The influence of Bacillus subtilis 87Y isolated from Eisenia fetida on the growth of pathogenic and probiotic microorganisms

B. subtilis 87Y strain, isolated from E. fetida decreases the growth of pathogenic Salmonella spp. and S. aureus strains and promotes the growth of probiotic Lactococcus spp. Preserving viability in acidic conditions as well as in bile salts, B. subtilis 87Y meets two of the conditions of the probiotic strain. Thanks to the production of the biosurfactant surfactin, B. subtilis 87Y limits the growth of gram-positive bacteria S. aureus. In the presence of sucrose, B. subtilis produces levan which contributes to promoting the growth of other probiotics. Our in vitro studies justify their continuation with solid state fermentation using B. subtilis 87Y solid waste as rapeseed meal to enrich it with high-value animal feed supplements.


Introduction
Solid-state fermentation (SSF) exhibits numerous advantages, including lower production energy, cost, water equipment and less waterwaste (Pandey 2003). Fermentation process improves nutritional and functional properties compared to original raw materials. Bacillus subtilis is widely used in SSF processes (Seo and Cho 2016;Singh and Bajaj 2016;Dai et al. 2017). Among them, traditional Japanese food "natto" is produced by fermentation of soybeans by B. subtilis strains (Chen et al. 2012).
B. subtilis is known Gram-positive, spore-forming bacterium. Spores of B. subtilis exhibit a wide range of tolerance properties like acid conditions or thermal tolerance (Setlow 2006)). Thus, the survival of spores in the stomach permits live bacteria to reach the intestines (Bernardeau et al. 2017). Probiotic effects of B. subtilis have been often reported (Terada et al. 2012;Yang et al. 2015), for example, the composition of Bifidobacterium in fecal flora was improved by consumption of traditional Japanese food "natto" (Terada et al. 2012). Co-culture of B. subtilis MA139 and Lactobacillus reuteri inhibited the growth of pathogenic Escherichia coli K88.
Moreover, Lactobacillus cultured alone was less toxic towards E. coli than in co-culture with B.
Bacillus spp. is also known for its ability to secrete extracellular enzymes, thus Bacillus spp.
have improving effects for the growth of probiotic bacteria (Falck et al. 2013;Horie et al. 2017).
Lactobacilli cannot use starch as a carbon source. Digesting starch into sugars by α-amylase from B. subtilis allows probiotic bacteria to utilize glucose or maltose (Horie et al. 2017). Bacillus spp. is also a great producer of lignocellulose-degrading enzymes like xylanases and cellulases.
Xylooligasaccharides received from hardwood and cereal xylans were reported to be used by Lactobacillus brevis and Bifidobacterium adolescentis (Falck et al. 2013).
In this work, we have isolated from Eisenia fetida and characterized B. subtilis 87Y strain with probiotic properties that can be used to transform solid waste (for example rapeseed meal) into the enriched animal feed.

Acid and bile salt tolerance
Acid tolerance of B. subtilis 87Y was determined according to Lee (2017), with modifications. 1 mL aliquot of the overnight culture of B. subtilis 87Y was centrifuged at 8000xg for 10 min. at room temperature. The pellets were washed in sterile PBS (100 mM, pH 7.4) and resuspended in 10 mL of sterile PBS (100 mM, pH 2.0). The bacterial suspensions were incubated at 37 ⁰C with agitation 180 rpm for 3 h. During the incubation, an A600 was measured at 1, 2 and 3 h.
Bile salt tolerance of the strain was performed similarly, an 1 mL aliquot of an overnight culture of B. subtilis 87Y was inoculated into 10 mL of LB broth containing 0.3% ox gall and incubated at 37 ⁰C for 12 h with agitation 180 rpm. During the incubation, an A600 was measured at 4, 8 and 12 h.

Antibacterial activity of SU
The antimicrobial activity of SU was determined against S. aureus ATCC 6538 by the 96-well microdilution assay, according to CLSI (2012), with modifications (Giurg et al. 2017). Here, A490 was measured using ASYS UVM 340 microplate reader (Biogenet; Poland). The viability was determined by normalizing A490 in control conditions (0 µg/mL SU) as 100%.

Co-culture on agar plates
S. aureus ATCC 6538 and B. subtilis 87Y inocula were prepared by resuspending freshly grown (18 h, LB agar) colonies in 0.9% NaCl solution to A490 = 0.125. S. aureus inoculum was streaked on LB agar plate, and B. subtilis inoculum was spotted on the agar plate. After incubation (24 h, 37 °C) the plates were photographed, using FastGene B/G GelPic imaging box (Nippon Genetics; Germany).

