Aflatoxin B1 Degradation by Stenotrophomonas Maltophilia and Other Microbes Selected Using Coumarin Medium

Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins in animal production and food industry. A safe, effective and environmentally sound detoxification method is needed for controlling this toxin. In this study, 65 samples were screened from various sources with vast microbial populations using a newly developed medium containing coumarin as the sole carbon source. Twenty five single-colony bacterial isolates showing AFB1 reduction activity in a liquid culture medium were selected from the screen. Isolate 35-3, obtained from tapir feces and identified to be Stenotrophomonas maltophilia, reduced AFB1 by 82.5% after incubation in the liquid medium at 37 °C for 72 h. The culture supernatant of isolate 35-3 was able to degrade AFB1 effectively, whereas the viable cells and cell extracts were far less effective. Factors influencing AFB1 degradation by the culture supernatant were investigated. Activity was reduced to 60.8% and 63.5% at 20 °C and 30 °C, respectively, from 78.7% at 37 °C. The highest degradation rate was 84.8% at pH 8 and the lowest was only 14.3% at pH 4.0. Ions Mg2+ and Cu2+ were activators for AFB1 degradation, however ion Zn2+ was a strong inhibitor. Treatments with proteinase K, proteinase K plus SDS and heating significantly reduced or eradicated the degradation activity of the culture supernatant. The results indicated that the degradation of AFB1 by S. maltophilia 35-3 was enzymatic and could have a great potential in industrial applications.

exhibited strong degradation activity thus was further identified and characterized. Factors affecting degradation efficiency of the isolate were also investigated.

Screening for AFB 1 degradation microbes
Twenty five single-colony bacterial isolates were obtained from 65 samples collected from various sources ( Table 1). All these isolates were able to reduce concentrations of AFB 1 in the liquid medium tested after 72 h incubation at 37 °C with various degrees of effectiveness. Sixteen isolates reduced AFB 1 in the medium by over 50%. Isolate 35-3 was the most effective and reduced AFB 1 by 82.5% (Table 1).
Volkl et al. [32] has proposed that biological degradation of mycotoxins occurs in nature since many mycotoxins are chemically stable but do not appear to accumulate in natural environments. Therefore, environmental samples rich in microorganisms, such as animal feces, decayed barks, soils and cereal grains, were chosen as sources for selection of microorganisms that degrade AFB 1 . To identify active isolates from the vast microbial populations of environmental samples, an effective selection method is very much needed. In this study, a medium containing coumarin (CM) as the sole carbon source was developed for the first time and was used for the microbial selection. The microorganisms grew slowly and only very few colonies appeared on the medium. Single colonies were picked up after incubation of 3-7 days and transferred to fresh CM plates three times sequentially. Only 25 single colonies were selected out of huge populations with great diversities in the collected samples, and none was false positive. The results clearly indicated that this newly developed method was not only extremely selective but also accurate.
Aflatoxins are a group of bisfuranocoumarin derivatives and the lactone ring in the common coumarin structure plays an important role in its toxicity and mutagenicity [33]. Coumarin is the basic molecular structure of all aflatoxins ( Figure 1) [34,35]. Therefore, microorganisms that could utilize coumarin as their carbon source might also be able to use aflatoxins, in this case, AFB 1 . The metabolizing processes should result in degradation of the mycotoxin. Coumarin is a phytochemical, which is widely used in flavor industry for sour. Compared with AFB 1 , it is much safer for users, easier to obtain and cheaper to buy. The developed coumarin method provided an inexpensive,

Coumarin
Aflatoxin B 1 feasible and effective tool for selecting AFB 1 degradation microorganisms. The method should also be useful in research targeting other aflatoxins.

Identification of isolate 35-3
Isolate 35-3 appeared on nutrient agar as round straw yellow colored colonies. It is a gram-negative bacterium. The isolate grew well at 37 °C, but not at 10 °C or 55 °C. It was able to use most single sugars including glucose, maltose and sucrose as a sole carbon source. The isolate could hydrolyze gelatin and Tween 80 but not amylum ( Table 2). Determination of the 16S rRNA gene sequence revealed that the isolate belonged to genus Stenotrophomonas ( Figure 2). The closest relationship (99% sequence similarity) obtained with the type of a described species was Stenotrophomonas maltophilia U62646, which is an aerobic gram-negative bacterium. It has been reported that isolates from this genus possess function in degradation of polycyclic aromatic hydrocarbons (PAHs) and enzymes are involved in these processes [36][37][38]. However, this is the first report indicating that a bacterium in this genus possesses function in mycotoxin degradation.

