Research Article Received: 31 August 2013
Revised: 14 November 2013
Accepted article published: 30 November 2013
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6507
Ochratoxin A biocontrol and biodegradation by Bacillus subtilis CW 14 Lei Shi,a,b Zhihong Liang,a∗ Junxia Li,a Junran Hao,a Yuancong Xu,a Kunlun Huang,a,b,c Jingjing Tian,a Xiaoyun Hea and Wentao Xua,b,c∗ Abstract BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species. In this study a strain of Bacillus subtilis was tested for its effects on OTA-producing Aspergillus and OTA degradation. The mechanisms of the effects were also investigated. RESULTS: A strain of Bacillus spp. isolated from fresh elk droppings was screened out using the methods described by Guan et al. (Int J Mol Sci 9:1489–1503 (2008)). The 16S rRNA gene sequence suggested that it was B. subtilis CW 14. It could inhibit the growth of the OTA-producing species Aspergillus ochraceus 3.4412 and Aspergillus carbonarius, with inhibition rates of 33.0 and 33.3% respectively. At 6 µg mL−1 OTA, both viable and autoclaved (121 ◦ C, 20 min) cells of CW 14 bound more than 60% of OTA. In addition, OTA was degraded by the cell-free supernatant of CW 14. By high-performance liquid chromatography, the cell-free supernatant degraded 97.6% of OTA after 24 h of incubation at 30 ◦ C, and no degradation products were produced. The fastest degradation occurred during the first 2 h. In 3 g samples of contaminated maize, 47.1% of OTA was degraded by 50 mL inocula of overnight cultures of CW 14. CONCLUSION: These findings indicated that B. subtilis CW 14 could both prevent OTA contamination and degrade OTA in crops. c 2013 Society of Chemical Industry Keywords: biocontrol; biodegradation; ochratoxin A; Bacillius subtilis CW 14
INTRODUCTION Each year, approximately 25% of the world’s commodities are contaminated by mycotoxins according to the Food and Agricultural Organization (FAO). Among the more than 300 known mycotoxins, ochratoxin A (OTA) is one of the most important. It is nephrotoxic, hepatotoxic, teratogenic and carcinogenic in animals and is also involved in the pathogenesis of Balkan endemic nephropathy. In 1993 the International Agency for Research on Cancer (IARC) classified OTA as a possible human carcinogen (group 2B). OTA is produced by several Aspergillus and Penicillium species. Aspergillus ochraceus was the first OTA producer discovered by Van Der Merwe et al.,1 and it affects crops in the tropics and subtropics. Aspergillus carbonarius is another major source of OTA contamination in fruits, especially in grapes and grape products.2 Penicillium verrucosum has better OTA production ability than A. carbonarius and mainly contaminates crops in cold regions.3 In the present experiments, A. ochraceus 3.4412 and A. carbonarius were chosen for inhibition assays. The best approach to reduce OTA levels in crops is to prevent fungal growth and OTA production in crops both before and after harvest. Biological control appears to be a more promising method than physical and chemical methods. Munimbazi and Bullerman4 reported that antifungal metabolites produced by Bacillus pumilus inhibited the mycelial growth of many species of Aspergillus, Pencillium and Fusarium, as well as inhibiting the production of aflatoxins, cyclopiazonic acid, OTA and patulin. Some non-toxigenic Aspergillus strains have been used to reduce J Sci Food Agric (2013)
aflatoxin contamination by 70–90%,5,6 and this inhibition is based on competitively excluding naturally toxigenic strains in the same niche and competing for crop substrates. Although the ideal solution for management of mycotoxins is to prevent them from contaminating foods, contamination cannot be completely avoided, so degradation approaches must also be taken. Biological degradation is the method of choice to deactivate mycotoxins. Such degradation comprises toxin binding by adsorptive materials and microbial inactivation by specific microorganisms or enzymes. Various organisms have been employed for this purpose, including lactic acid bacteria,7 Bacillus spp.,8 Phaffia rhodozyma9 and mycetes.10,11 Moreover, some enzymes were also reported to hydrolyze OTA. Carboxypeptidase A (CPA) (EC 3.4.17.1) from bovine pancreas was the first enzyme
∗
Correspondence to: Zhihong Liang; Wentao Xu, Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China. E-mail: [email protected], xuwentaoboy@ sina.com
a Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China b The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing, 100083, China c Beijing Key Laboratory of Nutrition, Health and Food Safety, Beijing, 100083, China
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www.soci.org found to hydrolyze the amide bond of OTA to L-β-phenylalanine and ochratoxin α (OTα), which was less toxic than OTA.12 Bacillus subtilis is a Gram-positive, rod-shaped and endosporeforming aerobic bacterium. It is found in soil and rotting plant material and is non-pathogenic. The formed spores can survive in adverse conditions and are resistant to many physicochemical factors. Bacillus subtilis also produces many inhibitory metabolites.13 Several strains related to B. subtilis are used in the commercial production of extracellular enzymes such as α-amylase. Other strains produce insect toxins, peptide antibiotics and antifungals, some of which have been used in agricultural crop protection. In this study a strain of B. subtilis CW 14 isolated from fresh elk droppings was tested for its ability to remove OTA, and a preliminary investigation of the mechanisms of OTA biocontrol and degradation was conducted.
