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

Bacillus subtilis strain 87Y, isolated from the earthworm Eisenia fetida, decreases the growth of pathogenic Salmonella spp. and Staphylococcus aureus 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 requirements of a probiotic strain. Thanks to the production of the biosurfactant surfactin, B. subtilis 87Y limits the growth of the Gram-positive bacterium 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 the continuation of enriching rapeseed meal waste from solid-state fermentation with B. subtilis 87Y, to produce high-value animal feed supplements. Graphical abstract Graphical abstract


Introduction
Solid-state fermentation (SSF) exhibits numerous advantages over submerged fermentation, including lower production energy, improved cost and water equipment efficiency, and less wastewater [1]. The fermentation process improves the nutritional and functional properties of the original raw materials. Bacillus subtilis is widely used in the SSF processes [2][3][4]. For example, the traditional Japanese food "natto" is produced through fermentation of soybeans by B. subtilis strains [5].
B. subtilis is a Gram-positive, spore-forming bacterium. Spores of B. subtilis exhibit tolerance to a wide range of conditions, including acidic conditions and thermal stress [6]. Spores are thereby able to survive within the stomach, permitting live bacteria to reach the intestines [7]. Probiotic effects of B. subtilis have frequently been reported [8,9], for example, improvements in the composition of Bifidobacterium in the faecal flora following consumption of the traditional Japanese food "natto" [8]. 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. subtilis spores [9].
Bacillus spp. are known for their ability to secrete extracellular enzymes, which may improve the growth of probiotic bacteria [10,11]. For example, Lactobacilli cannot use starch as a carbon source, yet the metabolism of starch into sugars by α-amylase secreted by B. subtilis allows probiotic bacteria to utilize glucose or maltose [11]. Bacillus spp. are also prolific producers of lignocellulose-degrading enzymes including xylanases and cellulases. Xylooligasaccharides metabolized from hardwood and cereal xylans are reported to be utilized by Lactobacillus brevis and Bifidobacterium adolescentis [10].
In this work, we isolate and characterize a B. subtilis strain, 87Y, from earthworm Eisenia fetida, as having probiotic properties that can be used to transform solid waste (for example, rapeseed meal) into enriched animal feed.

Strains and growth conditions
Bacillus subtilis 87Y was isolated from heat-treated samples of Eisenia fetida gut microflora. The enzymatic activities of the isolate were characterized with an analytical profile index (API) 20E test. Species assignment was performed by analysing 16S rRNA sequences.

Acid and bile salt tolerance
Acid tolerance of B. subtilis 87Y was determined according to Lee et al. [22], with following modifications: 1 mL aliquot of the overnight culture of B. subtilis 87Y was centrifuged at 8000×g 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°C with agitation 180 rpm for 3 h. During the incubation, A 600 was measured at 1, 2, and 3 h.
Bile salt tolerance of the strain was performed similarly, with following modifications: a 1 mL aliquot of an overnight culture of B. subtilis 87Y was inoculated in 10 mL of LB broth containing 0.3% ox gall and incubated at 37°C for 12 h with agitation at 180 rpm. During the incubation, A 600 was measured at 4, 8, and 12 h.

Antibacterial activity of SU
The antimicrobial activity of SU was determined against S. aureus ATCC 6538 with a 96-well microdilution assay, according to Clinical and Laboratory Standards Institute (CLSI) ( [25]), with modifications introduced by Giurg et al. [26]. Here, the plates were inoculated to a starting A 490 = 0.01. After 24-h incubation at 37°C, A 490 was measured using an ASYS UVM 340 microplate reader (Biogenet; Poland). The viability was determined by normalizing A 490 to 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 A 490 = 0.125. S. aureus inoculum was streaked onto an LB agar plate, and B. subtilis inoculum was spotted on an agar plate. After incubation (24 h, 37°C), the plates were photographed using a FastGene B/G GelPic imaging box (Nippon Genetics; Germany).

Co-culture insert method
The assay was performed with a modified tissue culture protocol [27]. A 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 A 490 = 0.1 (L compartment) or A 490 = 0.4 (M or U compartment). Following co-culture inserts have been used, 24-well 0.4 μm (Greiner Bio-one, Austria) between U and L compartments ( Fig. 1a) and between U and M compartments (Fig. 1b); 12-well 0.4 μm (Greiner Bio-one, Austria) between L and M compartment (Fig. 1b). Twenty-four or 12-well sterile plates (Greiner Bio-one, Austria) have been used for the culture (Fig.  1a, b, respectively). After incubation (24 h at 37°C), A 490 was measured using an Odyssey DR/2500 spectrophotometer (Hach, USA). The viability was determined by normalizing A 490 under control conditions as 100%.

