Biological degradation of aflatoxin M1 by Bacillus pumilus E‐1‐1‐1

Abstract Aflatoxin M1 (AFM1) is a potent mycotoxin which causes serious health concerns in developing countries, where it is mainly found in milk, meat, and other foods. Biological detoxification is a promising method for eliminating AFM1. The aim of this work was to search for AFM1‐degrading bacterial strains from animal waste, soil, and activated sludge. High‐performance liquid chromatography and Fourier‐transform infrared spectroscopy were used to analyze the AFM1 degradation products. A strain designated E‐1‐1‐1 was obtained from African elephants feces, with the degradation ratio of AFM1 reaching 89.55% in 12 hr. Based on morphology, physiological and biochemical tests, and 16S rRNA gene sequence analysis, strain E‐1‐1‐1 was identified as Bacillus pumilus. The culture supernatant of B. pumilus E‐1‐1‐1 degraded AFM1 effectively, whereas the cells and cell extracts of B. pumilus E‐1‐1‐1 were far less effective. Carbon and nitrogen sources had highly significant effects on the degradation of AFM1 by B. pumilus E‐1‐1‐1. The AFM1‐degrading strain, B. pumilus E1‐1‐1, could have great potential in industrial applications.

Strategies for eliminating aflatoxins in contaminated commodities include physical, chemical, and biological approaches. Various chemical and physical methods have been developed for removal of AFM 1 . However, these methods have many weaknesses, such as the high cost, the loss of nutrition and so on. Biological degradation of aflatoxins is an efficient, specific, and environmentally friendly approach to reduce or eliminate the aflatoxins in foods and feeds (Wu et al., 2009). Many researchers are entering the field of biodetoxification, and furthermore, the process of biological detoxification is considered a promising method for eliminating AFM 1 (Jebali et al., 2015;Kabak & Var, 2004).
In this study, an AFM 1 -degrading strain, Bacillus pumilus E1-1-1, was isolated from feces samples of African elephants. As far as we know, this is the first study to report an AFM 1 -degrading strain of B. pumilus.

| Chemicals and media
AFM 1 was obtained from Fermentek Corporation (Israel). The DNA purification kit and LA Taq DNA polymerase were purchased from TaKaRa (Otsu, Japan). The DNA Mini kit was purchased from Omega Bio-Tek (Norcross, GA). Coumarin medium (CM) included (per L): beef extract 3 g, NaCl 10 g, glucose 6 g, and peptone 5 g.

| Isolation of AFM 1 -degrading strains
Forty-two samples were collected from animal waste, soil, and activated sludge in Baoding (Hebei, China). Five grams of each sample were individually cultured in 50 ml sterilized CM at 37°C for 72 hr for the enrichment and isolation of AFM 1 -degrading strains (Guan et al., 2008). Single colonies were chosen for further research.
The strains obtained from preliminary screening were transferred to second screening medium and incubated at 37°C for 24 hr.
The fermentation liquid (0.9 ml) and AFM 1 (0.1 ml, 400 ng/ml) were added to sterile tubes. After incubation at 37°C for 72 hr, residual AFM 1 was measured according to the standard method. The strains that had the capacity of degrading AFM 1 were target strains. All strains were stored at −80°C before use. A strain which showed the greatest ability to degrade AFM1 was selected for further analysis.

| Identification of the isolates
Biochemical analysis, physiological tests, and 16S rRNA gene sequence analysis were carried out to identify the isolate. The Biolog automated bacterial identification system was utilized to test the general physiological and biochemical characteristics of the strain.
Genomic DNA of the strain was extracted using the method as described previously (Hesham, 2014). A universal primer set consisting of 27F and 1492R was used to amplify the 16S rRNA gene (Lane, 1991;Topp et al., 2000). The nucleotide sequence was determined by direct sequencing and compared with available 16S rRNA gene sequences in the GenBank database using the BLAST program (National Library of Medicine, Bethesda, MD). A phylogenetic tree was reconstructed using MEGA 5.0 software (Kumar, Nei, Dudley, & Tamura, 2008).

| Degradation of AFM 1 by strain E-1-1-1 in liquid cultures
The isolated strain was cultured in Luria-Broth medium (LB) containing AFM 1 (400 ng/ml) at 37°C for 14 hr. The AFM 1 degradation experiment was carried out by inoculating 900 μl of the culture in 24 ml LB containing 100 μl AFM 1 (400 ng/ml), final concentration of 40 ng/ml, and subsequently incubating at 37°C for 0, 2, 4, 6, 8, 10, and 12 hr. The AFM 1 residue in the medium was determined by highperformance liquid chromatography (HPLC). Sterile LB containing AFM 1 was used as a control.