MATERIALS AND METHODS Materials Standard OTA was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Aspergillus ochraceus 3.4412 was purchased from the Institute of Microbiology, Chinese Academy of Sciences. Aspergillus carbonarius was isolated from grapes in our laboratory. Methanol and acetonitrile of high-performance liquid chromatography (HPLC) grade were purchased from Fisher Scientific (Pittsburgh, PA, USA). Isolation, screening and identification of microorganisms Fresh elk droppings were collected from Beijing Zoo. A 1 g sample was suspended in 9 mL of phosphate-buffered saline (PBS, pH 7.4), mixed well by vortex and then agitated for 30 min. Appropriate dilutions (up to 107 colony-forming units (CFU) mL−1 ) were made with PBS. Each dilution (100 µL) was spread on Luria–Bertani (LB)nutrient agar (10 g L−1 pancreatic peptone, 5 g L−1 yeast extract, 10 g L−1 NaCl, 15 g L−1 agar, pH 7) and incubated at 37 ◦ C for 24 h. Colonies appearing on the plates were harvested and maintained on LB-nutrient agar at 4 ◦ C. Culture stocks were prepared by cultivation on LB-nutrient broth (10 g L−1 pancreatic peptone, 5 g L−1 yeast extract, 10 g L−1 NaCl, pH 7) and stored at −80 ◦ C after addition of sterile glycerol to 200 g L−1 . Two hundred colonies were first characterized by Gram’s staining technique and morphological observation. The Grampositive bacteria grown well were inoculated in LB-nutrient agar with 0.6 and 3 µg mL−1 OTA at 37 ◦ C for 24 h and stored at −80 ◦ C after addition of sterile glycerol to 200 g L−1 . Four Bacillus spp. were selected as the most effective using isocoumarin as the sole carbon source.14 To identify the bacterial strains, the 16S rRNA gene sequences of one interesting colony were amplified using the methods described by Upadhaya et al.15 Growth inhibition of A. ochraceus 3.4412 and A. carbonarius by B. subtilis CW 14 and its cell-free supernatant Bacillus subtilis CW 14 was initially studied for antagonism against A. ochraceus 3.4412 and A. carbonarius using the dual culture technique. The culture media used were LB-nutrient agar and potato dextrose agar (PDA, 200 g L−1 potato, 20 g L−1 glucose, 15 g L−1 agar).8 The media were poured into 90 mm Petri dishes. A single colony of CW 14 was inoculated at one-third of the diameter, and A. ochraceus 3.4412 or A. carbonarius was inoculated at two-thirds of the diameter. The plates were incubated at 28 ◦ C
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for 7 days. Triplicates were used throughout for each combination. Radial growth reduction was calculated according to the formula8 mycelial inhibition (%) =
r − r /r × 100
where r (mm) is the growth of the fungus from the center of the colony towards the edge of the Petri dish and r (mm) is the growth of the fungus from the center of the colony towards the center of the bacteria tested. The antifungal activity of the cell-free supernatant of B. subtilis was studied using an agar well assay. Bacillus subtilis CW 14 was incubated in LB-nutrient broth at 37 ◦ C overnight. The freshly grown culture was then centrifuged at 6000 × g for 5 min at 4 ◦ C and the supernatant was transferred to a new sterile centrifuge tube. A plug of fungi was plated at the center of a Petri dish containing 25 mL of PDA and incubated at 28 ◦ C for 2 days. After this incubation, three wells with a diameter of 6 mm were cut into the agar using a sterile cork-borer. Cell-free supernatant (100 µL) was added to the wells, and the plate was incubated for a further 5 days. Each test was conducted in triplicate and the mycelial inhibition was calculated as above. Next, the hyphae of A. ochraceus 3.4412 and A. carbonarius were harvested for microscopic examination. Briefly, the hyphae facing the B. subtilis CW 14 colony were sectioned uniformly (∼0.25 mm thickness) with a sterilized stainless blade. The hyphae were stained with methyl blue and observed with an optical microscope (DM2500, Leica, Wetzlar, Germany) and further assessed using a scanning electron microscope (S-3400 N, Hitachi, Tokyo, Japan). OTA degradation by B. subtilis CW 14 Bacillus subtilis CW 14 was cultured in LB-nutrient broth at 37 ◦ C in an orbital shaker at 200 rpm for 24 h. The cells were harvested by centrifugation at 6000 × g for 10 min at 4 ◦ C and divided into two portions for use as viable (untreated) or autoclaved (121 ◦ C, 20 