Statistical analysis
Statistical significance was determined using a binomial, unpaired Student t test.

fetida produces various enzymes and surfactin
For the present study, we have chosen B. subtilis 87Y strain, which has been isolated from the earthworm Eisenia fetida microflora. E. fetida is very efficient at composting organic waste and converting it into vermicompost, which is full of nutrients and has a lower level of toxicants [28]. Thus, we expected the B. subtilis 87Y strain to possess enzymatic properties, which in turn may lead to production of highly additive secondary metabolites. B. subtilis 87Y was examined using a API 20E test to determine basic physiology and enzymatic properties [29]. B. subtilis 87Y was positive for betagalactosidase; acetoin production; gelatin utilization; and fermentation of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose. Conversely, it was negative for arginine dihydrolase, lysine decarboxylase, ornitin decarboxylase, citrate utilization, hydrogen disulfide production, urease production, tryptophane deaminase, and indol production.
B. subtilis is known for producing various biosurfactants [14]. Using UHPLC, we have confirmed that B. subtilis 87Y is a great producer of lipopeptide surfactin (Fig. 2). Depending on the culture media used, B. subtilis produces a wide range of surfactin analogues that vary in hydrophobic and in hydrophilic moieties [23] (Table 1). During cultivation on Landy's rich medium, containing amino acids, B. subtilis 87Y produced many analogues (Fig. 2b) which were also found in the surfactin standard (Fig. 2c). In turn, mainly C13-C16 analogues were identified on basic LB medium (Fig. 2a).

B. subtilis 87Y is viable after acid and bile salt treatment
Spores of Bacillus spp. have been reported to be resistant to a broad range of environmental conditions. Among them, acid pH and bile salt tolerance were shown in the case of B. amyloquefaciens and B. subtilis [22,30]. Spinosa et al. [30] showed the presence of B. subtilis spores in the intestinal tract of mice. This indicates that spores can reach the intestine in a viable state after withstanding acidic conditions in the stomach. Lee et al. [22] showed a direct influence of acidic pH and bile salts on B. amyloliquefaciens LN growth. Viability of the LN strain after 3 h of acid pH, and after 12 h of bile salt treatment, decreased by approximately 20% and 10%, respectively. We noticed similar growth of B. subtilis 87Y during 3-h incubations in both neutral and acidic conditions (Fig. 3a). After 12 h of bile salts treatment, B. subtilis 87Y viability decreased fourfold in comparison with control conditions (LB medium), but we observed the doubling of optical density in comparison with the initial conditions (0.3% ox gall in LB, 0 h) which indicates slow cell growth (Fig. 3b).

