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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20

Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1 ab

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Xiaojiao Chang , Zidan Wu , Songling Wu , Yanshi Dai & Changpo Sun

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Cereals and Oils Microbiology Research Group, Academy of State Administration of Grain, Beijing, China b

Department of Food Science and Technology, Henan University of Technology, Zhengzhou, Henan, China Published online: 17 Dec 2014.

Click for updates To cite this article: Xiaojiao Chang, Zidan Wu, Songling Wu, Yanshi Dai & Changpo Sun (2014): Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.991948 To link to this article: http://dx.doi.org/10.1080/19440049.2014.991948

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Food Additives & Contaminants: Part A, 2014 http://dx.doi.org/10.1080/19440049.2014.991948

Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1 Xiaojiao Changa,b, Zidan Wua, Songling Wua, Yanshi Daia and Changpo Suna* a Cereals and Oils Microbiology Research Group, Academy of State Administration of Grain, Beijing, China; bDepartment of Food Science and Technology, Henan University of Technology, Zhengzhou, Henan, China

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(Received 15 July 2014; accepted 22 November 2014) Ochratoxin A (OTA) is widely found in food and feed products as a mycotoxin contaminant. It is produced by Penicillium species and several Aspergillus species. The identification OTA detoxification microorganisms is believed to be the best approach for decontamination. In this study, we isolated ASAG1, a bacterium with the ability to degrade OTA effectively, from grain depot-stored maize. A 16S rDNA sequencing approach was used to identify this strain as Bacillus amyloliquefaciens ASAG1. The degradation of OTA was detected in both medium and cell-free extracts after incubation with a culture of B. amyloliquefaciens ASAG1 cells. Subsequently, a hydrolysed enzyme (carboxypeptidase) related to the enzymatic conversion of OTA was cloned from the B. amyloliquefaciens ASAG1 genome. Using the Escherichia coli Expression System, we successfully expressed and purified this carboxypeptidase. When this enzyme was incubated with the engineered recombinant E. coli cells, the concentration of OTA was dramatically degraded. Our data therefore indicate that the carboxypeptidase produced by B. amyloliquefaciens ASAG1 is likely responsible for the biodegradation of OTA. Keywords: ochratoxin A; Bacillus amyloliquefaciens; biocontrol; carboxypeptidase

Introduction Ochratoxin A (OTA) is one of the most harmful secondary metabolites produced by toxigenic fungi, and it has the potential to contaminate many feed and food products, including cereals, figs, grapes, spices, coffee beans, cocoa beans, pork meat and dried vine fruits (Ringot et al. 2006). In addition, this toxin is transmitted to humans along the food chain. Epidemiological studies have shown that OTA may be a liver toxin, nephrotoxin, immune suppressant, carcinogen and potent teratogen in certain animal species (O’Brien & Dietrich 2005; Abrunhosa et al. 2010; Anzai et al. 2010; PfohlLeszkowicz & Manderville 2012). Biological approaches to remove mycotoxin contamination have received increased attention worldwide, as biological methods are safer, more efficient and more environmentally friendly than physical and chemical strategies for contamination control. Several microbes and enzymes have been reported to be able to degrade OTA, and some practical biological strategies have already been exploited (Böhm et al. 2000; Stander et al. 2000, 2001; Varga et al. 2000, 2005; Abrunhosa et al. 2002, 2010; Cubaiu et al. 2012; Armando et al. 2013; Shi et al. 2014). However, the identification of new microbes with the ability to degrade mycotoxins and their mechanisms of degradation will lead to improved ways to remove mycotoxins. The present work aimed to investigate the biological control of OTA contamination by microorganisms and *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

their active substances. To this end, we isolated B. amyloliquefaciens ASAG1, which exhibits a high OTA degradation activity. The goals of this study were (1) to isolate and identify strains of corn stored in a grain depot with the ability to degrade OTA levels effectively and inhibit the growth of toxigenic fungi; (2) to optimise the conditions of OTA degradation by B. amyloliquefaciens ASAG1; and (3) to identify, purify and test the biological detoxification effects of the B. amyloliquefaciens ASAG1 enzyme responsible for OTA degradation. Importantly, the strain and enzyme could be used as an important post-harvest strategy to degrade the OTA contamination of food and feed products. Materials and methods Materials Strains and growth conditions B. amyloliquefaciens ASAG1 was used to inhibit the growth of toxigenic fungi and degrade toxins isolated from maize collected in a grain depot. The ASAG1 cells were grown in No. 4 nutrient culture media containing 20% potatoes, 4% lactose, 1.5% peptone, 0.05% NaNO3 and 0.4% MgSO4 at pH 7.0. A. ochraceus (CGMCC 3.4520) was purchased from China General Microbiological Culture Collection (CGMCC). A. niger and A. carbonarius were stored in our laboratory. The OTA production abilities of the three toxigenic strains, A. ochraceus, A. niger and A. carbonarius, were found

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to be 4.3, 1.2 and 3.7 ng ml−1, respectively, when cultivated on maize. All the fungi had the ability to produce OTA. The pEB vector was used to clone the gene encoding the OTA degradation enzyme, and the protein was expressed in E. coli Rosetta.

