SAFETY OF BACILLUS SUBTILIS AS PROBIOTIC /ORAL USE
SAFETY OF BACILLUS SUBTILIS AS PROBIOTIC /ORAL USE
SYSTEMATIC LITERATURE SEARCH
Different steps below are followed to gather all documents related to the substance of Bacillus subtilis as oral supplement food probiotic:
History and pattern and human use
Use in therapeutic goods (Australian and International)
Australia and New Zealand have no specific regulations on probiotics, nor a definition of probiotics. Microorganisms, including probiotics, are considered novel foods according to FAO/WHO FOOD STANDARDS PROGRAMME CODEX COMMITTEE ON NUTRITION AND FOODS FOR HEALTH OR DIET.
Brazil, Colombia and Ecuador have adopted a definition of probiotics that is aligned with the definition proposed by FAO/WHO. In addition, Brazil has implemented a protocol for the evaluation of a probiotic as a food ingredient. The Southern Cone region and Caribbean countries include requirements for “probiotic” microorganisms on food labelling. In Europe, there is no regulatory status, no guidelines defining the category of probiotics, and no commonly accepted list of individual probiotic strains and/or species. A small number of EU member states, including Italy, have developed certain requirements for the qualification of specific strains as probiotics. In the United States, probiotics can be considered a food or an ingredient. Safety is demonstrated using the “Generally Regarded As Safe (GRAS)” process or by voluntary notification to the food ingredient regulatory body or using the “New Dietary Ingredients (NDI)” process regarding use in food supplements, if any. Canada has developed a guidance document to explain the acceptable use of health claims for microorganisms represented as probiotics on food labels and in advertising.
Use in food, Traditional use and History of safe use
As natural inhabitants of the human gut, many strains of Lactobacillus spp. and Bifi-dobacterium spp. have an established history of safe use in dietary supplements and foods such as yogurt, kefir, and cheese. While spore-forming Bacillus species have traditionally been described as soil-borne bacteria, they too have been described in the naturally occurring human gut microbiota, albeit less represented in commercially available probiotic products. Multiple independent studies have reported the presence of Bacillus spp., and specifically Bacillus subtilis, in intestinal and human fecal samples, independent of any probiotic supplementation. Collectively, these data show that Bacillus spp. occurs in the human gut in large enough numbers to be a resident gut commensal bacterial species [8–10]. Additionally, Bacillus subtilis has been safely used in traditional fermented foods of many east Asian cultures for centuries. Producers of natto, a traditional Japanese fermented soybean food, have utilized Bacillus subtilis var. natto for commercial production since the early 1900s [11]. Bacillus subtilis strains are naturally present in Korean kimchi, Egyptian kishk, and in a variety of cultural adaptations of fermented soy including miso and thuanao [12–15]. Sequencing and characterization of these strains support the safe use of Bacillus subtilis in foods and dietary supplements [16,17]. Safe use of Bacillus subtilis strains is supported not only in healthy adults [18,19] but also in pediatric populations [20].
Spears JL, Kramer R, Nikiforov AI, Rihner MO, Lambert EA. Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements. Nutrients. 2021 Feb 25;13(3):733. doi: 10.3390/nu13030733. PMID: 33668992; PMCID: PMC7996492.
Pharmacokinetic studies addressing:
Presence of Bacillus subtilis DE111® in the Small Intestine
Absorption
Tissue distribution and storage
Metabolism
Spores of B. subtilis DE111® (6.4 × 104 ± 1.3 × 105 CFU/g) were detected in the small intestinal tract 3 h following ingestion of the probiotic capsule (Figure 2; Supplementary Table 1). An increase in the number of spores over time was seen and reached a peak at 6 h following ingestion (9.7 × 107 ± 8.1 × 107 CFU/g). The same concentration of spores continued to be present in the ileal effluent at each time point assessed until end of the study session at 8 h following ingestion. Over the course of the 8-h study session, a total of 3.0 × 109 ± 6.8 × 109 CFU of the originally inoculated spores were recovered from the small intestinal effluent.
FIGURE 2
Figure 2. B. subtilis DE111® concentration in the small intestinal tract of healthy individuals with an ileostomy stoma. Vegetative DE111® (●), DE111® spores (■), and placebo (▲). Values are average concentrations (n = 11) ± standard deviation.