AFB 1 degradation by S. maltophilia 35-3
Culture supernatant of S. maltophilia 35-3 showed strong AFB 1 degrading activity and it was more effective (P<0.05) than viable cells and cell extracts ( Figure 3). Culture supernatant was able to degrade 78.7% AFB 1 after 72 h incubation compared to 17.5% and 9.6% by viable cells and cell extracts, respectively. Culture supernatant of Rhodococcus erythropolis degraded AFB 1 with 33.2% residual after 72 h incubation, and the degradation was proved to be enzymatic by using proteinase K and SDS treatments [25]. Similarly, the original culture supernatant of Flavobacterium aurantiacum degraded 74.5% of AFB 1 in 24 h and it could only degrade 34.5% of AFB 1 after being treated with proteinase K (0.1 mg/mL) [28]. The active ingredient in the culture supernatant was considered to be a protein or perhaps an enzyme [25,28]. In this study, the activity of AFB 1 degradation was mainly in the culture supernatant of S. maltophilia 35-3 rather than its cells or cell extracts. Degradation of AFB 1 by the culture supernatant produced without pre-exposure to AFB 1 indicated that the degradation was achieved during the normal growth of the bacterium, suggesting that the degradation was a constitutive activity of S. maltophilia 35-3. Culture supernatant treated with proteinase K displayed significantly reduced degradation ability (23.8%). When culture supernatant was treated with proteinase K plus SDS and heat (boiling water bath for 10 min), respectively, no degradation activity was observed. All these results implied that a protein or enzyme might be involved in the degradation by S. maltophilia 35-3. Degradation of AFB 1 by the culture supernatant of S. maltophilia 35-3 was a relatively rapid and continues process, with 46.3% AFB 1 degraded in the first 12 h and 78.7% degraded after 72 h ( Figure  4). Similar results were obtained elsewhere. Alberts et al. [25] reported a 66.8% reduction of AFB 1 from 0 to 72 h when incubated with culture supernatant of R. erythropolis; Hormisch et al. [26] indicated that liquid cultures of Mycobacterium strain FA4 could reduce AFB 1 level by 70 to 80% within 36 h and completely degrade AFB 1 in 72 h. In comparison, most of the lactic acid bacteria that were able to bind AFB 1 could rapidly remove the toxin from liquid; however, they would release AFB 1 to some extent in the prolonged incubation period [19,22]. The continuous increase in detoxification by S. maltophilia 35-3 with time indicated that binding might not play important role in the AFB 1 reduction. AFB 1 degradation was strongly affected by metal ions (Figure 5). Ions Mg 2+ and Cu 2+ showed effect on stimulating AFB 1 degradation at the concentration of 10 mM compared with control (78.7%). Their degradation rates were 85.4% and 85.0%, respectively. However, Li + ions at 10 mM reduced the degradation to 53.3% and Zn 2+ ions inhibited the activity even more significantly, with only 1.4% of AFB 1 degraded after 72 h. These results agreed with studies of D'Souza and Brackett [29] in effects of Mg 2+ on AFB 1 degradation by F. aurantiacum. Additions of 0.1, 1 and 10 mM Mg 2+ increased AFB 1 degradation after 48 h incubation. The explanation could be that Mg 2+ might stabilize membranes, maintain structural integrity of proteins and act as enzyme activator. Also, AFB 1 degradation by F. aurantiacum was significantly inhibited (P<0.05) after incubation with 10 mM Zn 2+ for 4, 24 and 48 h [30], which was similar to that was noticed in our study. Zn 2+ might be able to alter the enzyme  AFB 1 degradation by the culture supernatant was pH sensitive ( Figure 6). The highest degradation (84.8%) was observed at pH 8.0 and it decreased gradually as the pH value went down, to the lowest at pH 4.0 (14.3%). However, the culture supernatant maintained its degradation ability (76.9%) in basic condition at pH 9.0. No degradation was detected in controls with different pH values. The effect of pH on degradation of AFB 1 by cell extracts of F. aurantiacum showed a similar trend [31]. The degradation of AFB 1 was approximately 25% at pH 5, increased to 50% at pH 6 and 70% at pH 7, and decreased to 50% at pH 8. The correlation of AFB 1 degradation with pH values is typical for enzymatic reactions. Enzymes have an optimal pH range for maximal activities. At pH values outside of the optimum, enzymatic activity decreases due to the ionization of a critical amino acid residue within the catalytic site [39]. The maximal AFB 1 degradation by S. maltophilia 35-3 in this study was observed at a basic pH (pH 8), indicating the enzyme produced by the isolate had a higher optimal pH compared to enzyme in cell extracts of F. aurantiacum [31]. The AFB 1 degradation by S. maltophilia 35-3 culture supernatant varied under different temperatures (Figure 7). The degradation was lower at 20 °C (60.8%) and 30 °C (63.5%) than at 37 °C (78.7%) (P<0.05). The isolate 35-3 was originated from feces of South American tapir; temperature at 37 °C should be more suitable for the survival and growth of the bacterium, thus optimal for its enzyme system. Teniola et al. [27] reported that AFB 1 degradation by cell extracts of R. erythropolis and M. fluoranthenivorans were about the same in between 10-40 °C (> 90%). They proposed either that the enzymes in the extracts had a wide temperature range of activity or that other factors were involved in the degradation.