min) cells. Cell pellets were washed twice with 3 mL of sterile PBS and resuspended in 2.9 mL of sterile PBS to which 0.1 mL of OTA was added. The final concentration of OTA was 6 µg mL−1 . The cell suspension was incubated at 30 ◦ C and 200 rpm for 24 h in the dark. Bacillus subtilis CW 14 was grown in LB-nutrient broth at 37 ◦ C and 200 rpm for 24 h. The cell-free supernatant was harvested by centrifugation at 6000 × g for 10 min at 4 ◦ C. The cell pellets were washed twice with 3 mL of PBS, resuspended in 4 mL of PBS and sonicated for 10 min with an ultrasonicator (JY92-I2N, SCIENTZ, Ningbo, China). The resuspended solution was centrifuged at 11 000 × g for 20 min at 4 ◦ C. The intracellular supernatant and the cell-free supernatant were aseptically filtered using 0.22 µm sterile cellulose pyrogen-free disposable filters. Aliquots (3 mL) of the cell-free supernatant or intracellular supernatant containing 6 µg mL−1 of OTA were incubated at 30 ◦ C and 200 rpm for 24 h. The extraction of OTA was performed according to Abrunhosa et al.11 Briefly, the suspensions were acidified with 2 mol L−1 HCl and extracted twice with 1 mL of chloroform. The extracts were centrifuged at 6000 × g for 5 min at 4 ◦ C to recover OTA. Determination of OTA Thin layer chromatography (TLC) analysis was performed according to Liang.16 Briefly, chloroform extracts (30 µL) were spotted on silica gel plates and developed in toluene/ethyl acetate/formic acid (6:3:1 v/v/v). OTA was examined under UV light.
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Ochratoxin A biocontrol and biodegradation
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Figure 1. Sequence comparison between CW 14 and Bacillus subtilis (Genbank No. GU191901).
For HPLC analysis the chloroform extracts were filtered using 0.22 µm cellulose pyrogen-free disposable filters, evaporated, redissolved and diluted with methanol. Aliquots of 20 µL were used for HPLC analyses. Kinetic studies were repeated three times. The HPLC equipment consisted of a solvent delivery system with a fluorescence detector (λex = 334 nm, λem = 460 nm). BST Rutin C18 BD HPLC columns (250 mm × 4 mm, particle size 10 µm; BioSeparation Techniques, Budapest, Hungary) were used. OTA was eluted with acetonitrile/water/acetic acid (100:100:1 v/v/v) as the mobile phase at a flow rate of 1 mL min−1 .11 The relative standard deviation of this analysis method is 4–5%. The rate of OTA degradation was calculated using the formula OTA degradation (%) = (1 − OTA peak area in treatment /OTA peak area in control) × 100 Effects of B. subtilis CW 14 on OTA production in contaminated maize Aspergillus ochraceus 3.4412 was chosen as the toxin-producing fungus. A spore suspension of A. ochraceus 3.4412 was prepared as follows. Aspergillus ochraceus 3.4412 was induced to sporulate on a PDA plate at 28 ◦ C for 7 days. Spores were harvested by adding 10 mL of sterile water to the plate and gently joggling and dislodging spores into a 50 mL Erlenmeyer flask with glass beads. The Erlenmeyer flask was shaken for 12 h and the spore suspension was filtered through four layers of sterile cheesecloth to remove mycelial debris. The concentration of spores was determined using a hemocytometer and adjusted to 107 spores mL−1 . The maize used in this study was purchased from local supermarket outlets. Autoclaved maize (30 g) was inoculated with 0.5 mL of the spore suspension (107 spores mL−1 ) described above. The final moisture content of the maize was adjusted to 180 g kg−1 J Sci Food Agric (2013)
with sterile water. After incubation in the dark at 28 ◦ C and 200 rpm for 21 days, the maize was sterilized at 121 ◦ C for 20 min and then 3 g of maize was mixed with 50 mL of the overnight culture of B. subtilis. The mixture was incubated at 30 ◦ C and 200 rpm for 72 h and extracted for OTA according to the method described by Liang.16 The extract was diluted with an appropriate volume of methanol and stored at −20 ◦ C for HPLC analysis. As a control, maize without B. subtilis was prepared. All tests were performed in triplicate. Statistics Statistical analyses were performed on repeated measurements by one-way analysis of variance (ANOVA) using Prism Version 5.0 (GraphPad Software, Inc., San Diego, CA, USA), followed by Bonferroni’s multiple comparison test. P values less than 0.05 were considered statistically significant.
