In Vitro Efficacy of Myxococcus fulvus ANSM068 to Biotransform Aflatoxin B1

Aflatoxin B1 (AFB1) is commonly found in cereals and animal feeds and causes a significant threat to the food industry and animal production. Several microbial isolates with high AFB1 transformation ability have been identified in our previous studies. The aim of this research was to characterize one of those isolates, Myxococcus fulvus ANSM068, and to explore its biotransformation mechanism. The bacterial isolate of M. fulvus ANSM068, isolated from deer feces, was able to transform AFB1 by 80.7% in liquid VY/2 medium after incubation at 30 °C for 72 h. The supernatant of the bacterial culture was more effective in transforming AFB1 as compared to the cells alone and the cell extract. The transformation activity was significantly reduced and eradicated after the culture supernatant was treated with proteinase K, proteinase K plus SDS and heating. Culture conditions, including nitrogen source, initial pH and incubation temperature were evaluated for an optimal AFB1 transformation. Liquid chromatography mass spectrometry (LCMS) analyses showed that AFB1 was transformed to a structurally different compound. Infrared analysis (IR) indicated that the lactone ring on the AFB1 molecule was modified by the culture supernatant. Chromatographies on DEAE-Ion exchange and Sephadex-Molecular sieve and SDS-PAGE electrophoresis were used to determine active components from the culture supernatant, indicating that enzyme(s) were responsible for the AFB1 biotransformation. This is the first report on AFB1 transformation by a strain of myxobacteria through enzymatic reaction(s).

biotransformation ability. In our previous studies, several microbes with AFB 1 biotransformation ability were isolated by using a newly developed coumarin medium method [22]. One of those microbes that was isolated from deer feces exhibited high AFB 1 biotransformation ability in liquid culture. It was further identified as a bacterial strain of Myxococcus fulvus and designated M. fulvus K2 It was later renamed as M. fulvus ANSM068 according to the laboratory's regulations.
The objective of this study was to further characterize M. fulvus ANSM068 as an AFB 1 biotransformation agent, to optimize the biotransformation conditions and to explore the biotransformation mechanism.

AFB 1 Biotransformation by M. fulvus ANSM068
The bacterial culture of M. fulvus ANSM068 reduced AFB 1 by 80.7% after co-incubation at 30 °C for 72 h. The culture supernatant of M. fulvus ANSM068 was able to reduce AFB 1 by 76.6%. However, no significant reduction was observed in the treatments with the cells and cell extracts ( Figure 1). Culture supernatant treated with proteinase K (Prok) displayed significantly decreased reduction ability, from 76.6 to 18.9%. No reduction of AFB 1 concentration was observed when the culture supernatant was treated with Prok plus SDS or with heating (boiling water bath for 10 min) ( Figure 1). Myxobacteria became well-known as prolific producers of secondary metabolites during the past few decades [37,38]. Thus far, no pathogenic myxobacteria have been observed and many bioactive components and drugs were isolated from myxobacteria [39][40][41]. Myxococcus fulvus was first documented and characterized in 1969 [42]. It is a species of myxobacteria, a group of Gram-negative eubacteria with rod-shaped cells. The colonial and cell morphology of M. fulvus ANSM068 are shown in Figure 2. It was previously found to be capable of reducing AFB 1 [22]. Up to now, no study has focused on mycotoxin control by using myxobacteria. This is the first report on mycotoxin biotransformation by a bacterial strain belonging to myxobacteria. Reduction of AFB 1 by culture supernatant produced without pre-exposure to AFB 1 suggested that it was achieved during the normal growth of the bacterium, indicating that the reduction was a constitutive activity of M. fulvus ANSM068. Results from proteinase K, proteinase K plus SDS and heating treatments implied that the reduction of AFB 1 was due to biotransformation instead of cell binding. Protein or enzymes in culture supernatant might be involved in AFB 1 biotransformation by the bacterial strain.

