number of decimal reductions (γ) of BC caused by exposure to simulated GIT conditions was ≥0.89 in orange juice and ≥1.17 in yogurt. No differences (p ≥ 0.05) were observed on the survival of BC among the samples collected over the digestion in juice or yogurt, or between these matrices. After the in vitro digestion, BC counts were ≥7 log CFU/mL or g. Results show the great survival of BC under GIT conditions and suggest both, juice and yogurt as appropriate carries for delivering this probiotic to the diet. The semi-dynamic in vitro system was easily built and to operate, comprising an intermediate approach to assess the resistance of probiotic or potentially probiotic strains under simulated gut conditions.

Assessment of different Bacillus coagulans strains for l-lactic acid production from defined media and gardening hydrolysates: Effect of lignocellulosic inhibitors.

Cellulose valorisation has been successfully addressed for years. However, the use of hemicellulosic hydrolysates is limited due to the presence of C5-sugars and inhibitors formed during pretreatment. Bacillus coagulans is one of the few bacteria able to utilize both C6- and C5-sugars to produce l-lactic acid, but its susceptibility to the lignocellulosic inhibitors needs further investigation. For such a purpose, the tolerance of different B. coagulans strains to increasing concentrations of inhibitors is studied. The isolated A162 strain reached the highest l-lactic acid productivity in all cases (up to 2.4 g L-1  h-1), even in presence of 5 g L-1 of furans and phenols. Remarkably, most of furans and phenolic aldehydes were removed from defined media and hemicellulosic gardening hydrolysate after fermentation with A162. Considering the high productivities and the biodetoxifying effect attained, A162 could be pointed out as a great candidate for valorisation of mixed sugars from hemicellulosic hydrolysates with high inhibitors concentration, promoting the implementation of lignocellulosic biorefineries.

Simultaneous fermentation and hydrolysis to extract chitin from crayfish shell waste.

Chitin is the second-most abundant bioresource and widely used in the food, agricultural, textile, biomedical, and pharmaceutical industries. However, an efficient, environmentally friendly, and economically feasible process for chitin extraction from shellfish waste remains to be explored. This study aimed to extract chitin from crayfish shell waste powder (CSP) by removing Ca2+ and protein, using Bacillus coagulans LA204 and proteinase K. A simultaneous enzymatic hydrolysis and fermentation process was conducted at 50 °C with 5% (w/v) CSP, 5% (w/v) glucose, 1000 U proteinase k g-1 CSP, and 10% inoculation of B. coagulans LA204 in a 5-L bioreactor under non-sterile conditions. After 48 h of fermentation, the deproteinization efficiency, demineralization efficiency, and chitin recovery reached 93%, 91%, and 94%, respectively. 1 mol additional glucose efficiently removed 0.91 mol calcium carbonate and 93% of the removed protein was hydrolyzed to acid-soluble protein. Simultaneous enzymatic hydrolysis and fermentation was a new strategy and a competitive biological method for chitin extraction.

Evaluation of probiotic Bacillus coagulans MTCC 5856 viability after tea and coffee brewing and its growth in GIT hostile environment.

In recent years, probiotic functional foods have gained quite a popularity and become a preferred choice among consumers, due to their positive effects on the gut microbiota and overall health. However, it is imperative for a probiotic strain to remain live and active at the time of consumption in high enough population density, in order to provide such health benefits. Thus, this study aimed to investigate the Bacillus coagulans MTCC 5856 spore stability after tea and coffee brewing and its subsequent growth in gastrointestinal tract (GIT) hostile environment. B. coagulans MTCC 5856 showed remarkable survival (94.94% and 99.76% in unroasted green coffee and tea, respectively) after brewing conditions and was able to grow in GIT hostile conditions using tea and coffee as a sole nutritional source. B. coagulans MTCC 5856 inclusion in tea and coffee after brewing did not significantly (P > .05) alter the sensory profile when compared to that without the probiotic inclusion. Moreover, B. coagulans MTCC 5856 growth was significantly (P < .05) higher when water soluble fibers were added during brewing, suggesting a synergistic property. It showed over 99% viability (P > .05) in tea and coffee powder at room temperature up to 24 months of storage. This study demonstrated the stability of the tested probiotic strain B. coagulans MTCC 5856 after tea and coffee brewing and its growth in GIT hostile environment, thereby suggesting functional probiotic use in tea and coffee.

Spores of Bacillus coagulans GBI-30, 6086 show high germination, survival and enzyme activity in a dynamic, computer-controlled in vitro model of the gastrointestinal tract.

The aim of this study was to assess the germination, survival and metabolic activity of the probiotic Bacillus coagulans GBI-30, 6086 [GanedenBC30] (BC30) in a dynamic, computer controlled in vitro model of the gastrointestinal (GI) tract, simulating human adults. Experiments were performed in the presence of a meal to maximise germination, due to the presence of germination-triggers. Both an upper GI tract (stomach and small intestine; TIM-1) and a colon model (TIM-2) were used, where material exiting TIM-1 was added to TIM-2. Spores of BC30 were introduced in the gastric compartment of TIM-1 and samples were taken immediately after the pylorus. Moreover, for 6 h, every hour the ileal efflux was collected and a subsample was plated for viable counts (spores and germinated cells). The remainder of the sample was fed to TIM-2, and after 24 h another sample was taken and tested for viable counts. In addition, samples were taken from the dialysates of the model and analysed using LC-MS/MS to determine bacterial metabolites and digestion products. Survival after transit through the gastric compartment was high (97%) and most cells were still in the spore form (76%). Survival after transit through TIM-1 was on average 51%, meaning that on average half of the orally provided spores was found back as cfu on the agar plates. Of these on average 93% were germinated cells and only 7% were spores. 24 h after the start of the experiments germination had increased in TIM-2 to 97% vegetative cells, and only 3% spores. No further loss of viability was observed in TIM-2. In terms of metabolic activity, increased levels of amino acids, dipeptides and citric acid cycle metabolites were found compared to experiments in the absence of BC30. In conclusion, BC30 spores germinate to a large extent (>90%) in the presence of germination triggers in the small intestine in a model that closely mimics the physiological conditions of human adults. Of the oral dose, as much as half of the cells survived transit through the upper GI tract, and based on the metabolite profile, these cells were metabolically active. Either these cells or the enzymes released from the dead cells aided in digestion of the meal. These insights help explain some of the observations in previous experiments, and support the understanding of the mechanism of action of the probiotic BC30.

