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.

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.

Genomic-, phenotypic-, and toxicity-based safety assessment and probiotic potency of Bacillus coagulans IDCC 1201 isolated from green malt.

Probiotics are beneficial microorganisms, and the evaluation of their safety for human use in the food industry has become critical. This study examines the safety of Bacillus coagulans IDCC 1201 isolated from green malt by analyzing its genomic and phenotypic characteristics and determining its toxicity. The presence of antibiotic resistance and toxigenic genes and gene transferability were investigated using whole-genome analysis. The strain's hemolytic and enzyme activities, minimum inhibitory concentrations of antibiotics, and biogenic amine and D-lactate production were also examined. Furthermore, the principal properties of B. coagulans IDCC 1201 as probiotics, such as resistance to abiotic stress and intestinal adhesion, were studied. The whole-genome analysis demonstrated that B. coagulans IDCC 1201 had no antibiotic resistance or toxigenic genes; the strain was susceptible to the nine antibiotics proposed by the European Food Safety Authority. Moreover, this strain lacked hemolytic and ß-glucuronidase activities. Additionally, it was confirmed that B. coagulans IDCC 1201 produced undesirable metabolites, including biogenic amines or D-lactate, at a safe level. Finally, the strain exhibited functional potential as a probiotic in terms of abiotic tolerance, such as bile tolerance and intestinal adhesion in in vitro experiments. In conclusion, B. coagulans IDCC 1201 can be considered as a safe probiotic with regard to human health.

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.

Genome based safety assessment for Bacillus coagulans strain LBSC (DSM 17654) for probiotic application.

The present study on Bacillus coagulans strain LBSC (DSM 17654) describes the use of whole genome sequencing, in correlation with the phenotypic properties to assess the safety of the strain. Analysis of the 16S rRNA sequence of the B. coagulans strain LBSC (DSM 17654), showed 100% homology with 99% coverage with B. coagulans strain HM-08. BLAT (BLAST Like Analysis Tool) analysis for whole genome comparison with B. coagulans ATCC 7050, B. coagulans HM-08 and B. coagulans Slac showed 96%, 99% and 99% sequence identity respectively. Whole genome sequencing results demonstrated a single scaffold of 36,35,902 bp and 3331 coding sequences. Gene ontology segregated the proteins as those with molecular function, cellular component and biological process of the predicted genes from assembled genome. Risk associated sequences like antibiotic resistance genes, biogenic amine producing genes, virulence factor genes and other safety related genes were identified with focus on horizontal gene transfer and its non-functionality. The absence of mobile elements in the vicinity of the genes, render it non-transferable and non-toxic phenotypic properties confirm the non-functionality of the genes. Absence of functional genes of concern and confirmation of absence of mobile elements in the vicinity of other non-clinically significant genes indicated no safety concern. The absence of complete and functional prophage sequences which are deleterious for the genome stability and presence of CRISPR system which are advantageous for genome stability by acting as a barrier to entry of foreign DNA elements indicated the stability of the genome. The molecular approach used in this study satisfies the requirements for the safety assessment of the probiotic strain which could indicate it to be potentially safe.

In Silico Prediction and In Vitro Assessment of Multifunctional Properties of Postbiotics Obtained From Two Probiotic Bacteria.

In this study, a global metabolite profile using Raman spectroscopy analysis was obtained in order to predict, by an in silico prediction of activity spectra for substance approach, the bioactivities of the intracellular content (IC) and cell wall (CW) fractions obtained from Lactobacillus casei CRL 431 and Bacillus coagulans GBI-30 strains. Additionally, multifunctional in vitro bioactivity of IC and CW fractions was also assessed. The metabolite profile revealed a variety of compounds (fatty acids, amino acids, coenzyme, protein, amino sugars), with significant probable activities (Pa > 0.7) as immune-stimulant, anti-inflammatory, neuroprotective, antiproliferative, immunomodulator, and antineoplastic, among others. Moreover, in vitro assays exhibited that both IC and CW fractions presented angiotensin-converting enzyme-inhibitory (> 90%), chelating (> 79%), and antioxidant (ca. 22-57 cellular antioxidant activity units) activities. Our findings based on in silico and in vitro analyses suggest that L. casei CRL 431 and B. coagulans GBI-30 strains appear to be promising sources of postbiotics and may impart health benefits by their multifunctional properties.

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%

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Cost-effective simultaneous saccharification and fermentation of l-lactic acid from bagasse sulfite pulp by Bacillus coagulans CC17.

The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous ß-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol.

Integrate genome-based assessment of safety for probiotic strains: Bacillus coagulans GBI-30, 6086 as a case study.

Probiotics are microorganisms that confer beneficial effects on the host; nevertheless, before being allowed for human consumption, their safety must be verified with accurate protocols. In the genomic era, such procedures should take into account the genomic-based approaches. This study aims at assessing the safety traits of Bacillus coagulans GBI-30, 6086 integrating the most updated genomics-based procedures and conventional phenotypic assays. Special attention was paid to putative virulence factors (VF), antibiotic resistance (AR) genes and genes encoding enzymes responsible for harmful metabolites (i.e. biogenic amines, BAs). This probiotic strain was phenotypically resistant to streptomycin and kanamycin, although the genome analysis suggested that the AR-related genes were not easily transferrable to other bacteria, and no other genes with potential safety risks, such as those related to VF or BA production, were retrieved. Furthermore, no unstable elements that could potentially lead to genomic rearrangements were detected. Moreover, a workflow is proposed to allow the proper taxonomic identification of a microbial strain and the accurate evaluation of risk-related gene traits, combining whole genome sequencing analysis with updated bioinformatics tools and standard phenotypic assays. The workflow presented can be generalized as a guideline for the safety investigation of novel probiotic strains to help stakeholders (from scientists to manufacturers and consumers) to meet regulatory requirements and avoid misleading information.