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.

ARTP mutation and adaptive laboratory evolution improve probiotic performance of Bacillus coagulans.

Bacillus coagulans is a thermophilic, facultative anaerobic, spore-forming Gram-positive bacterium, which is used as a probiotic in animal feed and human dietary supplements. In the present study, a bile-resistant thermophilic B. coagulans WT-03 strain was isolated and genetically identified. Atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the probiotic performance of B. coagulans WT-03. After 15 s of ARTP mutation and 40 days of ALE culture, a mutant artp-aleBC15 was obtained and showed the improved tolerance to pH 2.5 and 0.3% bile salt with a survival rate of 22.4%. Further studies showed that the artp-aleBC15 mutant exhibited a relatively stable morphology, lower permeability, and higher hydrophobicity of cell membrane compared with the parent strain of B. coagulans. Additionally, artp-aleBC15 could maintain homeostasis with an intracellular pH of over 4.5 and had the altered contents of saturated fatty acids/unsaturated fatty acids in the cell membrane at pH 2.5. Our study proved that ARTP mutation combined with ALE is an efficient mutagenesis strategy to improve the probiotic performance of B. coagulans for potential industrial use.Key Points• A B. coagulans strain that can grow at 80 °C and 0.3% bile salt was screened.• ARTP combined with ALE effectively mutated B. coagulans WT-03.• B. coagulans artp-aleBC15 mutant showed an improved probiotic performance.• The mutant exhibited the lower permeability and altered fatty acid contents in the cell membrane.

Difference in the antibacterial action of epigallocatechin gallate and theaflavin 3,3'-di-O-gallate on Bacillus coagulans.

AIMS: To study the mechanism of the antibacterial action of tea polyphenols such as catechins and theaflavins against Bacillus coagulans, and the interaction of epigallocatechin gallate (EGCg) or theaflavin 3,3'-di-O-gallate (TFDG) with the surface of B. coagulans cells was investigated. METHODS AND RESULTS: The antibacterial activities of EGCg and TFDG against B. coagulans cells were measured by counting of the viable cells after the mixing with each polyphenol. Bactericidal effect of TFDG was shown at the concentration of greater than or equal to 62·5 mg l-1 ; however, at the same concentration, EGCg did not. According to the results of two dimensional (2D)-electrophoresis analysis, TFDG seemed to interact with cytoplasmic membrane proteins. The activity of the glucose transporters of the cells decreased 40% following the treatment with TFDG of 62·5 mg l-1 ; however, this decrease was only slight in case of EGCg. This result was in accordance with the strength of their bactericidal activities. CONCLUSION: Our results suggest that the direct interaction between membrane proteins and TFDG is an important factor in the antibacterial activity of polymerized catechins, affecting their functions and leading to cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Tea polyphenols can effectively use the prevention of product spoilage in the food and beverage industry.

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.

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly.

The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS) and inulin both stimulated bifidobacteria compared to other treatments (p<0.05). Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05). IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05). To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters.

Precultivation of Bacillus coagulans DSM2314 in the presence of furfural decreases inhibitory effects of lignocellulosic by-products during L(+)-lactic acid fermentation.

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in L(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.

Leguminose green juice as an efficient nutrient for l(+)-lactic acid production.

Lactic acid is one of the most important building blocks for the production of bioplastic. Many investigations have been conducted to reduce the lactic acid production costs. In this work, the focus was put on the application of legume pressed juice or green juice as nutrient source. The pressed juice was utilized directly without prior pre-treatment and sterilization. Using two different alfalfa green juices and a clover green juice from two different harvest years as sole nutrients, non-sterile fermentations were performed at 52°C and pH 6.0 with a thermotolerant strain Bacillus coagulans AT107. The results showed that alfalfa green juices generally were more suitable for high lactic acid production than clover green juices, presumably due to the higher nitrogen content. A final titer of 98.8g/L after 30h with l(+)-lactic acid purity of >99% was obtained.

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.