Vegetative cells of B. subtilis DE111® were also evident after 3 h (4.7 × 104 ± 1.1 × 105 CFU/g; Figure 2), revealing germination of the spore in the small intestine. Vegetative B. subtilis DE111® concentrations in the ileal effluents reached a peak concentration 7 h after ingestion (7.3 × 107 ± 1.4× 108 CFU/g), with the final concentration of 1.2 × 107 ± 1.4 × 107 CFU/g at the final time point.
All participants had both spores and vegetative cells present in their ileal effluent although the rate at which they first presented and persisted varied among individuals (Supplementary Table 1; Table 2). Presence of spores and vegetative cells was seen from 3 h after ingestion, with spores identified in 36% of participants and vegetative cells in 27% of samples at this point (Table 2). Four hours following ingestion, 80% of participants had spores in their ileal effluents and 60% had vegetative cells. All participant samples had spores present 5 h after ingestion and spores remained present in the effluents until the end of the 8-hour study session (Table 2). Detection of vegetative B. subtilis DE111® in ileum effluents was 82% after 5 h, 91% at 6 h, and remained similar until the end of the study. All participants had vegetative cells present in their ileal effluent at some time throughout the session (Table 2).
TABLE 2
Table 2. Bacillus subtilis DE111® relative spore and vegetative cell concentration (% of total DE111® counts) in ileal effluents of individual participants (A–K) over the course of the study session (0–8 h).
Discussion
A majority of human intervention studies examining Bacillus probiotic behavior in the gut involve samples recovered from the end of the intestinal tract via feces (Hanifi et al., 2015). Confirming the presence of Bacillus vegetative cells in the small intestinal tract is challenging. To date, germination of Bacillus spores in the small intestine of humans has only been characterized in artificial gastrointestinal models (Bernardeauet al., 2017). A majority of studies examining the fate of Bacillus spores administered orally have been carried out in animal models and reveal a disparity of results. In one study, mice inoculated with spores of B. subtilis and B. clausii had no vegetative cells detected in the intestinal tract (Spinosa et al., 2000). In comparison, another mouse model study investigating the spore germination of two different strains of B. subtilis revealed the presence of vegetative cells in the jejunum 12–18 h following ingestion (Tam et al., 2006). Molecular approaches based on competitive reverse transcription-PCR, targeting a gene uniquely expressed by vegetative B. subtilis cells, detected 1–12% germination of spores in the jejunum and ileum of mice (Casula and Cutting, 2002). In contrast, 70–80% germination of B. subtilis and B. licheniformis spores was observed in the proximal intestinal tract of pigs (Leser et al., 2008). Taken together, these studies suggest the fate of spore-forming probiotics in the gut is strongly strain dependent and may differ dramatically depending on the model organism used for the study. This highlights the necessity of strain-specific studies that are performed in the target population to collect accurate data regarding the behavior of the probiotic strain in the gut.