Optimal Culture Conditions for AFB 1 Biotransformation
Nitrogen sources have a dramatic effect on AFB 1 biotransformation by M. fulvus ANSM068. Yeast extract was found to be the best nitrogen source for AFB 1 biotransformation and resulted in 76.6% AFB 1 biotransformation ( Figure 3). In comparison, AFB 1 biotransformation was significantly (P < 0.05) lower with ammonium nitrate (25.8%). Among the four levels of yeast extract tested (0.2, 0.5, 0.8 and 1.2%), 0.5% was optimal for AFB 1 biotransformation (76.6%). Nitrogen in substrates is one of the most important factors influencing myxobacteria growth and production of metabolites. Chun [43] studied the effect of different nitrogen sources on the production of antifungal metabolites by myxobacteria in liquid culture and found that yeast extract improved secretion of antifungal metabolites but fish meal, soybean powder and inorganic nitrogen treatments showed no function. A similar trend was also observed in this study. Yeast extract was most favored in AFB 1 biotransformation by M. fulvus ANSM068 and inorganic nitrogen was far less effective. The reasons could be that in natural environments these myxobacteria feed on other organisms such as eubacteria or yeasts by bacteriolysis or cellular lysis [44]. The nutrients ratio such as amino acid and vitamins in yeast extract might be more suitable to the bacterial strain for its growth and production of AFB 1 biotransformation components.  The initial pH value in culture medium showed a significant effect on AFB 1 biotransformation ( Figure 4). The highest AFB 1 biotransformation rate was detected at pH values of 6.5 to 7.5 (P < 0.05). It has been well documented that the optimum pH range for myxobacteria is 6.8-7.8 in liquid culture [45]. Ahn et al. [46] studied the effect of pH value on antifungal metabolites produced by a Myxococcus isolate and found that a pH value between 6.5 and 7.6 was favored and the optimal pH value was 7.2.
The effect of temperature on AFB 1 biotransformation by M. fulvus ANSM068 is shown in Figure 5. The % biotransformation exhibited was significantly lower (P < 0.05) at 15 °C (11.0%) and 40 °C c (18.9%) than at 30 °C (76.6%). Since the bacterial strain originated from deer feces, a temperature at 30 °C should be more suitable for the survival and growth of the bacterium, thus optimal for its enzyme system. Figure 5. Effect of temperature on AFB 1 biotransformation by M. fulvus ANSM068. The reactions were performed at 30 °C for 72 h. Each value is a mean ± SE of three replicates. Means within a row with no common letters differ (P < 0.05).

AFB 1 Biotransformation Product Analysis
In the analysis by LCMS, AFB 1 standard was identified at m/z = 313 for the protonated cation [M + H] + , m/z = 335 for the sodium adduct of AFB 1 [M + Na] + , and at m/z = 351 for the potassium adduct [M + K] + ( Figure 6A); VY/2 medium supplemented with AFB 1 clearly showed the above three distinct ions ( Figure 6B). However, the distinct AFB 1 ion at m/z =351 disappeared while the ions at m/z = 313 and m/z = 335 significantly decreased after AFB 1 was co-incubated with M. fulvus ANSM068 culture supernatant for 72 h ( Figure 6C). These results suggest AFB 1 level was significantly reduced although it was still present.
Additionally, the HPLC chromatograph ( Figure 7) confirmed that AFB 1 (peak eluting at 8.27 min) was biotransformed to a new product eluting at 3.17 min. The IR spectrum showed that there were three major modifications in absorption peaks between the standard AFB 1 and the product resulting from culture supernatant treatment ( Figure 8). The absorption peak at 1728 cm -1 in AFB 1 standard disappeared after the treatment, which indicates that the lactone attached to the benzene ring was modified. The two peaks in the area between 1658 and 1634 cm -1 in AFB 1 standard changed into one peak after treatment. The change at 2930 cm -1 indicates modification of methyl group.   Alberts et al. [19] explored the AFB 1 biotransformation product with Rhodococcus erythropolis extracellular fractions by electro spray mass spectrometry and liquid chromatography mass spectrometry analysis. By comparing the three distinct AFB 1 peaks among treatments, AFB 1 was still present but at a lower concentration. No breakdown products were revealed, which suggested that AFB 1 was most likely metabolized to biotransformation products with chemical properties different from that of AFB 1. Liu et al. [12] compared the infrared spectrum between AFB 1 standard and fungal enzyme treated AFB 1 . Opening of the difuran ring was proposed since the group of five bands in the area between 1000 cm -1 and 1200 cm -1 did not appear in the spectrum of the enzyme treated AFB 1 . Marisa and Das [4] analyzed the biotransformed AFB 1 structure by fluorescence spectra measurements and TLC. Treatment of AFB 1 with purified enzyme resulted in a decrease in fluorescence intensity, suggesting the enzymatic cleavage of the lactone ring. The lactone ring was confirmed to be responsible for toxicity and mutagenicity in AFB 1 [47,48]. In the current study, results from LCMS, HPLC and IR analysis confirmed AFB 1 biotransformation by M. fulvus ANSM068 instead of cell binding. Modification of the lactone ring on the AFB 1 molecule may result in detoxification although further investigation needs to be done. This result implies the potential applications of M. fulvus ANSM068 or its metabolites in detoxifying AFB 1 in contaminated food or feed.