The samples were cultivated at 31°C and 220 r min−1 for 24, 48 and 72 h. OTA was added to the control (2). The samples were then extracted using dichloromethane (Curtui & Gareis 2001), filtered and analysed by HPLC.

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Standard OTA standard (purity degree 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Strain identification A 16S rDNA sequencing approach was used to identify the antifungal strain that was isolated from the maize. Genomic DNA of ASAG1 was extracted for 16S rDNA amplification with PCR primers Bs16sR and Bs16sF (Barbosa et al. 2005). The PCR product was cloned into the PMD 18-T Vector (TaKaRa). DNA sequencing confirmed that the 16S rDNA fragment was successfully subcloned. Next, the strain was characterised according to its morphological, physiological and biochemical features, including its Gram type, growth properties, morphology, anaerobic growth capacity and oxidase activity (Pathak & Keharia 2013).

Optimisation of the culture conditions To determine the most appropriate culture conditions, cellfree extracts of ASAG1 cultured for 8, 12, 16, 24, 30, 36, 48, 60, 72 and 96 h were tested by inhibition zone measurements with A. ochraceus, A. niger and A. carbonarius. To determine the optimum temperature, different fermentation temperatures (25, 27, 29, 31, 33, 35 and 37°C) were tested in triplicate. The effect of the cell-free extract was evaluated by measuring the inhibition zone (Yuan et al. 2012). The inhibition test was carried out after ASAG1 cultivation for 3 days at different pH values of liquid media, which were adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0.

OTA degradation Degradation of OTA by B. amyloliquefaciens ASAG1 in liquid cultures B. amyloliquefaciens ASAG1 was grown in No. 4 nutrient medium. The sample was inoculated with a 10% inoculum of B. amyloliquefaciens ASAG1 cultivated at 31°C for 10 h and OTA at a final concentration of 1 µg ml−1. The following samples were used as controls: (1) sterile No. 4 nutrient medium supplemented with OTA and (2) No. 4 nutrient medium supplemented with a 10 h inoculum of B. amyloliquefaciens ASAG1 diluted 1:10 (without OTA).

To prepare the fresh cell-free extracts, 1% of a 24-h culture medium was transferred to fresh sterile medium, incubated at 31°C for 72 h, and filtrated sequentially with a 0.45 µm and a 0.22 µm filter (PES). A volume of 600 μl OTA with an initial concentration of 50 µg ml−1 was added to 30 ml fresh cell-free extract. The mixture was incubated at 31°C with gentle shaking for 0, 1, 2, 4, 12 and 24 h to determine the optimal incubation time. The controls included the following: (1) sterile water with OTA (at a final concentration of 1 µg ml−1) and (2) cellfree extracts without OTA. The experiments were performed in duplicate. When the samples were prepared, OTA was added to the control (2), and the samples were extracted with dichloromethane, evaporated under nitrogen gas, dissolved in methanol and filtered with a 0.45 µm membrane (Nylon). The remaining OTA was then analysed by HPLC. HPLC analysis of OTA with fluorescence detection According to Cubaiu et al. (2012), the HPLC instrument (Agilent 1100 Series, Santa Clara, CA, USA) contained a fluorescence detector set at 333 nm excitation and 460 nm emission wavelengths. The analysis was carried out with an Agilent Eclipse XDB-C18 column (150 × 4.6 mm I.D., 5 μm particle size; Agilent) at a column temperature of 30°C, an injection volume of 20 μl and a flow rate of 1.0 ml min−1. The mobile phase consisted of water–acetonitrile–acetic acid in a ratio of 99:99:2, by volume. The samples were filtered using a 0.45 μm nylon filter and injected directly.