This current study is, to the best of the authors knowledge, the first time in which the fate of a probiotic in the small intestine was investigated. A novel clinical intervention trial in healthy human participants with an ileostomy was developed, enabling real-time, direct access to effluent at the end of the ileum (small intestine). Using this method, the ability of the spore-based probiotic B. subtilis DE111® to survive gastrointestinal transit and germinate in the small intestine was evaluated. Three hours following the ingestion of a commercially available capsule of B. subtilis DE111®, spores and vegetative cells were found to be present in the ileum. The number of both spores and vegetative cells increased over the course of 6 h in ileum effluxes and remained constant through to the end of the time course (8 h). The counts in this study are representative of non-adhered cells, with in-situ numbers potentially being higher as Bacillus species are known to adhere to intestinal mucus (Elshaghabee et al., 2017) and specifically, DE111® has been seen to adhere to Caco2 cells (unpublished data). While spores of B. subtilis have been shown to enhance host immunity in the small intestine (Huang et al., 2008), additional host benefits are only possible if the vegetative form of the bacteria is also present. Detecting vegetative cells of B. subtilis DE111® in the small intestinal tract suggest metabolically active bacteria are present, producing key beneficial molecules and supporting a healthy microbiome and gut (Elshaghabee et al., 2017). This finding is significant, as for a probiotic to produce enzymes and small molecules to assist in digestion and exert maximal immune benefits it needs to be in the vegetative form and proliferate in the small intestinal tract (Vighi et al., 2008; Santaolalla et al., 2011; Aidy et al., 2015). The time after ingestion at which vegetative cells were first seen in the small intestinal tract varied between individuals, with a proportion having vegetative cells evident 3 h following ingestion of the probiotic. Widespread presence of vegetative cells across participants was observed after 5 h and remained reasonably constant until the end of the study session. It has been shown that in healthy adults transit time, from ingestion through to the end of the ileum, can range from 157 to 240.5 min (O’Grady et al., 2020). Therefore, the variations observed in the timing of initial presence of B. subtilis DE111 in ileum effluxes may be attributed to inherent differences in transit times for each participant. The small intestinal microbiota is dynamic, reflecting the complexity of the environment (Kastl et al., 2020). Recent studies showed daily intake of B. subtilis DE111® results in subtle shifts in some key genera, ultimately supporting a healthy microbiome in children aged 2–6 years old (PaytuvÃ-Gallart et al., 2020). Future investigations including metagenomic profiling specifically of the small intestinal microbiota during ingestion of the spore-based probiotic may help further elucidate the beneficial effects of B. subtilis DE111® in this region of the gastrointestinal tract.
In conclusion, a unique real-time intervention trial was developed which allowed proof of the survivability of the probiotic B. subtilis DE111® through the upper gastrointestinal tract and subsequent germination in the human small intestine. Interestingly, while germination of spores was seen in all participants, the timeline of when vegetative cells first emerged in the ileal effluent was individual dependent. Further studies examining the presence and vegetation of B. subtilis over an extended intervention period would be interesting and offer insight into efficacy, metabolic activity, colonization, and re-sporulation of this spore-based probiotic in the small intestinal tract.
Presence and Germination of the Probiotic Bacillus subtilis DE111® in the Human Small Intestinal Tract: A Randomized, Crossover, Double-Blind, and Placebo-Controlled Study
Joan Colom1†,
Daniela Freitas2†,
Annie Simon1,
Andre Brodkorb2,
Martin Buckley3,
John Deaton4*and
Alison M. Winger1
Pharmacodynamies
Mode and extent of excretion or elimination
For substances that are systemically absorbed (or cannot be excluded), pharmacology information addressing:
•Safety pharmacology to study the effects of the substance on the following vital functions:
-Central nervous system
-Cardiovascular system
-Respiratory system
The use of Bacillus species as probiotics has gained recent interest due to the fact that spore-forming bacteria, in contrast to vegetative cells, possess a number of interesting characteristics. Specifically, spores are extremely resistant to heat and desiccation allowing Bacillus probiotics to be stored at room temperature as dried powdered formulations for extended periods. Secondly, a wide range of pH stability allows spores to survive passage through the low pH gastric barrier of the stomach resulting in the delivery of high numbers of viable microbes to the lower intestine (Cutting, 2011). Indirect introduction of Bacillus species to the food supply has occurred for decades through their use in animal production as feed additives (e.g., B. subtilis) without evidence of untoward effects on humans (Hong et al., 2005; EFSA, 2014). Probiotic preparations containing Bacillus species (e.g., B. clausii, B. coagulans, B. subtilis) have been reported to be effective in preventing gastrointestinal disorders such as childhood diarrhea when used prophylactically (Hong et al., 2005), decreasing the duration of respiratory infections in children (Marseglia et al., 2007), and overcoming symptoms associated with irritable bowel syndrome (Tompkins et al., 2010). Bacillus probiotics are currently more widely marketed in South East Asia than in Europe and the United States (U.S.), but interest in the West is increasing rapidly (Cutting, 2011).
Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans Marie Lefevre a, * , Silvia M. Racedob , Muriel Denayrolles b , Gabrielle Ripert b , Thomas Desfougeres a , Alexandra R. Lobach c , Ryan Simon c , Fanny Pelerin a , Peter Jüsten a , Maria C. Urdaci b a Lesaffre Human Care, Lesaffre Group, 137 Rue Gabriel Peri, F-59700, Marcq-en-Baroeul, France b University of Bordeaux, UMR 5248, Bordeaux Sci Agro, Gradignan, France c Intertek Scientific & Regulatory Consultancy, 2233 Argentia Rd., Suite 201, Mississauga, ON, L5N 2X7, Canada
KNOWN PHARMACODYNAMIE DRUG INTERACTIONS
Drug Interactions, Safety and Efficacy of Probiotics Khaling Mikawlrawng1*, Suresh Kumar1 and KartikiBhatnagar1
The use of probiotics has considerably increased and their potential domain of application has extended into bowel inflammatory diseases or infectious diseases, protection against diarrhea, Helicobacter pylori infection, lactose intolerance, hypercholesterolemia, systemic diseases. The clinical utility of probiotics may even further extend to fields such as allergic disease and cancer [17-20]. Due to its wide applications, it is therefore important to understand the interaction of probiotic with other drugs. Studies have recommended that administration of antibiotics and bacteria-derived probiotics be at least separated by two hours [21,22]. Saccharomyces boulardii present in probiotics might interact with antifungals, reducing the efficacy of this probiotic [23]. According to the manufacturer of Florastor, a probiotic which contains S. boulardii, it is instructed not be taken with any antibiotics and antifungals like clotrimazole (Mycelex Troche), ketoconazole (Nizoral), griseofulvin (Gris-PEG), and nystatin (Mycostatin). Probiotics should also be used cautiously in patients taking immunosuppressants, such as cyclosporine, tacrolimus, azathioprine, and chemotherapeutic agents, since probiotics could cause an infection or pathogenic colonization in immunocompromised patients [21-23]. Histamine has been reported to cause histamine intoxication, while tyramine has been reported to affect hypertensive problems in individuals who are administered with monoamine oxidase inhibitors [24-26]. Considering these outcomes, it is important to note that only amine-negative isolates are selected as probiotics, dietary adjuncts, and starter cultures [27].
TOXICOLOGICAL DATA
Information from in vitro studies, animal studies, human clinical studies or other information (or a combination) addressing:
•Maximum daily dosage
•Duration of use
•Genotoxicity
•Carcinogenicity (if continuous use of at least 6 months intended)
Repeat dose toxicity, carcinogenicity, genotoxicity, reproductive toxicity, developmental toxicity and neurotoxicity studies were not performed as it was a microbial preparation according to the delegates of the Secretary to the Department of Health Australiangood.
•Reproductive and developmental toxicity (if there are no restrictions proposed in the application that Iimit use of the substance for use in pregnant or lactating females, or in a paediatricpopulation< 18 years)
Bacillus subtilis QST 713 Supplementation during Late Gestation in Gilts Reduces Stillbirth and Increases Piglet Birth Weigh
During late gestation, the richness of microbiota in intestine of the sows reduced [1, 2] while fecal endotoxin increased [1]. Besides, an increase in gut permeability results in increased plasma endotoxin concentration in sows [1]. These changes may influence sows’ digestion, absorption, and nutrient metabolism which may affect the birth weight of piglets [3]. Recently, there is an increase in studies investigating effectsof probiotic supplementation at late gestation on reproductive performance of sows [4–8]. Some researchers have shown that sows supplemented with probiotic had larger litter sizes [5, 8], higher numbers of piglets born alive [5, 8, 9], and heavier litter weights [7, 8, 10]. Zhang et al. [5] also found that probiotic supplementation could reduce farrowing duration and birth interval.