Characterization of Active Components from Culture Supernatant
Since the culture supernatant of M. fulvus ANSM068 exhibited high AFB 1 biotransformation activity, it was selected for active components extraction. Precipitates from 80% saturated ammonium sulfate showed higher AFB 1 biotransformation activity, with 54.1% of AFB 1 biotransformed. Chromatography was used to purify enzyme from the precipitates. Two peaks were obtained by ion exchange chromatography on DEAE A-50 column and UV detection at 280 nm ( Figure 9A). The second peak, which showed AFB 1 biotransformation activity (35.5%), was applied to molecular sieve chromatography on a Sephadex G-100 column ( Figure 9B). SDS-PAGE showed that the active components consist of at least three proteins with a relatively small molecular weight between 20 and 66 kDa ( Figure 9C). To date, there are only a few reports on extraction or purification of mycotoxin biotransformation active components from microbial metabolites. Liu et al. [27] extracted and purified an intracellular enzyme from fungi Armillariella tabescens by ammonium sulfate precipitation, ion exchange chromatography and chromatofocusing chromatography. The AFB 1 detoxification enzyme was then characterized to have a molecular mass of 51.8 kDa by SDS-PAGE and it exhibited a specific activity of 7.09 nmol min/mg at pH 6.0 and 28 °C. Motomura et al. [6] purified an aflatoxin biotransformation enzyme from the edible mushroom Pleurotus ostreatus by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. This study has confirmed that the AFB 1 biotransformation components exist in the culture supernatant and are very likely enzyme(s). Further purification and identification of the enzyme(s) are undergoing.

Bacterium, Mycotoxin and Medium
Myxococcus fulvus ANSM068 was isolated and purified from deer feces collected from the Beijing Zoo in July, 2006. It was identified using physiological and biochemical tests and 16S rRNA gene sequence analysis by the China General Microbiological Culture Collection Center (CGMCC) and has been deposited there as CGMCC #3194. It was preserved at -80 °C before use (China Agriculture University, Beijing, China). AFB 1 was obtained from Sigma Chemical Co. (Bellefonte, USA). The medium used for liquid culture, VY/2 medium, contains 5 g yeast extract, 1 g CaCl 2 , 0.5 g MgSO 4 , 0.5 mg VB 12 in 1 L distilled water at room temperature.

AFB 1 Quantification
For AFB 1 analysis, the HPLC procedure by AOAC [49] 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 was 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 derivatized 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 biotransformation was calculated using the following formula: (1 -AFB 1 peak area in treatment/AFB 1 peak area in control) × 100%

Efficacy of AFB 1 Biotransformation by M. fulvus ANSM068
Biotransformation of AFB 1 by M. fulvus ANSM068 was carried out in liquid culture. The bacterial isolate was cultured in VY/2 medium for 24 h and used to inoculate VY/2 (50 mL) in a 300 mL flask, which was incubated at 30 °C with agitation at 160 rpm for 50 h in a Gyrotary shaker incubator without AFB 1 (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 bacterial cultures of 0.8 mL for a final concentration of 100 ppb. The biotransformation tests were conducted in the dark at 30 °C without shaking for 72 h. After incubation, bacterial cells were removed by centrifugation at 9,300 g for 10 min (TGL-16C centrifuge, Beijing Medical Centrifuge Inc., China). Sterile VY/2 medium was used to substitute the bacterial culture in the control.
Biotransformation of AFB 1 by cells, culture supernatant and intracellular cell extracts was conducted according to the procedure of Shu et al. [22]. Cells were pelleted using a centrifuge at 9,300 g, 4 °C for 10 min. The pellets were washed twice with a phosphate buffer (50 mM; pH 7.5) before being resuspended in the phosphate buffer (5 mL). Culture supernatant was obtained by centrifuging 50 h liquid culture at 9,300 g, 4 °C for 10 min. Intracellular cell extracts were harvested by the following procedures. The cell suspension was disintegrated twice (work every other 5 s for 33 min) by using ultrasonic cell disintegrator on ice. The disintegrated cell suspension was centrifuged at 9,300 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. The phosphate buffer solution (50 mM; pH 7.5) was used to substitute intracellular cell extracts and cells in the controls. 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 into a boiling water bath for 10 min. 0.2 mL of AFB 1 stock solution was added to 0.8 mL of each reaction liquid for a final concentration of 100 ppb. The biotransformation tests were conducted in the dark at 30 °C without shaking for 72 h. After incubation, bacterial cells were removed by centrifugation at 9,300 g for 10 min (TGL-16C centrifuge, Beijing Medical Centrifuge Inc., China).The untreated culture supernatant was used as control.