Cloning and expression of carboxypeptidase in Escherichia coli Bacteria strains and primers B. amyloliquefaciens ASAG1, which was cultivated in No. 4 medium at 30°C for 8 h with shaking at 220 r min−1, was used as a template for cloning. The E. coli JM109 strain was used for cloning, and the Rosetta strain was used for expression; these strains were grown in Luria Bertani (LB) medium at 30°C. The sequences of the primers used for PCR amplification are shown below. The forward primer contained a TTG start codon and a BamHI restriction enzyme cleavage site (underlined), while the reverse primer contained a HindIII restriction enzyme cleavage site. Following digestion with BamHI/HindIII, the PCR product

Food Additives & Contaminants: Part A was subcloned into the pEB vector adjacent to the coding sequence of the 6-His tag, which was subsequently used for affinity purification: Primer sequences: Forward primer: 5ʹ-CGC GGA TCC GTT GAA CAT CAC GAA ATG GAA-3ʹ Reverse primer: 5ʹ-CCC AAG CTT AAA CCA GCC TGT TAC CG-3ʹ

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DNA preparation and transformation The coding sequence of carboxypeptidase was amplified by PCR using B. amyloliquefaciens ASAG1 genomic DNA as the template and DAIR/DAIF as the primers. The primer sequences were located in the conserved regions of FZB42 (GenBank Accession No. CP000560). The PCR reaction was performed in a total reaction volume of 40 μl. The template DNA was initially denatured for 5 min at 94°C. The PCR amplification consisted of 30 cycles with 30 s denaturing at 94°C, 30 s annealing at 50°C, 1 min extension at 68°C and a final 10 min extension at 68°C. Double-restriction enzyme digestion reaction was performed at 37°C for 3 h following the PCR reaction. The digestion reaction contained 10 μl PCR product, 4 μl 10× digestion universal buffer, 2 μl restriction endonuclease BamHI (12 units μl−1, TaKaRa) and 2 μl HindIII (12 units μl−1, TaKaRa). The pEB vector was also digested using BamHI/HindIII. Following digestion with BamHI/HindIII and purification by DNA gel electrophoresis, the PCR product was subcloned into the pEB vector, yielding the final transformation vector, pEB-CP. The pEB-CP construct was transformed into E. coli JM109 using the heat-shock method. The transformed bacteria were then plated onto an agar plate containing ampicillin. After 16 h at 37°C, positive colonies were selected, and 10 ml of cells were cultured to purify the recombinant bacmid. The purified bacmid was then transformed into E. coli Rosetta competent cells for over-expression (Galluccio et al. 2012).

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C. OTA was added to the supernatant fluid at a final concentration of 1 μg ml−1, incubated at 37°C for 12 h, extracted by dichloromethane and analysed by HPLC.

Purification of carboxypeptidase A volume of 200 ml of induced recombinant cells was harvested by centrifugation at 8000 r min−1 for 10 min at 4°C and resuspended at a ratio of binding buffer (0.02 mol l−1 sodium phosphate, 0.5 mol l−1 NaCl, and 0.02 mol l−1 imidazole, pH 7.4) volume to cell pellet weight of 5:1, disrupted by sonication for 10 min and centrifuged at 12 000 r min−1 for 10 min at 4°C. They were then filtered using a 0.22 μm filter (PES). Then, the supernatant was transferred to a 1 ml HisTrap HP column (GE Healthcare, Shanghai, China), which had been equilibrated with 5 ml binding buffer. The target protein was obtained by washing with 5 ml elution buffer (0.02 mol l−1 sodium phosphate, 0.5 mol l−1 NaCl, 0.5 mol l−1 imidazole, pH 7.4). It was detected by 5–12% SDS-PAGE.

DNA and protein sequence analysis Protein sequence analysis tools at the ExPASy Proteomics website (http://us.expasy.org/tools) were used to deduce the amino acid sequence from the corresponding cDNA sequence as well as the characteristics of the amino acid sequence.

Degradation activity of the protein purified Transformed Rosetta (pEB-CP) and non-transformed Rosetta (pEB) were induced by IPTG at 18°C with shaking at 150 r min−1 overnight. The crude enzyme extracts were prepared after the ultrasonic disruption of the cell plate and suspended in Tris-Cl (pH 7.0). The supernatant of the crude enzyme was co-cultivated overnight with OTA at a terminal concentration of 10 µg ml−1. The positive control, the ASAG1 fermented extract and protein purified by a HisTrap HP column were analysed with the same OTA concentration as the co-culture.