Results Among 94 gilts enrolled in the study, 10 gilts farrowed in late evening, so were not supervised and discarded from the study. Among 84 gilts remained, 41 gilts were in the treatment and 43 gilts were in the control groups. In total, 939 piglets were born from 84 gilts. Among them 910 (96.91%) piglets were born alive, 26 (2.77%) were stillborn, and 3 (0.32%) were born as mummies. )The overall incidence of stillbirth at farrowing level was 21.43% (18/84), and the birth weight was 1269.76 ± 154.09 g. )e farrowing duration and birth interval were 167.44 ± 44.38, and 16.45 ± 9.14 minutes, respectively. Bacillus subtilis QST 713 supplementation did not influence gestation length (116.12 vs. 116.56 days), farrowing duration (174.39 vs. 160.81 minutes), birth interval (16.32 vs. 16.59 minutes), incidence of stillbirth at litter level (14.63 vs. 27.91%), SDBW (85.07 vs. 94.65 g), and CVBW (6.42 vs. 7.85%). Bacillus subtilis QST 713 supplementation increased litter size (11.85 vs. 10.67, p �0.03), number of piglets born alive (11.71 vs. 10.23, p �0.008), and the litter weight (15473.06 vs. 13174.86, p �0.002). Supplementation of Bacillus subtilis QST 713 decreased stillbirth rate (1.26 vs. 4.37%, p � 0.035) and dead born rate (1.46 vs. 4.78, p � 0.028), and increased birth weight of piglets (1303.94 vs. 1234.09, p � 0.007) (Table 2). 4. Discussion )e present study revealed that Bacillus subtilis QST 713 supplementation both decreased the stillbirth rate and increased the birth weight. Interestingly, the increased birth weight was simultaneously present with a higher litter size and higher number of piglets born alive. Moreover, despite the larger litter size, the within litter variation of piglet birth weight depicted as SDBW and CVBW in the treatment group was numerically lower than that in the control group. )The larger litter size in the treatment group in this study reflected the results of some previous studies [5, 8]. Other studies also reported a numerically higher litter size in probiotic supplementation groups in comparison with that in control groups [6, 7]. However, as discussed by Zhang et al. [5] the increased litter size in the treatment group is independent of probiotic supplementation because the litter size had already been determined before the use of probiotics. By contrast, the number of piglets born alive (NBA) which is the result of total born minus the number of piglets born dead (NBD) might be influenced by the probiotic supplementation. )The increased NBA in the treatment group was in agreement with some previous findings [5, 8, 9]. However, many other studies reported nonsignificant effect of probiotic supplementation on this criterion [4, 6, 7, 10, 14–20]. )Thepositive effect of the probiotic supplementation on NBA may be partly attributable to the fact that it reduced stillbirth rate. Previous studies demonstrated that probiotic supplementation did not reduce piglet stillbirth rate [4–9]. It has been shown that stillbirth decreased when birth weight increased [21–24]. )Therefore, the reduced stillbirth rate in the treatment group may be due to the increased birth weight (1303.9± 161.5 g vs. 1234.1± 137.3 g). It is interesting that Bacillus subtilis QST 713 increased both litter size/NBA and birth weight despite the fact that birth weight is negatively associated with litter size [25, 26]. Some previous studies failed to find any beneficial effects of probiotic supplementation at late gestation on birth weight of piglets [7–9, 20] while other studies even found a reduced birth weight in the probiotic supplementation group where litter and/or NBA increased in comparison with that in the control [5, 14]. Two previous studies found that birth weight was increased while litter sizes were unaltered when probiotic was supplemented throughout 2 successive estrus cycles [27] or late gestation [28]. However, in the former study, the positive effect of probiotic on birth weight only exhibited in the second estrus cycle [27], and in the latter one, the increased birth weight seemed to be attributable to isomaltooligosaccharide rather than probiotic [28]. It is well documented that the litter size positively correlates with within litter variation of piglet birth weight [29–31]. In other words, a larger litter size results in a larger variation of piglet birth weight. However, in the present study, the probiotic supplementation increased litter size while did not increase within litter variation of piglet birth weight. )Thisfinding can be partly explained via the increased birth weight in the treatment group because birth weight has been found negatively associated with CVBW [29]. In the present study, the effect of Bacillus subtilis QST 713 supplementation on farrowing duration and birth interval is diverse from the finding of the solely existed study that evaluated this aspect [5]. In their study Zhang et al. [5] speculated that the sows and piglets in the treatment group might be more physically stronger, therefore, the farrowing duration and birth interval were reduced. It is well established that litter sizes are positively associated with farrowing duration [26, 32], and birth weight positively correlated with birth interval [25]. )Therefore, the increased litter size and birth weight might mask any potentially beneficial effect of Bacillus subtilis QST 713 supplementation on farrowing duration and birth interval resulting in nonsignificant difference in the present study. ) The mechanism of action of Bacillus subtilis QST 713 supplementation on gilt reproductive performance is not totally clear. Previous studies showed that Bacillus subtilis improve the intestinal immune status [33], and immunological response to vaccination [34, 35]. Bacillus spp. can