B. subtilis 87Y inhibits S. aureus growth through SU production
Amphipathic compounds such as lipopeptides are highly toxic towards Gram-positive bacteria [31]; thus, Grampositive bacteria are more vulnerable to SU [32]. We observed inhibition of S. aureus growth by SU (Fig. 4a), ranging 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 on co-culture with B. subtilis 87Y on agar plates (Fig. 4b). However, we 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 [33,34].
In subsequent experiments, we applied the direct coculture insert method (Fig. 1a), which was validated by testing the influence of SU and B. subtilis 87Y on the growth of S. aureus (Fig. 4c). At 4 μg/mL, SU inhibited the growth of S. aureus by 50% (Fig. 4c), which is in agreement with results obtained with the modified CLSI method (Fig. 4a). B. subtilis 87Y inhibited the growth of S. aureus by 20% ( Fig. 4c) while S. aureus did not influence the growth of B. subtilis 87Y (data not shown). Table 1 Surfactin analogues identified in the SU standard and in the cultivation broths (Fig. 2). (+)-peak found, (ND)-peak not detected  The fermentation process provides improved nutritional and functional properties compared with the original raw materials. B. subtilis is widely used in SSF processes [3,4,35]. Supplementing poultry feed with B. subtilis leads to an increase in the number of lactic acid bacteria (LAB) in the gastrointestinal tract [36]. L. lactis stimulates the growth of broilers [37,38] and is one of the dominant LAB species isolated from the faeces of broilers [39]. In this study, in LB medium, B. subtilis 87Y did not stimulate a significant increase in L. lactis growth (Fig. 5a). However, B. subtilis 87Y growth was promoted by the presence of L. lactis (Fig. 5c).
Al-Hijazeen and Al-Rabadi [40] reported that a sucroserich diet positively affected the quality of broilers' meat.
Here, the addition of sucrose resulted in~10% growth promotion in case of B. subtilis 87Y and~14, 26, or 52% promotion of L. lactis, ATCC 11454, ATCC 19435, and ATCC 49032, respectively. In co-cultures, the significant promotion of the growth of two L. lactis strains by B. subtilis 87Y was also observed (Fig. 5b). Most likely, this effect occurred since sucrose is a substrate for the synthesis of levan, a prebiotic polymer produced by B. subtilis [41]. The growth of B. subtilis 87Y increased by~10% in the presence of L. lactis (Fig. 5c). The lower level promotion of B. subtilis 87Y growth on LB + 5% sucrose by L. lactis, in comparison with growth on LB might result from stronger L. lactis growth under those conditions (Fig. 5b). 3.5 B. subtilis 87Y and L. lactis inhibit Salmonella spp.
B. subtilis has been reported to inhibit the growth of pathogenic Gram-negative bacteria in poultry feeding [42,43]. In this study, both B. subtilis 87Y or L. lactis strains inhibited S. enteritidis and S. typhimurium (Fig. 6a). The effect of probiotic bacteria was similar, regardless of the Salmonella strain or species. B. subtilis 87Y displayed the greatest inhibitory activity (~40% growth inhibition); whereas L. lactis ATCC 49032 and ATCC 11454 showed lower inhibitory activity (~20% growth inhibition).
The addition of sucrose resulted in significant inhibition of the growth of S. enteritidis ATCC 13076 by both B. subtilis 87Y and L. lactis (Fig. 6b). S. enteritidis ATCC 49223 and S. typhimurium ATCC 14028 were inhibited to a lesser extent than when cultured without sucrose (Fig. 6a). The reduction in percentage growth of both Salmonella spp. strains were still significant in the case of co-culture with B. subtilis 87Y or L. lactis ATCC 19435 (Fig. 6b).
Yang et al. [9] reported that the combination of Lactobacillus spp. and B. subtilis had a higher inhibitory effect towards E. coli than Lactobacillus spp. alone. Here, we The % of growth of S. aureus (L compartment) untreated (control), in the presence of B. subtilis 87Y (U compartment, here: + 87Y) or with 4 μg/ mL SU (U compartment, here: + SU), n = 6 ± SD. Statistical analysis was performed comparing control conditions and + 87Y and + SU conditions (*P < 0.05; **P < 0.01; ***P < 0.001) applied a triple co-culture insert method (Fig. 1b) to check the effect of the presence of B. subtilis 87Y and L. lactis on Salmonella growth (Fig. 7).
For the experiment, we used L. lactis ATCC 19435 as the strain demonstrating the greatest inhibitory effect on Salmonella spp. (Fig. 6) among other L. lactis strains, as well being promoted at the highest rate by B. subtilis 87Y (Fig. 5). S. typhimurium ATCC 14028 was chosen due to its wide animal host range, including poultry, cattle, and pigs [44]. S. enteritidis is associated mostly with poultry [44].
We o b s e r v e d a~2 0 % g r o w t h i n h i b i t i o n o f S. typhimurium when co-cultured with B. subtilis 87Y and L. lactis, regardless of sucrose concentration (Fig.  7a), which was similar across strains (Fig. 6). However, the promotion rate of L. lactis (Fig. 7b) in triple inserts was much higher than in the case of co-culture with B. subtilis 87Y alone (Fig. 5). This effect most likely occurred due to the presence of S. typhimurium, which is in agreement with data reported by Denkova et al. [45]. During co-culture with Lactobacillus acidophilus and Salmonella spp., an increase in viable L. acidophilus cells was observed [45].
The external B. subtilis 87Y supplementation should not lead to B. subtilis overgrowth in animal guts. Thus, we examined the viability of B. subtilis 87Y in coculture with L. lactis and S. typhimurium, which decreased by~30 or 10% on LB, and LB + 5% sucrose, respectively (Fig. 7c). The reason for this may have been interspecies competition for nutrients. B. subtilis 87 Y and L. lactis were grown in compartment U and Salmonella spp. in compartment L. Statistical analysis was performed using control conditions as reference values (*P < 0.05; **P < 0.01; ***P < 0.001)

Conclusions
B. subtilis strain 87Y, which can be isolated from E. fetida (earthworm), may be a promising strain for use in the production of fermented animal food. The following probiotic characteristics were noticed in our in vitro studies: (1) B. subtilis 87Y produces surfactin and a variety of enzymes, which may contribute to improving gastrointestinal digestion and nutrient assimilation.
(2) B. subtilis 87Y would be able to survive gastrointestinal conditions due to its viability under low pH and bile salts.
(3) B. subtilis 87Y promotes the growth of L. lactis in the presence of sucrose and is promoted by the presence of L. lactis.
(4) B. subtilis 87Y inhibits the growth of Salmonella spp. directly and indirectly through the promotion of L. lactis.
Funding information This work was partially supported by POIR grant 01.02.00-00-0064/17 and Wrocław Center for Biotechnology (WCB) the Leading National Research Centre (KNOW).
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