Degradation of OTA by recombinant Rosetta (pEB:CP) The recombinant cells and non-transformed Rosetta (pEB) were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, Dingguo, China) at a 0.8 mmol l −1 terminal concentration at 18°C with shaking at 150 r min−1 overnight in 200 ml LB media supplemented with 100 μg ml−1 ampicillin and 100 μg ml−1 chloramphenicol. After 2 h at 37°C and 220 r min−1, the cells were harvested by centrifugation at 8000 r min−1 at 4°C for 10 min. Then, the cell pellets were resuspended in 10 ml cold 20 mmol l−1 Tris-HCl buffer (pH 7.0). The cells were then disrupted by sonication for 10 min and centrifuged at 12 000 r min−1 for 5 min at 4°

Results and discussion Isolation and identity of ASAG1 The physiological, biochemical and morphological properties of the bacteria ASAG1 were determined. The strain is Gram positive, aerobic, short-rod and endospore-forming (Figure 1a), starch hydrolysed (Figure 1b), and oxidase reactions. The 16 S rDNA sequence amplified using primers Bs16sR and Bs16sF was approximately 1.5 kb. It was submitted to GenBank under Accession No. FJ597542. The phylogenetic tree of 16 S rDNA sequences was consistent with the phylogeny of some Bacillus spp.,

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Figure 1. (colour online) Biochemical and morphological properties of ASAG1: (a) Gram stain and (b) decomposition of the starch test: (1) B. amyloliquefaciens ASAG1 and (2) B. subtilis 168. The test is performed on the modified starch plate and a colour change of the iodine indicates that starch is decomposed.

such as B. subtilis, B. amyloliquefaciens and B. polyfermenticus. The alignment results indicated that the strain has high homology with B. amyloliquefaciens. Considering physiological and morphological characteristics, a biological test and the 16S rDNA sequence, the strain used in this study was confirmed to be B. amyloliquefaciens. It was deposited with the China General Microbiological Culture Collection Center (CGMCC).

Optimisation of the culture conditions ASAG1, which we isolated from maize stored in a grain depot, shows a high level of antifungal activity, especially compared with A. niger, A. ochraceus and A. carbonarius (Figure 2). A. niger was chosen as the indicator strain due to its rapid growth and the ease at which it is observed. The maximal inhibition activity of the cell-free fermented liquid was obtained when the cells were cultivated

Food Additives & Contaminants: Part A

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Figure 2.

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(colour online) Antagonism to the OTA toxigenic fungi: (a) A. niger, (b) A. ochraceus and (c) A. carbonarius.

in medium at pH 7.0 and at 31°C for 48 h. The diameter of the inhibition zone was not apparent until ASAG1 was fermented for 24 h. There was no longer a dramatic increase in the inhibition activity when the strain was cultivated for more than 48 h (Figure 3a). In the testing of different cultivation temperatures, no apparent difference in the active antifungal material produced by ASAG1 was observed between 25 and 37°C (Figure 3b). The active inhibition substances can be produced at intermediate pH values of 5.0–11.0, but the inhibition zone is the largest at a pH of 7.0 (Figure 3c). The results illustrate that the active substances could be produced by the strain at a wide range of medium pH values.

OTA degradation The HPLC analysis showed that the strain has the capacity to degrade OTA. The percentages of OTA degradation by B. amyloliquefaciens ASAG1 in liquid cultures and cellfree extracts as well as the level of degradation were determined. The level of OTA was degraded by 98.5% during the first 24 h, and there was almost no OTA remaining after 72 h. This change can be interpreted as the degree of degradation by the liquid cultures of B. amyloliquefaciens ASAG1. The OTA degradation could occur through two possible paths: adsorption by the cell wall or degradation by the active substances produced by the strain. During the first 24 h, the number of bacteria was small, and the concentration of the active substances was low. Thus, the OTA level was degraded due to degradation rather than adsorption by the cells. As time passed and more of the active substances were produced, the OTA adsorbed by the cells was eliminated, such that little OTA remained after 72 h. After cultivation in a 72-h fermenting solution without cells, degradation of approximately 99.7% of the OTA occurred within 1 h (Figure 4). Abrunhosa and Venâncio (2007) isolated a novel carboxypeptidase enzyme, which appears to be the hydrolytic enzyme of OTA. We found that carboxypeptidase is also produced by B. amyloliquefaciens FZB42. In this study, we therefore cloned and expressed this gene

from B. amyloliquefaciens ASAG1 and isolated the pure protein, which was expected to be the active material.