| AFM 1 degradation by cell-free supernatant, cells and cell extracts
Strain E-1-1-1 was incubated at 37°C for 24 hr in LB medium. The induced cultures were centrifuged at 12,000 g for 20 min at 4°C to collect cells. The cells were washed three times with phosphate buffer (pH 7.0). The cells and heat-killed cells were incubated with AFM 1 (100 μl, 400 ng/ml) at 37°C for 12 hr, respectively. Residual AFM 1 was measured by HPLC.
For analysis of cell extracts, strain E-1-1-1 was incubated at 37°C for 24 hr in the LB medium. The cultures were centrifuged at 4°C, 12,000 g for 20 min to collect cells. The cells were washed three times in phosphate buffer (pH 7.0), and then disrupted by ultrasonic vibration for 10 min. The intracellular extracts (900 μl) were incubated with AFM 1 (100 μl, 400 ng/ml) at 37°C for 12 hr, and residual AFM 1 was measured by HPLC.
For analysis of cell-free supernatants, strain E-1-1-1 was incubated at 37°C for 24 hr in the LB medium. The cultures were centrifuged at 4°C, 12,000 g for 20 min to remove cells and undissolved materials. The cell-free supernatants (900 μl) were incubated with AFM 1 (100 μl, 400 ng/ml) at 37°C for 12 hr, and residual AFM 1 was measured by HPLC. Sterile LB with AFM 1 (100 μl, 400 ng/ml) was used as a control.

| Effect of carbon and nitrogen sources on degradation of AFM 1
After cultivation of strain E-1-1-1 in LBC medium or LBN medium at 37°C for 14 hr, the cell supernatant was collected by centrifugation (6000 g, 10 min, 4°C) and tested for AFM 1 degradation ratio in different nitrogen (N) sources and different carbon (C) sources. The LBC medium was supplemented with yeast extract (5 g/L), tryptone (10 g/L), NaCl (5 g/L), and different carbon sources: glucose, fructose, sucrose, maltose, lactose, soluble starch, and trehalose at a final concentration of 6 g/L. The LBN medium was supplemented with glucose (6 g/L), NaCl (5 g/L), and different nitrogen sources in place of yeast extract and tryptone: peptone, yeast extract, beef extract, tryptone, NH 4 NO 3 , casein and Mix N (tryptone:NH 4 NO 3 , 1:1) at a final concentration of 10 g/L.

| Isolation of AFM 1 degradation products
Before analysis by HPLC, the samples were extracted: 900 μl of sample was incubated with 100 μl AFM 1 in sterile tubes at 37°C for 12 hr; in the control, sterile water with AFM 1 (100 μl, 400 ng/ ml) was used. Five milliliters of methanol-water (70:30, v/v) was added to the sample tubes and was extracted three times with chloroform (5 ml). The lower layer fluid was collected and dried by rotary evaporation at 35°C. The dried sample was dissolved in 1 ml acetonitrile solvent.

| HPLC analysis of AFM 1 determination
Before analysis by HPLC, the samples were extracted: 900 μl of sample was incubated with 100 μl AFM 1 in sterile tubes at 37°C for 12 hr; in the control, sterile water with AFM 1 (100 μl, 400 ng/ml) was used. Five milliliters of methanol-water (70:30, v/v) was added to the sample tubes and was extracted three times with chloroform (5 ml). The detoxification ratio of AFM 1 was calculated using the following formula: Y = (1 − X 1 /X 2 ) × 100%, where X 1 is the AFM 1 content in the treated group, X 2 is the AFM 1 content in the negative control, and Y is the detoxification ratio

| Statistical analysis
All assays in this study were carried out in triplicate. Data analysis was performed using SAS software (SAS Institute Inc., Gary, NC).

