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In vitro evaluation of Bacillus licheniformis and Bacillus subtilis enzyme activity, Clostridium perfringens Type A inhibition, and biofilm formation.

A. Segura

Events

06-23-2020

Abstract:

233
In vitro evaluation of Bacillus licheniformis and Bacillus subtilis enzyme activity, Clostridium perfringens Type A inhibition, and biofilm formation.
A. Segura*1, N. Milora1, O. Queiroz1, M. D. Cantor2, G. Copani1. 1Animal Health and Nutrition, Chr. Hansen A/S H�rsholm, Denmark, 2R&D Discovery, Chr. Hansen A/S H�rsholm, Denmark.

The use of Bacillus as direct-fed microbials (DFM) has been investigated due to their ability to produce antimicrobials and synthetize enzymes. In ruminants, Bacillus supplementation has been reported to improve performance and limit diseases. The aim of this study was to investigate (1) the enzymatic activity profile of 2 Bacillus strains (B. licheniformis [BL], DSM 5749, and B. subtilis [BS], DSM 5750), (2) their inhibitory capacity toward Clostridium perfringens Type A (CPa, DSM 756), and (3) their biofilm synthesis capacity. Enzyme activity was measured qualitatively with insoluble chromogenic substrates and on milk agar for protease activity. CPa inhibition was evaluated qualitatively by using an agar diffusion assay. In addition, the Ankom RF Gas Production System was used to measure gas production of CPa with or without BL or BS incubated in a Brain Heart Infusion (BHI) broth (37�C, 24h, in triplicate). Biofilm formation was assessed by crystal violet staining after overnight incubation in BHI broth at 37�C. Statistical analysis was performed using the one-way ANOVA with the Dunnett's post-hoc test (GraphPad Prism). Table 1 reports the enzyme activities and shows that Bacillus differed in their capacity to produce enzymes. BL and BS were able to generate similar inhibition zones against CPa. The quantitative method also revealed that BL and BS significantly reduced CPa gas production (20%, P < 0.001, and 12%, P < 0.01, respectively). Finally, BL appeared to be a strong biofilm producer (OD595nm > 2) while BS weakly produced biofilm (OD595nm < 1). The results show that the enzyme activity, and the ability to produce antimicrobials and biofilm appear to be specific features for each Bacillus. In vivo studies need to be conducted to test if BL and BS could enhance ruminant performance and health.Table 1. Bacillus licheniformis and B. subtilis enzymatic activity profile1

SpeciesProteaseCellulaseAmylaseXylanaseGalactomannanaseGalactanaseX-gal
B. licheniformisxx(x)(x)x(x)(x)
B. subtilisxxxxxxxx(x)
1Where (x), x, and xx represent increasing zones of clearance/more enzyme activity; — = none.

Keywords: Bacillus, direct-fed microbial (DFM), ruminant.