Culture Conditions Affecting AFB 1 Biotransformation
Unless specifically indicated, all biotransformation experiments were conducted by using culture supernatant at 30 °C for 72 h with aeration. Culture supernatant was obtained by centrifuging 50 h culture with 9,300 g at 4 °C for 20 min.
To detect the appropriate nitrogen source for the bacterial growth with optimum AFB 1 biotransformation, 0.5% of tryptone, peptone, beef extract, yeast extract, fish peptone and ammonium nitrate (NH 4 NO 3 ) were chosen as the nitrogen source in the culture medium.

AFB 1 Biotransformation Product Analysis
The biotransformation products of AFB 1 after 72 h incubation with culture supernatants were determined by liquid chromatography mass spectrometry (LCMS) and infrared analysis (IR).
For LCMS, the following samples were analyzed: (a) AFB 1 standard, (b) VY/2 medium supplemented with AFB 1 , (c) AFB 1 treated with culture supernatants for 72 h. The samples were extracted with chloroform, dried under nitrogen, suspended in methanol: water (1:1, v/v) and analyzed by LCMS using a Phenomenex 2.0 × 150 mm C 18 column and methanol: acetonitrile: water (1:1:2, v/v/v) as solvent, at a flow rate of 100 µL/min.The HPLC system was equipped with a Finnigan Spectra System UV6000LP ultraviolet (UV) detector. Atmospheric pressure chemical ionization (APCI) positive ion mode was used for MS detection (ThermoFinnigan, San Jose, CA, USA).
For IR analysis, 2 mL chloroform was added to the incubation mixture and vortexed for 30 seconds. The phases were separated by low-speed centrifugation and the organic phase was evaporated to dryness at 20 o C under N 2 . The samples were purified by HPLC before IR analysis. IR analysis was carried out with a KBr pellet using an IR spectrophotometer (NEXUS-470F TIR, Nicolet, Japan). Sterile VY/2 medium was used to substitute culture supernatant in the control.

Precipitation of Active Components
Methanol, acetone, ethanol and solid ammonium sulphate were used to precipitate the active components. For the organic solvents, 500 mL of each of the chilled solvent was added to 150 mL of the culture supernatant. For ammonium sulphate, 150 mL of the culture supernatant was supplemented to 20, 40, 60 and 80% saturation respectively under constant stirring. The above solutions were stored at 4 o C for 4 h followed by centrifugation at 9,300 g for 20 min. The precipitates were dissolved in 2 mL of 50 mM sodium phosphate buffer (pH 7.5). The ammonium sulphate treatments were dialysed over night against distilled water before being applied to chromatography.

Purification of the Active Components by Chromatography
The dialyzed samples were applied to a DEAE A-50 Ion exchange column (Pharmacia Biotech, Sweden) pre-equilibrated with 50 mM sodium phosphate buffer pH 7.5. Elution was performed with the same buffer and by a gradient of 0.5 M NaCl in the buffer. Fractions of 2 mL were collected in separate tubes at a flow rate of 0.4 mL/min. The protein concentration in each eluted fraction was detected at 280 nm using a UV-visible spectrophotometer. The fractions with AFB 1 biotransformation activity were combined and applied to a Sephadex G-100 Molecular sieve column (Pharmacia) pre-equilibrated with 50 mM sodium phosphate buffer containing 0.2 M NaCl. Proteins were eluted with the same buffer and fractions of 2 mL were collected in separate tubes at a flow rate of 0.2 mL/min. After dialysis against buffer, the AFB 1 biotransformation activity of each fraction was tested.

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE was performed in 12.5% polyacrylamide gels according to the method of Motomura et al. [6]. The separated proteins were stained with Coomassie Brilliant Blue R-250 (Fluka, Switzerland) and their molecular weights were determined by comparison with low range molecular weight markers (Pharmacia Biotech, Uppsala, Sweden).

Statistical Analyses
The results were average of triplicates and were expressed as mean ± standard error. Data were 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
The present study demonstrated the ability of M. fulvus ANSM068 to effectively biotransform AFB 1 . Furthermore, optimal conditions for biotransformation were determined as follows: 0.5% of yeast extract as nitrogen source; initial pH value at 7.5; temperature at 30 °C. The results indicated that enzyme(s) in the culture supernatant was responsible for the biotransformation although further confirmation is needed. Research is underway to purify the effective enzyme(s) and to identify metabolites produced during the biotransformation processes. The AFB 1 biotransformation enzymes, once identified, may be mass-produced by the bacterial isolate and be used to treat materials contaminated with AFB 1 .