produce a wide range of antimicrobial substances which were found to inbibit the growth of many pathogenic bacteria such as Clostridium difficile, Campylobacter jejuni, Streptococcus pneumoniae, Campylobacter coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhi [36, 37]. ) These inhibitions might lead to lowered sera endotoxin concentration in sows supplemented with Bacillus subtilis during the late stage of gestation [5]. Tactacan et al. [11] reported that Bacillus subtilis QST 713 could also produce some antimicrobials although the names of these products were not specified. In chicken, Bacillus subtilis QST 713 stimulated the development of intestinal mucosa and villi, and the growth of Lactobacillus spp., and inhibited Escherichia coli and Enterococcus spp. [12]. In the pig, Bacillus subtilis QST 713 prevented the growth of Escherichia coli, and promoted the development of beneficial bacteria such as Bulleidia [13]. )The beneficial effects of Bacillus spp., Bacillus subtilis, and Bacillus subtilis QST 713 lead to the suggestion that supplementation of Bacillus subtilis QST 713 during the late gestation might enhance the intestinal immune status and the health of gut microbiota, increase the digestion and absorption of nutrients in treated gilts. )These promoting effects resulted in enhanced nutrient delivery to the swine fetuses which subsequently increased birth weight, and decreased stillbirth rate. )The present study had a limitation. We used commercially industrialized diets in which only some main ingredients are listed without any further information. )This led to the difficulty of formulation of such diets in the future studies. Nevertheless, the promising beneficial effect of Bacillus subtilis QST 713 on reproductive performance of gilts/sows deserves further investigation in the future.
Conclusion and Recommendations
the present study suggested that supplementation of Bacillus subtilis QST 713 at the dose of 4 ×108 cfu/meal/day from day 85 of gestation to farrowing in gilts could decrease stillbirth rate and increase the birth weight. )the increase birth weight and decreased stillbirth rate were simultaneously present with a larger litter size/NBA. Bacillus subtilis QST 713 is a promising probiotic supplement in gilts during late gestation for improvement of reproductive performance
Bacillus subtilis QST 713 Supplementation during Late Gestation in Gilts Reduces Stillbirth and Increases Piglet Birth Weigh.
LOCAL TOLERANCE
Oral administration of MB40 to male and female Crl:CD(SD) rats at dosage levels of 500, 1000, and 2000 mg/kg bw/day (equivalent to 1.71 − 2.18 × 1010 , 3.38 − 4.33 × 1010 , and 6.84 − 8.51 × 1010 CFU per day) for 14 consecutive days was well tolerated at all dosage levels tested. The no-observed-adverse-effect level (NOAEL) for MB40 after was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1010 CFU/kg bw/day), the highest dose tested.
[Article Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements Jessica L. Spears 1,*, Richard Kramer 1 , Andrey I. Nikiforov 2 , Marisa O. Rihner 2 and Elizabeth A. Lambert 3]
ADVERSE REACTIONS
Oral Toxicity Study in Rats
Oral administration of MB40 to male and female Crl:CD(SD) rats at dosage levels of 500, 1000, and 2000 mg/kg/day equating to 1.71 − 2.18 × 1010, 3.38 − 4.33 × 1010, and 6.84 − 8.51 × 1010 CFU per day for 14 consecutive days resulted in no test article-related effects at any dosage level tested. No mortality and no test article-related effects were reported for any of the evaluated parameters at any dose of MB40. There were no test article-related clinical observations. Body weights (Tables S4 and S5) and food consumption (Tables S6 and S7) were unaffected by test article administration. Some statistically significant differences in hematology and coagulation parameters were reported when the control and test article-treated groups were compared but were considered non-test-article related because they were not dose dependent and were generally within the laboratory’s historical range (Table 3). Slightly higher mean prothrombin times were noted in all test article-treated male groups and the low-dose female group, but these increases did not occur in a dose-related manner and group means were generally within the laboratory’s historical control range of study means (14.1–18.1 seconds for males and 13.7–17.5 seconds for females). Higher mean alanine aminotransferase (ALT) values were noted in all test article-treated female groups (statistically significant at 500 and 2000 mg/kg bw/day) (Table 4) but there was no dose–response relationship and group means (26 to 29 U/L) were within the laboratory’s historical control range of study means (24–71 U/L). There was no effect of treatment on urinalysis of males or females (Table 5). There were no test article-related macroscopic findings at the scheduled necropsy. All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test article administration. Higher mean adrenal gland weights (absolute and/or relative to brain weight) were noted in the test article-treated male groups at 500 and/or 2000 mg/kg bw/day (Table 6) but were considered non-test article related because of the lack of a dose–response relationship and because group means were within the laboratory’s historical control range (absolute adrenal weight 0.0406–0.0795 g; relative to brain weight: 2.1072–3.9678 g). Additionally, higher mean testes weights (absolute and relative to brain weight) were noted in the 500 mg/kg/day group males. This difference also was not considered test article related due to the lack of a dose–response relationship and a mean value within the range of historical controls (absolute testes weight: 2.59–3.87 g; relative to brain weight: 132.998–194.337 g].