Samples.
Sixty-five samples were screened for AFB 1 degradation activity. The samples consisted of thirty nine feces of wild animals collected from Beijing Zoo, Beijing, China; nineteen cereal grains, obtained from Beijing Huilongguan foodstuff market; five soil samples and two decayed tree bark samples collected from farmland in China Agricultural University, Beijing, China. All these samples were airdried at room temperature.

Isolation.
Samples (0.5 g) were ground with 5.0×10 5 IU nystatin (Beijing Chemical Inc., China) before being homogenized in sterilized distilled water (9.0 mL). After incubation at room temperature on a rotary shaker for 12 h, the supernatant was serially diluted with sterilized distilled water to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 folds. Diluted aliquots (0.2 mL) were plated on plates of CM medium, which were incubated at 37 °C for 3-7 days until visible colonies appeared. Single colonies were isolated and subsequencially transferred to fresh CM plates for three times. Colonies that were able to grow on the medium were selected and preserved as pure isolates on NA, and tested for AFB 1 degradation.

Tests of AFB 1 degradation
Degradation of AFB 1 by the selected isolates was carried out in liquid cultures. The microbial isolates were cultured in NB. For inoculation, 12 h culture broth (2.5 mL) was transferred to NB (50 mL) in a 300 mL flask. The microbes were grown at 37 °C with agitation at 140 g for 24 h in a Gyrotary shaker incubator (Haerbin Donglian Electronic Equipment Inc., China). AFB 1 standard solution (Sigma Chemical Co., Bellefonte, USA) was diluted with methanol (Beijing Chemical Inc., Beijing, China) to a stock solution of 500 ppb, of which 0.2 mL was added to microbial cultures of 0.8 mL for a final concentration of 100 ppb. The degradation tests were conducted in the dark at 37 °C without shaking for 72 h. After incubation, cells of microbes were removed by centrifugation at 10,000 g for 10 min (Beijing Medical Centrifugator Inc., China). Sterile NB was used to substitute microbial culture in the control.
For AFB 1 analysis, the HPLC procedure by AOAC [40] was used with slight modifications. The reaction mixtures were extracted three times with chloroform. The chloroform extracts were evaporated under nitrogen at room temperature, the residue were dissolved in 50% methanol in water (1:1, v/v) and analyzed by HPLC. HPLC analysis was performed using a LiChroCART RP-C18 (250-4 Hypersil ODS (5 μm), Merck) column with a guard column (LiChroCART 4-4 RP-C18 (5 μm), Merck). The mobile phase was methanol: water (1:1, v/v) isocratic at a flow rate of 1 mL/min. AFB 1 was derived by a photochemical reactor (AURA, USA) and measured by a fluorescence detector. The excitation and detection wavelengths were set at 360 and 440 nm, respectively. The percentage of AFB 1 degradation was calculated using the following formula: (1 -AFB 1 peak area in treatment / AFB 1 peak area in control) × 100%

Physiological and biochemical tests
Physiological and biochemical tests were carried out following the method of Holt et al. [41].

Phylogenetic analyses
The generated DNA sequences and sequences derived from GenBank were aligned using the ClustalX program [42]. Neighbour joining analysis and calculation of bootstrap values were done according to the MEGA program [43].

Degradation of AFB 1 by S. maltophilia 35-3
Stenotrophomonas maltophilia isolate 35-3 was selected for further study owing to its high degradation efficiency. Unless specifically indicated, all degradation experiments were conducted under 37 °C for 72 h with aeration.

