Exploring the hemicellulolytic properties and safety of Bacillus paralicheniformis as stepping stone in the use of new fibrolytic beneficial microbes

Abstract

Bacillus strains from the Moroccan Coordinated Collections of Microorganisms (CCMM) were characterised and tested for fibrolytic function and safety properties that would be beneficial for maintaining intestinal homeostasis, and recommend beneficial microbes in the field of health promotion research. Forty strains were investigated for their fibrolytic activities towards complex purified polysaccharides and natural fibres representative of dietary fibres (DFs) entering the colon for digestion. We demonstrated hemicellulolytic activities for nine strains of Bacillus aerius, re-identified as Bacillus paralicheniformis and Bacillus licheniformis, using xylan, xyloglucan or lichenan as purified polysaccharides, and orange, apple and carrot natural fibres, with strain- and substrate-dependent production of glycoside hydrolases (GHs). Our combined methods, based on enzymatic assays, secretome, and genome analyses, highlighted the hemicellulolytic activities of B. paralicheniformis and the secretion of specific glycoside hydrolases, in particular xylanases, compared to B. licheniformis. Genomic features of these strains revealed a complete set of GH genes dedicated to the degradation of various polysaccharides from DFs, including cellulose, hemicellulose and pectin, which may confer on the strains the ability to digest a variety of DFs. Preliminary experiments on the safety and immunomodulatory properties of B. paralicheniformis fibrolytic strains were evaluated in light of applications as beneficial microbes' candidates for health improvement. B. paralicheniformis CCMM B969 was therefore proposed as a new fibrolytic beneficial microbe candidate.

Figure 1

Detection of lichenase and xylanase activities of Bacillus CCMM strains grown in BSS agar medium supplemented with 0.5% (w/v) lichenan or xylan for 48 h at 37 °C. Congo red staining revealed clear halos around the streaks in the presence of enzymatic activities.

(a) Neighbor-joining phylogenetic tree based on 16S rRNA sequences of Bacillus isolates. The phylogenetic tree was inferred using the Neighbor-Joining method. The percentage of replicate trees in which associated taxa clustered in the bootstrap test (1000 replicates) is shown next to the branches (bootstrap values ≥ 60). Evolutionary analyses were conducted in MEGA X. The bar indicates 0.0020 substitution per site. rRNA gene sequences are presented with the corresponding species name and strain, or with the CCMM reference. (b) Multi Locus Sequence Typing (MLST) analysis of B. licheniformis and B. paralicheniformis strains. The phylogenetic tree was generated in iTOL v4 integrated into the PubMLST database, based on the internal fragments of six housekeeping genes. Seven Bacillus CCMM strains were submitted to the database. Bacillus isolates are presented with the corresponding species and strain names.

Figure 3

Detection of xylanase, xyloglucanase and lichenase activities of Bacillus CCMM strains, using agar-well plate assays with concentrated extracellular proteins from cultures in BSS medium and 0.5% natural substrates. Agar plate BSS medium was supplemented with 0.1% (w/v) lichenan, xylan or xyloglucan; Plates were incubated overnight at 37 °C. Clear and blue halos around the wells indicate a positive sample for the corresponding glycoside hydrolase activity.

Figure 4

Enzymatic activity of extracellular proteins of B. paralicheniformis CCMM strains grown in BSS medium supplemented with carrot, apple and orange peels. Activity was measured by quantification of reducing sugars released after enzyme incubations with lichenan or xylan substrate. One international unit (IU) was defined as the amount of enzyme which produced 1 µmol of equivalent glucose or xylose in 1 min.

Overview of GH families and their predicted target substrates from B. licheniformis (CCMM B943) and B. paralicheniformis (CCMM B940, B951, B774, and B969) genomes. Assignment of ORFs to GH families was done according to the CAZy database (http://www.cazy.org/).

Figure 6

(a) Heat map comparison, (b) Principal Component Analysis of expression patterns, and (c) Abundance of selected CAZymes detected in the secretomes of B. paralicheniformis CCMM969 during growth on different carbon sources. Groups with different letters exhibit significantly different growth rates at a 95% confidence interval according to a Kruskhal–Wallis rank sum test, followed by a Dunn posthoc pairwise comparison test.

Figure 7

(a) Scanning electron microscopy (FEG-SEM) of natural fibres without bacteria, and (b, c) after incubation with B. paralicheniformis CCMM B951 for 48 h at 37 °C. Observations were made at different magnifications. Photos @INRAE—Thierry Meylheuc, ISC MIMA2, MICALIS, INRAE Jouy-en-Josas, France.

Figure 8

Effect of B. paralicheniformis CCMM strains on (a) NF-κB activity in HT29 cells. NF-κB activation was measured by SEAP secretion and expressed as mean ± SEM fold change toward unstimulated cells. Data represent mean ± SD (n ≥ 3). The difference from the RPMI control group was analysed by t test; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 was considered as not significant. (b) Barrier integrity of Caco-2 cells. TEER was measured before adding bacterial suspensions at a ratio of 40 bacteria per cell. TEER was measured a second time at the end of the 24 h of coincubation.