Overview of Probiotic Strains of Weizmannia coagulans , Previously Known as Bacillus coagulans , as Food Supplements and Their Use in Human Health

: Weizmannia coagulans , previously known as Bacillus coagulans and before that as Lactobacillus sporogenes , is a spore-forming, lactic acid-producing, Gram-positive, bacillus-shaped bacterial species with several known probiotic strains, including GBI-30, 6086 Unique IS-2, MTCC 5856, LBSC (DSM 17654), TBC169, SNZ 1969, BC30, and T11. This review focusses on the health beneﬁt of these strains. A total of 53 clinical trials were found to use various strains of Weizmannia coagulans . However, 19 of these clinical trials did not provide strain information. Clinical evidence has shown that supplementation with strains of Weizmannia coagulans resulted in statistically signiﬁcant health effects in the probiotic groups compared to the placebo. Several health beneﬁts of the Weizmannia coagulans strains were found including relieving symptoms of irritable bowel syndrome, constipation, diarrhoea, and other gastrointestinal symptoms, function recovery treatment of non-fatty liver disease, after surgery or in patients with rheumatoid arthritis, quality of life and glucose-and lipid-related biomarkers related to overweight or obese participants or diabetic patients, absorption of protein or muscle integrity and improvement of peri-and post-menopausal symptoms. The main mechanism of action is the modulation of the intestinal microbiota and host immunity. However, in terms of several clinical studies involving small patient populations, others did not provide strain information. Larger, well-designed clinical studies are warranted to support the health beneﬁts of Weizmannia coagulans strains.


Introduction
A special group of beneficial microbes is probiotics, which are by definition 'live microorganisms that, when administered in adequate amounts, confer a beneficial effect on the host' [1].The most common probiotics are strains of the following genera: Lacticaseibacillus, Limosilactobacillus, Lactobacillus, Lactiplantibacillus Latilactobacillus, Levilactobacillus, Limosilactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, Bacillus, Clostridium, Propionibacterium, Escherichia, and Saccharomyces [2].For a probiotic strain to confer a beneficial effect on the host, it must have certain characteristics including survival at relevant body sites, such as adhesion to mucus or intestinal epithelial cells, interaction with human immune cells, resistance to digestive enzymes, bile or acid, antibacterial activity via competitive exclusion or production of bacteriocins or hydrogen peroxide [3].
Spore-forming bacteria are a wide group of bacteria that contain many pathogens and also beneficial bacteria and belong to the phyla Bacillota (previously known as Firmicutes) [4].The most common genera are Bacillus and Clostridium; both genera have been recently divided and renamed [5,6], with some new names of genera including Weizmannia, Geobacillus, Alkalihalobacillus, Priestia, and Clostridioides.Endospores or shortly spores are robust, metabolically inert, and incredibly resilient structures that are resistant to environmental stresses, such as UV, desiccation, heat, many disinfectants, high temperatures, and antibiotics [7].The robust properties of the spore are due to its multi-layered structure.The dense core in Clostridioides difficile (previously Clostridium difficile) contains the DNA that is surrounded by small proteins.Around this core is a thin layer of peptidoglycan with the same composition as in vegetative cells.This layer is surrounded by the cortex, which contains a much thicker layer of peptidoglycan and is followed by another membrane, derived from the mother cell and a lamellar coat that contain highly crosslinked proteins.The final outmost coat or exosporium of Clostridioides difficile contains an amorphous structure.Other spore formers also contain similar structures and a more hexameric organization of this outer membrane [7][8][9][10].Once the environmental conditions improve, the spore germinates back to vegetative cells.Germination is initiated when chemical signals indicate the spore is in an environment that is conducive to vegetative cell survival and growth [7].Due to their resistance to various conditions, many aerobic sporogenic bacteria are ubiquitous in the environment.
Several spore-forming bacteria are pathogens or opportunistic pathogens, including Clostridioides difficile, which is the most common cause of antibiotic-associated diarrhoea and pseudomembranous colitis [7], Clostridium tetani and Clostridium botulinum that cause tetanus and botulism, respectively [11], as well as Bacillus anthracis that causes anthrax [12], Bacillus cereus and Clostridium perfringens that cause food poisoning [13] and Bacillus thuringiensis that causes diarrhoeal illness [14].
As spores can survive the harsh conditions of the stomach with a very low pH due to gastric acid (hydrochloric acid) and bile acids (mainly derivates of cholic acid) and, upon transit into the duodenum, begin to germinate back to vegetative cells that colonize and proliferate in the colon [7,30], they are often considered to have an important potential as a probiotic if a health benefit is found.A strain can be defined as a probiotic if it is supported by at least one positive human clinical trial with a statistically significant beneficial effect on health in the probiotic group compared to the placebo group.
In this review, we focussed on the health effects established from published randomised controlled clinical trials of the sporogenic probiotic strains of Weizmannia coagulans, as a comprehensive review of these strains has not been published and many health traits are strain specific.