Co-culture insert method
The assay was performed by modifying a tissue culture protocol (Renaud and Martinoli 2016). The schematic representation of the testing conditions is given in Fig. 1. Inocula of bacteria were prepared by resuspending freshly grown (18 h, LB agar) colonies in LB to A490 = 0.1 (L compartment) or A490 = 0.4 (U compartment). After incubation (24 h, 37 °C), A490 was measured using Odyssey DR/2500 spectrophotometer (Hach, USA). The viability was determined by normalizing A490 in control conditions as 100%.

Statistical analysis
Statistical significance was determined using binomial, unpaired Student's t-test.

B. subtilis 87Y, isolated from E. fetida produces various enzymes and surfactin
Eisenia fetida is very efficient in composting organic waste and converting it into vermicompost, which is full of nutrients and with lower level of toxicants (Sharma and Garg 2018).
Among isolated microflora of E. fetida we found 96 bacteria strains. Four of 96 isolated microorganisms were sequenced by 16S RNA and assigned as B. subtilis strains.
Selected strain B. subtilis 87Y was then examined by API 20E test for basic physiology. B.
B. subtilis is known for producing various biosurfactants (Urdaci and Pinchuk 2004). Using Ultra-High Performance Liquid Chromatography (UHPLC), we have confirmed that strain B. subtilis 87Y is a great producer of lipopeptide surfactin (Fig.2). B. subtilis produces a wide range of surfactin analogues, that vary in hydrophobic as well as in hydrophilic moiety (Jajor et al. 2016). B. subtilis 87Y during cultivation on Landy's medium produced mainly analogues that differ between carbon chain length (Fig.2).

B. subtilis 87Y is viable after acid and bile salt treatment
Spores of Bacillus spp. were reported to be resistant to various conditions. Among them, acid pH and bile salt tolerance were shown in the case of B. amyloquefaciens and B. subtilis strains (Spinosa et al. 2000;Lee et al. 2017). Spinosa (2000) shows the presence of B. subtilis spores in the intestinal tract of mice. This indicates, that spores can reach intestines after acidic conditions in the stomach. Lee (2017) shows direct influence acidic pH and bile salts to B. amyloliquefaciens LN. Viability of LN strain after 3 h of acid pH, and after 12 h of bile salts treatment decrease viability of the strain about 20% and 10% respectively. We noticed similar growth of B. subtilis 87Y during 3 h incubation in both neutral and acidic conditions. (Fig. 3A). After 12 h of bile salts treatment, B.
subtilis 87Y viability decreased four-fold in comparison to control conditions (LB medium), but we observed the doubling the optical density in comparison to the initial conditions (0.3% ox gall in LB, 0 h) which indicates slow cells growth (Fig. 3B).

B. subtilis 87Y inhibits S. aureus growth due to SU production
Amphipathic compounds such as lipopeptides are highly toxic towards gram-positive bacteria (Silhavy et al. 2010), thus, gram-positive bacteria are more vulnerable towards SU (Jiang et al. 2016). We observed the inhibition of S. aureus growth, gram-positive by SU (Fig. 4A) from 20% (1 -2 µg/mL SU) to 80% (8 -16 µg/mL SU). Due to SU production (Fig. 2), an inhibition zone of S. aureus was observed in direct co-culture with B. subtilis 87Y on agar plates (Fig. 4B). However, we have noticed restricted SU diffusion through the agar [data not shown]. Restricted SU diffusion may result from restricted diffusion of non-polar compounds in the agar (Cleidson Valgas;Jenkins and Schuetz 2012).

B. subtilis 87Y and L. lactis promote their mutual growth in the presence of sucrose
Fermentation process improves nutritional and functional properties compared to original raw materials. B. subtilis is widely used in SSF processes (Seo and Cho 2016;Singh and Bajaj 2016;Dai et al. 2017).
Supplementing poultry feeding with B. subtilis leads to an increase in the number of lactic acid bacteria (LAB) in the gastrointestinal tract (Teo and Tan 2007). L. lactis stimulates the growth of broilers (Fajardo et al. 2012;Brzóska et al. 2013) and is one of the dominant LAB species isolated from faeces of broilers (Shazali et al. 2014). Here, in LB medium B. subtilis 87Y did not lead to a significant increase in L. lactis growth (Fig. 5A). However, B. subtilis 87Y was promoted by the presence of L. lactis (Fig. 5C).

Conclusions
B. subtilis 87Y strain isolated from E. fetida compost worm might be promising strain for production of fermented animal food. Following probiotic characteristics were noticed in our in vitro studies: 1. is viable in bile salts and low pH 2. inhibits S. aureus growth due to SU production 3. promotes the growth of L. lactis in the presence of sucrose 4. is promoted by the presence of L. lactis 5. inhibits the growth of Salmonella spp.