Comparison of high-titer lactic acid fermentation from NaOH- and NH3-H2O2-pretreated corncob by Bacillus coagulans using simultaneous saccharification and fermentation.

Lignocellulose is one of the most abundant renewable feedstocks that has attracted considerable attention as a substrate for biofuel and biochemical production. One such biochemical product, lactic acid, is an important fermentation product because of its great potential for the production of biodegradable and biocompatible polylactic acid. High-titer lactic acid production from lignocellulosic materials has been achieved recently; however, it requires biodetoxification or results in large amounts of waste washing water. In this study, we employed two alkaline pretreatment methods and compared their effects on lactic acid fermentation of pretreated corncob by Bacillus coagulans LA204 using fed-batch simultaneous saccharification and fermentation under non-sterile conditions. The lactic acid titer, yield, and productivity from 16% (w/w) NaOH-pretreated and washed corncob were 122.99 g/L, 0.77 g/g corncob, and 1.37 g/L/h, respectively, and from 16% NH3-H2O2-pretreated and washed corncob were 118.60 g/L, 0.74 g/g corncob, and 1.32 g/L/h, respectively. Importantly, the lactic acid titer, yield, and productivity from 18.4% NH3-H2O2-pretreated and unwashed corncob by using fed-batch simultaneous saccharification and fermentation reached 79.47 g/L, 0.43 g/g corncob, and 1.10 g/L/h, respectively, demonstrating that this method is possible for industrial applications and saves washing water.

Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans.

Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer.

Layer-by-Layer Encapsulation of Probiotics for Delivery to the Microbiome.

The gastrointestinal (GI) microbiome is widely investigated for its role in many diseases. However, technologies designed for microbiome delivery are lacking. Here, a layer-by-layer (LbL) approach is reported for probiotic encapsulation to protect probiotics against GI tract insults and improve their adhesion and growth on the intestines. These advantages translate to significantly enhanced survival of LbL-probiotics in vivo.

Desenvolvimento tecnológico do Bacillus coagulans BVB5 como potencial cepa probiótica / Technological development of Bacillus coagulans BVB5 as potential probiotic strains

Introdução: O aumento da demanda por alimentos funcionais, os quais incluem os suplementados ou fermentados por microrganismos probióticos, resultou no avanço da pesquisa e desenvolvimento de novas cepas potencialmente probióticas. Os microrganismos probióticos pertencentes ao gênero Bacillus spp. são atraentes devido a sua estabilidade inerente de bactérias formadoras de esporos. Os esporos permitem uma vida de prateleira prolongada e aumentam a capacidade do microrganismo de sobreviver às barreiras gástricas, que se revelam uma vantagem sobre os lactobacilos. Objetivo: Foram realizados experimentos para o desenvolvimento tecnológico de duas cepas formadoras de esporos de Bacillus coagulans BVB1 e BVB5 como potenciais microrganismos probióticos objetivando avaliar o potencial probiótico dos mesmos. Método: Como primeira etapa, as caracterizações fenotípica e genotípica identificaram que a cepa BVB1 não era B. coagulans e sim B. subtilis. Os estudos seguiram com a cinética da fermentação/esporulação apenas da cepa de Bacillus coagulans BVB5. Conclusão: O desafio da esporulação de Bacillus coagulans BVB5 foi vencido, fato que pode ser verificado na cinética da fermentação que apresentou resultados superiores a 99% de grau de esporulação

Introduction: The increased demand for functional foods, which include those supplemented or fermented by probiotic microorganisms, resulted in the advancement of research and development of new potentially probiotic strains. Probiotic microorganisms Bacillus spp. Are attractive because of their inherent stability of spore forming bacteria. Spores allow prolonged shelf life and increase the ability to survive gastric barriers, which prove to be an advantage over lactobacilli. Objective: Experiments were performed for the technological development of Bacillus coagulans BVB1 and BVB5 as potential probiotic microorganisms with two strains of Bacillus coagulans aiming to evaluate the efficacy of probiotic product composed of spore forming microorganism (Bacillus coagulans strains BVB1 and BVB5). Method: As a first step, phenotypic and genotypic characterization identified that the BVB1 strain was not B. coagulans but B. subtilis. Although Bacillus subtilis strain BVB1 presented a technological potential to be used as a probiotic strain in food additives, the studies followed with the kinetics of fermentation/sporulation of Bacillus coagulans BVB5, in agreement with the master's project originally proposed. Conclusion: The challenge of sporulation of Bacillus coagulans BVB5 was overcome, a fact that can be verified with the results of fermentation kinetic which presented sporulation degree more than 99%