Degradation of AFB 1 by S. maltophilia 35-3 cells
Fresh NB was inoculated with 12 h pre-cultured isolate 35-3 at 37 °C, agitation at 140 g for 24 h in a Gyrotary shaker incubator. Cells were pelleted using a refrigerated high-speed centrifuge (GL-20G-Ⅱ ， centrifugator Shanghai Anting Instrument Inc., China) at 10,000 g, 4 °C for 10 min. The pellets were washed twice with phosphate buffer (50 mM; pH 7.0) before resuspension in the phosphate buffer (5 mL) [19]. The AFB 1 degradation tests were performed as described in 3.3. Phosphate buffer was used to substitute bacterial cell suspensions in the control samples.

Degradation of AFB 1 by S. maltophilia 35-3 intracellular cell extracts
Cell pellets were prepared as described previously (3.5.1). Pellets were suspended in phosphate buffer (pH 7.0; 3 mL buffer per gram cell mass). The suspension was disintegrated twice (work every other 5 s for 33 min) by using ultrasonic cell disintegrator on ice (Ningbo Xinzhi Instruments Inc., China). The disintegrated cell suspension was centrifuged at 12,000 g for 20 min at 4 °C. The cell extracts were collected by filtering the supernatant aseptically using 0.2 µm pore size sterile cellulose pyrogen free filters (Beijing Biotech Inc., China). The AFB 1 degradation tests were performed as described in 3.3. Phosphate buffer solution was used to substitute intracellular cell extracts in the control.
3.5.3. Effects of incubation period, temperature, pH, metal ions and proteinase K treatment on AFB 1 degradation by S. maltophilia 35-3 supernatant Isolate 35-3 grown in NB for 24 h was centrifuged with 10,000 g at 4 °C for 20 min, and the resulting culture supernatant was tested for AFB 1 degradation. AFB 1 methanol stock solution (0.2 mL) was added to 0.8 mL culture supernatant in a 7 mL tube. The reaction mixture was incubated in the dark at 37 °C without shaking for 1, 12, 24, 48, 72 and 90 h, respectively. To determine the effect of temperature, the mixtures were incubated at 20, 30 and 37 °C, respectively for 72 h. Controls were set at the above temperatures by using NB medium.
In the pH tests, initial pH value was obtained by adjusting pH to 4.0, 5.0 and 6.0 with citrate acid buffer, and to 7.0, 8.0 and 9.0 by sodium phosphate buffer. Controls were set by adjusting NB medium to different pH values.
The effects of different metal ions on degradation were determined by adding Mg 2+ , Zn 2+ , Cu 2+ , Mn 2+ and Li + (in the form of MgCl 2 , ZnSO 4 , CuSO 4 , MnCl 2 and LiCl, respectively) to the reaction mixture respectively resulting in a final ion concentration of 10 mM. NB was used to substitute culture supernatant in the control.
The effect of protease treatment was determined by exposing the culture supernatant to 1 mg/mL proteinase K (Roche Diagnostics, Basel, Switzerland; specific activity ≥30 U/mg) for 1 h at 37°C; 1 mg/mL proteinase K plus 1% SDS for 6 h at 37 °C. The effect of heat treatment was determined by dipping the culture supernatant in boiling water bath for 10 min. The untreated culture supernatant was used as control.

Statistical analyses
Data was analyzed as a completely randomized single factor design by ANOVA using the general linear models procedure in SAS. Significant F tests at the 0.05 levels of probability are reported. When a significant F-value was detected, Duncan's Multiple Range Test was used to determine significant differences among means.

Conclusions
An innovative method with coumarin as a selective agent was developed and used to search for AFB 1 degradation microorganisms in this study. The results have proven that the method is effective and accurate; the method is also safe, practical and economical. Twenty-five purified isolates were obtained using this method and all were able to degrade AFB 1 . Isolate 35-3, a bacterium belonging to Stenotrophomonas maltophilia, was identified for the first time to have the function of degrading AFB 1 . Enzyme(s) in the culture supernatant of the isolate might be responsible for the degradation although further confirmation is needed. Research is underway to purify the effective enzyme(s) and to identify metabolites produced during the degradation processes. The AFB 1 degradation enzymes, once identified, may be mass-produced by the bacterial isolates and used to treat materials contaminated with AFB 1. Furthermore, identification of genes responsible for AFB 1 degradation can provide potential for AFB 1 control with genetically modified microbes and crop cultivars.