Safety Evaluation and antioxidant potential of new probiotic strain Bacillus subtilis (NMCC-path-14) in Balb/c mice by sub-acute repeated dose toxicity

Abstract

Probiotics have recently gained significant interest for their possible therapeutic effects in treating numerous health conditions. Probiotics containing Bacillus subtilis have been shown to have several health benefits, most notably in preventing diarrhea and gastrointestinal problems. A novel probiotic strain, Bacillus subtilis (NMCC-path-14), isolated from the rumen of a Nilli Ravi Buffalo, was evaluated for 28-day repeated dose toxicity in Balb/c mice. The NMCC-path-14 in low dose (1 × 10⁸ CFU/ml) and high dose (1 × 10¹⁰ CFU/ml) was administered to the mice through gavage regularly. After 28 days of treatment, it was discovered that the no-observed-adverse-effect level (NOAEL) for NMCC-path-14 wasgreater than 1 × 10¹⁰ CFU/animal/day. This study also revealed no treatment-related changes in clinical parameters, body weight, gross pathology, or histology. Food consumption, hemoglobin, hematocrit, red blood cell counts, and colon length increased, while total/differential leukocyte count and platelets remained unchanged. The administration of NMCC-path-14 also resulted in decreased bilirubin and creatinine levels. Furthermore, NMCC-path-14 also displayed a promising antioxidant potential by increasing the antioxidant enzymes (GST, GSH, and CAT) and decreasing oxidant enzyme (MDA and NO) levels in vital organs like the liver, kidneys, spleen, and colon. TheNMCC-path-14also decreased the pathogenic bacterial population while increasing the beneficial population. Given the lack of adverse effects observed after NMCC-path-14 treatment, this strain is safe and must be considered as a potential probiotic in humans.

Keywords: Antioxidant; Bacillus subtilis; Colon length; Feed intake; Probiotic safety study; Repeated dose toxicity.

Graphical abstract

Fig. 1

Effect of various doses of NMCC-path-14on a) Body Weight,b) Feed intake, and c) Relative organ weight in various groups. Each value represents mean ± S. D (n = 5), Analysed using One-way ANOVA using LSD for multiple comparisons. Where ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001: significant versus the NC. (N/S = 1 ml/day p. o given to NC group, B. subtilis 1 × 10⁸ CFU/day p. o given to BS-LD; while B. subtilis 1 × 10¹⁰ CFU/day p. o given to BS-HD) Abbreviations: Normal saline (N/S), Normal control (NC), Bacillus subtilis low dose (BS-LD), Bacillus subtilis high dose (BS-HD).

Fig. 2

Effect of various doses of NMCC-path-14on a) Gross morphology of liver, kidneys, and spleen and b) Gross morphology of colon along with changes in length in various groups. Each value represents mean ± S. D (n = 5), Analysed using One-way ANOVA using LSD for multiple comparisons. Where ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001: significant versus the NC(N/S = 1 ml/day p. o given to NC group, B. subtilis 1 × 10⁸ CFU/day p. o given to BS-LD; while B. subtilis 1 × 10¹⁰ CFU/day p. o given to BS-HD) Abbreviations: Normal saline (N/S), Normal control (NC), Bacillus subtilis low dose (BS-LD), Bacillus subtilis high dose (BS-HD).

Fig. 3

Histopathological analysis of the liver, kidneys, spleen, and colon after sub-acute toxicity test treated with different doses (1 × 10⁸ CFU/day p. o given to BS-LD; while1 x 10¹⁰ CFU/day p. o given to BS-HD) of NMCC-path-14inFemale Balb/c mice (40× magnification, scale bar: 20 for liver, kidneys and colon while 4× magnification, scale bar 200 was used for spleen). (In series 1: liver, where CV central vein, K kupffer cells, H hepatocytes, S sinusoids. In series 2: kidneys, where B Bowman's capsule, G glomerulus, PCT proximal convoluted tubule, DCT distal convoluted tubule. In series 3: spleen, WP white pulp, RPred pulp. In series 4: colon, G goblet cells, C crypt, LP lamina propria).

Fig. 4

Effect of various doses of NMCC-path-14ona) GST, b) GSH, c) CAT, d) MDA, and e) NO levels in liver, kidneys, spleen, and colon homogenates. Each value represents mean ± S. D (n = 5), Analysed using One-way ANOVA using LSD for multiple comparisons. Where ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001: significant versus the NC, ^#p < 0.05,^##p < 0.01 and^###p < 0.001: significant versus the BS-LD.(N/S = 1 ml/day p. o given to NC group, B. subtilis 1 × 10⁸ CFU/day p. o given to BS-LD; while B. subtilis 1 × 10¹⁰ CFU/day p. o given to BS-HD) Abbreviations: Normal saline (N/S), Normal control (NC), Bacillus subtilis low dose (BS-LD), Bacillus subtilis high dose (BS-HD).

Fig. 5

Microbiological analysis of fecal content of the animals receiving NMCC-path-14. Each value represents mean ± S. D (n = 5), Analysed using One-way ANOVA using LSD for multiple comparisons. Where ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001: significant versus the NC, ^#p < 0.05,^##p < 0.01 and^###p < 0.001: significant versus the BS-LD.(N/S = 1 ml/day p. o given to NC group, B. subtilis 1 × 10⁸ CFU/day p. o given to BS-LD; while B. subtilis 1 × 10¹⁰ CFU/day p. o given to BS-HD) Abbreviations: Normal saline (N/S), Normal control (NC), Bacillus subtilis low dose (BS-LD), Bacillus subtilis high dose (BS-HD).