| Screening of AFM 1 -degrading strains
In this research, 68 strains showing AFM 1 degradation were isolated from the 42 samples. These strains could grow well in CM (Table 1).
Forty-seven strains were isolated from animal feces, which had many intestinal floras that could decompose feed; eight strains were isolated from sludge; and 13 strains were isolated from soil. The strains obtained were used to analyze the detoxification ratio of AFM 1 . Ten isolates showed >60% detoxification ratio of AFM 1 ( Table 2). The strain E-1-1-1 showed the strongest ability to degrade AFM 1 , with an AFM 1 degradation percentage of 89.55%. Therefore, strain E-1-1-1 was chosen for further study.

| Identification of strain E-1-1-1
Colonies of strain E-1-1-1 were round, small and ivory-colored, the fringe was trim, and the surface was wet. Cells of strain E-1-1-1 were short pole and obtuse round under the microscope (Figure 1a). Gram staining was positive. According to physiological and biochemical tests, strain E-1-1-1 was most similar to B. pumilus (Table 3). The online BLAST search of the 16S rRNA gene sequence of strain E-1-1-1 showed that strain E-1-1-1 exhibited 99% similarity to B. pumilus. A phylogenetic tree was reconstructed with the 16S rRNA gene sequence of strain E-1-1-1 (Figure 1b), and strain E-1-1-1 was included

| AFM 1 degradation by B. pumilus
AFM 1 degradation by strain E-1-1-1 after different time intervals was studied ( Figure 2). The result showed the AFM 1 degradation ratio increased with incubation time. The degradation ratio of AFM 1 could reach 50% in the first 2 hr, while the maximum value of degradation ratio (89.55%) was reached after 12 hr.
The culture supernatant of strain E-1-1-1 could degrade 76.9% AFM 1 compared to <5% and 10% by cell and cell extracts, respectively ( Figure 3). These results showed that the detoxification mechanism of strain E-1-1-1 was degradation rather than adsorption.

| Effect of C source and N source on AFM 1 degradation
To determine the effect of C and N sources on AFM 1 degradation, strain E-1-1-1 was cultured in LBC medium or LBN medium. The resulting data are shown in Figure 4. Seven different N sources were tested. In general, higher AFM 1 degradation ratios were observed in media with N sources of peptone, yeast extract, beef extract, tryptone, and casein. Biomass was also strongly influenced by N sources; yeast extract, beef extract, tryptone, and casein could significantly promote the biomass of strain E-1-1-1. When strain E-1-1-1 was cultured in media containing seven different C sources, the highest AFM 1 degradation ratio was observed in media with the C source of maltose. Based on these results, maltose as C source and yeast extract as N source were selected as cultural conditions for production. F I G U R E 1 Phylogenetic relationship between strain E-1-1-1 and other strains based on 16S rRNA gene sequence analysis (a) Colonial and morphology of strain E-1-1-1. (b) Phylogenetic tree of strain E-1-1-1 based on 16S rRNA sequence.

Bacillus thuringiensis strain ATCC 10792
Bacillus cellulosilyticus strain DSM 2522  microorganisms in animal feces, soils, and other environments can be chosen as sources for the selection of microorganisms that degrade aflatoxins (Chang & Lynd, 1970).
Due to short degradation time and nonpigmentation in foods, microbial degradation is preferred in the food and feed industry (Teniola et al., 2005). Scientific reports have shown that, to date, numerous microorganisms are capable of degrading aflatoxins (Adebo, Njobeh, Gbashi, Nwinyi, & Mavumengwanal, 2015). These bacterial species include Nocardia corynebacteroides, Corynebacterium rubrum, and Rhodococcus spp. (Ciegler, Lillehof, Peterson, & Hall, 1966). Of all the bacteria used to detoxify aflatoxins, lactic acid bacteria (LAB) are the most studied (Oliveira, Zannini, & Arendt, 2014 The mechanism of biologically eliminating aflatoxins is by binding or degradation. Biological binding may be easily released and has a potential risk. Thus,the mechanism of strain E-1-1-1 degrading toxins needs further study.

| CON CLUS ION
In this study, we obtained an AFM 1 -degrading strain, B. pumilus E-1-1-1, where the degradation ratio of AFM 1 reached 89.55%. To the best of our knowledge, this is the first report that B. pumilus E-1-1-1 possesses the ability to degrade AFM 1 . The detoxification of AFM 1 by B. pumilus E-1-1-1 is a rapid process, and AFM 1 was decreased by 88.79% at 12 hr. Extracellular secretion from B. pumilus E-1-1-1 caused an apparent decrease in AFM 1 content. In conclusion, B. pumilus E1-1-1 could have great potential in the field of biological detoxification.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.