Oral administration of MB40 to male and female Crl:CD(SD) rats at dosage levels of 500, 1000, and 2000 mg/kg bw/day (equivalent to 1.71 − 2.18 × 1010 , 3.38 − 4.33 × 1010 , and 6.84 − 8.51 × 1010 CFU per day) for 14 consecutive days was well tolerated at all dosage levels tested. The no-observed-adverse-effect level (NOAEL) for MB40 after was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1010 CFU/kg bw/day), the highest dose tested.
[Article Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements Jessica L. Spears 1,*, Richard Kramer 1 , Andrey I. Nikiforov 2 , Marisa O. Rihner 2 and Elizabeth A. Lambert 3]
Human Clinical Safety and Tolerability Trial
Fifty-two healthy participants were screened for this study and 30 participants were enrolled. Three total participants discontinued participation from this study after week 1 (two participants) and week 2 (one participant) due to non-compliance with test article and completion of the study forms (Figure 1). The completed participants consisted of 12 males and 15 females, with an average age of 35.3 ± 11.2 years and an average weight of 75.6 ± 15.4 kg. The overall test article compliance of the subjects that completed this study was 99.2% ± 3.3%.
There were no serious adverse events (AEs). There were five reported AEs during this
study, all graded as level 1 (scale 1–4, with 4 being the most severe), and two were ascribed
as likely related to the administration of the probiotic, neither of which required treatment (one instance of vomiting and one of chills both reported by the same subject on the same day during the middle of the treatment period and resolving within 31 h ) they were no medically clinically significant changes based on physical examinations finding ,clinicals laboratory result and vital signs.
There were no significant changes in the number of bowel movements per subject per week between the placebo week (average of 11.1 ± 4.6) and the three subsequent weeks when the test article was administered (week 2: 10.7 ± 3.6; week 3: 10.7 ± 3.8; week 4: 11.2 ± 4.3). Each participant’s Bristol stool form description was scored Type 1 (hard) through Type 7 (watery). The Bristol stool form score was consistent across all of the study weeks for each subject (average for the placebo week 1: 3.8 ± 0.1; averages for treatment weeks, week 2: 3.9 ± 0.1; week 3: 3.9 ± 0.1; week 4: 3.9 ± 0.2). The symptoms reported on the daily GI questionnaires (e.g., abdominal bloating, abdominal pain, constipation, and flatulence) were few and of low severity (graded on a scale of 1—very mild to 10—extreme) such that statistical evaluation was not feasible. The symptoms during the treatment period generally occurred with similar or lower incidence and severity compared to the placebo week. None of the symptoms reported during the treatment period had an average severity over 6; typically, the average severity was less than 4 (Table 7). Additionally, while not statistically significant, an overall decrease in the average number of symptoms reported each week and the number of subjects reporting those symptoms was observed when comparing the treatment period to the placebo week.
[Article Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements Jessica L. Spears 1,*, Richard Kramer 1 , Andrey I. Nikiforov 2 , Marisa O. Rihner 2 and Elizabeth A. Lambert 3]
Substances of human or animal origin
Information on clearance of risk for transmissible spongiform encephalopathy (TSE) if substances of human or animal origin were used during manufacture
NA.
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