Search Strategy
We used the search strategy: "probiotics" AND (Bacillus coagulans OR Weizmannia coagulans OR Lactobacillus sporogenes) in various databases (PubMed, ScienceDirect) and included placebo-controlled clinical trials, which investigated the health benefits of the sporogene probiotic.Clinical trials on animals, without the full text available or in languages other than English were excluded.A total of 53 clinical studies investigating the health benefit of the probiotic strains of Bacillus coagulans (Weizmannia coagulans) were found (up to 1 July 2023).Clinical studies without placebo control or strain information were not added in results in tables, but only in descriptive form in the results and the discussion.
Table 1 depicts two cross-over studies by Nyangale and co-authors [31,32].The strain Weizmannia coagulans GBI-30, 608 was used in both cross-over clinical trials.The beneficial effect was connected to intestinal microbiota modulation.Five randomised controlled clinical trials [33][34][35][36][37] included various strains of Weizmannia coagulans (previously Bacillus coagulans) such as CGI314, Unique IS2, MY01, and SNZ 1969 as part of multi-strain probiotics.Safety and tolerance as well as the effect of examination stress on students, influence on functional dyspepsia symptoms, gastrointestinal discomfort, and overweight and obesity-related parameters were investigated and statistically significant health benefits were found in all clinical trials with multi-strain probiotics.These studies are described in Table 2. Weizmannia coagulans GBI-30, 6086, 1 × 10 9 cfu/day; 1 capsule per day for 28 days, followed by a 21-day washout period before switching to the other treatment.
Increase in beneficial groups of bacteria in the human gut and increase in production of anti-inflammatory cytokines.
Modulation of the faecal microbiota using in vitro batch culture fermenters.
Randomised controlled trials (RCT) are the most common studies to evaluate healthcare interventions.They consist of participants randomly allocated to either a treatment or intervention group or a control group.A control group allows for the most effective comparison of two groups that are initially similar.Randomisation prevents bias as it allows allocation blinding and statistical control.It is important that the design, conducting, and reporting are without selection, allocation, performance, attribution, or experimental bias.The reporting quality of an RCT should be transparent and clear, allowing readers to appropriately analyse and understand the study design and results that may change clinical practice [87][88][89].In a crossover clinical trial, participants are randomly allocated to receive treatment one first, followed by a washout period, when no treatment is received and then allocated to the other treatment and vice versa.Removing patient variation in this way makes crossover trials potentially more efficient than similar-sized, parallel-group trials in which each subject is exposed to only one treatment.And the treatment effects can be estimated with greater precision given the same number of subjects.However, long washout periods to prevent carryover can cause the study to lose effect [90].
Other investigated effects of supplementation with Weizmannia coagulans strains included function recovery treatment of non-fatty liver disease [45], after surgery [40], for maintaining power and short-term speed performance, while attenuating the inflammatory response during intense training [64], or in patients with rheumatoid arthritis [57].Weizmannia coagulans strains (previously B. coagulans or L. sporogenes) together with prebiotics were also effective in improving glucose-and lipid-related biomarkers related to overweight or obese participants or people with type 2 diabetes mellitus some metabolic parameters after supplementation [44,49,50,66,67,69,[71][72][73].Even certain peri-or post-menopausal symp-toms improved [74,77,81].The main mechanisms of action were modulation of the gut microbiota and host immunity.
Nineteen studies did not contain information on strains [63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81].One of the studies without strain information [63] investigated the influence of Clostridium butyricum and Bacillus coagulans and another study [76] investigated a multistrain probiotic without information on any of the strains.The rest of the studies without strain information utilised only Bacillus coagulans (or Lactobacillus sporogenes).The study by Hoffman and coauthors [64] also noted that an inactivated (or non-viable) probiotic was used.This is not in line with the beginning of the definition of probiotics, as they are 'live microorganisms' [1].Conflicting information by the authors was also the concentration of the daily serving as 10 9 cfu, as colony-forming units are produced by multiplication and metabolic activity of live active and viable microbes.Although this study may have used spores of Bacillus coagulans or postbiotics, this was not clear from the information.Also, the method of the inactivation of microbes was not evident.Authors of clinical studies on probiotics need to be more accurate when describing their clinical trials as, although several benefits are core benefits due to shared mechanisms [1,91], the action of probiotics is often strain-specific, especially when more specific ailments are researched [3].When authors do not accurately note the specific probiotic strain used in their clinical study, it is like generalising dog breeds and saying it does not matter which dog breed is used as all are efficient working dogs, when it is general knowledge that a German shepherd dog is probably a more efficient working dog than, for example, a pug.
Although this review focussed on sporogenic probiotic strains of Weizmannia coagulans (previously Bacillus coagulans or Lactobacillus sporogenes), there are many effective probiotic strains with or without spores as supported by a vast number of robust, well-designed clinical studies.Even if the survival of viable probiotic strains on their passage into the intestine is difficult, there are many novel applications, such as the manufacturing of double-coated capsules, that enable the survival of probiotic strains to the far parts of intestine without spores, where they can colonize the intestine, produce organic acids, bacteriocins, and other metabolites and modulate the intestinal microbiota to achieve eubiosis and modulate host immunity by regulating the functions of mucosal immune cells and intestinal epithelial cells.However, it is important to emphasize that the quality of clinical studies can be improved and that more large well-designed clinical trials are needed to support the many health claims of probiotics.

Table 1 .
Characteristics of two cross-over clinical trials using probiotic strains of the species Weizmannia coagulans (previously Bacillus coagulans), listed in descending chronological order.

Table 3 .
Characteristics of twenty-five randomised, placebo-controlled clinical trials using only a single strain probiotic of the species Weizmannia coagulans (previously Bacillus coagulans), listed in descending chronological order.