Cloning and expression of carboxypeptidase in Escherichia coli DNA preparation The sequence of the full-length carboxypeptidase gene, which was chosen due to its similarity to the carboxypeptidase gene of B. amyloliquefaciens FZB42, was amplified by PCR using genomic DNA as a template. An open reading frame (ORF) of 1332 bp encoded a putative polypeptide of 443 amino acid residues with a predicted molecular weight of 48.6 kDa and a theoretical isoelectric point of 5.42. The DNA sequence was deposited in GenBank under the Accession No. KP161493. Sequence alignments generated from analysis indicated that the protein sequence of B. amyloliquefaciens ASAG1 was most similar to those of B. amyloliquefaciens FZB42 (CP000560.1) and B. amyloliquefaciens DSM7 (FN597644.1), sharing identities of 99% and 97%, respectively. Similarly, the B. amyloliquefaciens ASAG1 protein sequence shared 86% identity with B. subtilis W23 (CP002183.1) and B. subtilis 168 (AL009126.3).

Protein expression and purification The obtained DNA fragment and prokaryotic expression vector pEB were digested completely with BamHI and HindIII. The recombinant plasmid was ligated using T4 DNA ligase and transformed into Rosetta. The plasmid could be identified by double digestion, and the recombinant strain also had the ability to degrade OTA by 23.9% after a 12-h cultivation. In contrast, no degradation took place in the Rosetta strain or the negative control. The B. amyloliquefaciens ASAG1 pEB-CP protein was produced at a high level in the E. coli Rosetta, producing a fusion protein containing a 6-His-Tag, which was isolated using a HisTrap affinity column, and the full-length of recombinant pEB-CP was carboxypeptidase with a His tag.

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Figure 3. Effect of different culture conditions on the inhibition activity and the growth of A. niger by ASAG1 fermentation broth: (a) different fermentation time, (b) different cultivation temperature and (c) different medium pH.

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Food Additives & Contaminants: Part A

Figure 4.

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HPLC analysis of OTA co-cultivated with ASAG1 medium for different periods of time (hours).

Degradation activity of the fermentation broth The HPLC analysis indicated that OTA decreased by 41% and 72% when co-cultivated with the supernatant of the crude enzyme and the purified protein of carboxypeptidase, respectively. The cell-free extract of ASAG1 and the purified carboxypeptidase expressed in E. coli showed strong inhibition of the growth of A. niger. The negative control consisting of LB medium and the B. subtilis 168 cell-free extract confirmed that there was no antagonism (Figure 5).

Previously, researchers isolated and purified carboxypeptidase from an A. niger species and bovine pancreas specimens (Stander et al. 2001; Abrunhosa & Venâncio 2007). In the present work, B. amyloliquefaciens ASAG1 showed a high level of inhibition against OTA-producing fungi. Meanwhile, carboxypeptidase, an active substance, was more efficient at OTA degradation in vitro than in the strain. Research conducted by Xiao et al. demonstrated that OTA was cleaved into the non-toxic ochratoxin α (OTα) and L-phenylalanine (Phe) by carboxypeptidase (1995). Moreover, the metabolism of OTA, along with that of the nontoxic OTα, hydrolysed by carboxypeptidase, was measured, and the kinetic data on OTA were clarified (Stander et al. 2001; Tozlovanu et al. 2012). It can be inferred that the derived metabolites of OTA were hydrolysed into OTα by carboxypeptidase produced by ASAG1. Microorganisms and biologic enzyme degradation approaches must be developed as complementary biomonitoring tools. However, further research should be performed to identify whether there are any other active substances that play roles in the detoxification process.

Conclusions

Figure 5. (colour online) Detection of the antagonism to A. niger: (1) LB medium, (2) B. subtilis 168, (3) ASAG1 medium cultivated for 72 h and (4) crude enzyme of carboxypeptidase.

OTA is a fungal carcinogen found in several foods and feed products. B. amyloliquefaciens ASAG1 was isolated from maize and found to be strongly antagonistic to the OTA of toxigenic fungi. The inhibition of active substances occurs over a broad range of pH values, from 5.0 to 11.0. At pH 7 and 37°C, the inhibition activity of the ASAG1 fermentation broth was higher than at any other conditions tested. Moreover, the product of the carboxypeptidase gene, which was cloned from ASAG1, shows substantial OTA degradation in vitro. The application of B. amyloliquefaciens ASAG1 to degrade the OTA

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content of contaminated agricultural products is the subject of current evaluation. Funding This study was supported by grants from the Ministry of Sciences and Technology of China [973 Program, grant number 2013CB127805].

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Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1.

Ochratoxin A (OTA) is widely found in food and feed products as a mycotoxin contaminant. It is produced by Penicillium species and several Aspergillus...
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