RESULTS AND DISCUSSION Bacillus isolation, screening and identification Two hundred colonies were isolated from the sample of fresh elk droppings on LB-nutrient agar. The results of Gram’s staining technique showed that about two-thirds of the strains were Gram-positive bacteria. About 60 Gram-positive bacteria were cultivated in LB-nutrient agar with OTA, of which 12 could grow well at 0.6 µg mL−1 and four at 3 µg mL−1 . To obtain the most effective strain, the latter four strains were cultivated again using isocoumarin as the sole carbon source, and only one strain, named CW 14, was able to use isocoumarin and grow well. OTA was a derivative composed of isocoumarin, phenylalanine and different structure groups, mainly methyl and ethyl groups. It was viable that isocoumarin as a selective agent was used for screening instead of
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A
B
C
D
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Figure 2. In vitro interactions between Aspergillus ochraceus 3.4412 and Bacillus subtilis CW 14 in dual culture at day 7: A and C, normal hyphal growth in a control (arrows); B and D, curly growth of A. ochraceus 3.4412 hyphae facing a B. subtilis CW 14 colony (arrows). A and B were imaged with a digital camera (Leica DFC295) connected to an optical microscope (Leica DM2500); C and D were imaged with a scanning electron microscope (Hitachi S-3400N). 120
Mycelial inhibition (%) OTA-producing species
B. subtilis CW 14
A. ochraceus 3.4412 A. carbonarius a
30.0 33.3
Cell-free supernatant 28.8 44.4
Samples were incubated at 28 ◦ C for 7 days under aerobic conditions.
OTA. Guan et al.14 used coumarin as the sole carbon and energy source to screen bacteria degrading aflatoxin B1 (AFB1), and 25 bacterial isolates reducing AFB1 were obtained from 65 samples. CW 14 was confirmed by 16S rRNA gene sequences and showed 99% sequence similarity to B. subtilis (Fig. 1). Bacillus subtilis CW 14 and its cell-free supernatant inhibit growth of A. ochraceus 3.4412 and A. carbonarius Bacillus subtilis CW 14 clearly inhibited the growth of A. ochraceus 3.4412 and A. carbonarius in dual test plates at rates of 30.0 and 33.3% respectively (Table 1). The inhibition rate was close to that of Bacillus spp. for Aspergillus westerdijkiae NRRL 3174 (34%)8 and less than that of Bacillus spp. for Aspergillus flavus (52%).8
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OTA adsorption (%)
Table 1. Effects of Bacillus subtilis CW 14 and its cell-free supernatant on growth of Aspergillus ochraceus 3.4412 and Aspergillus carbonariusa
80
40
0 Viable cells
Autoclaved cells
Figure 3. OTA adsorption by viable and autoclaved (121 ◦ C, 20 min) cells of Bacillus subtilis CW 14 (107 CFU mL−1 ) for 24 h. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 24 h under aerobic conditions in the dark.
However, the inhibitory effect might be due to competition for nutrients and/or space between B. subtilis CW 14 and the mycete17 or to diffusible inhibitory substances produced by B. subtilis CW 14 that suppressed the growth of the mycete.4,18 To further study the inhibition mechanism, an agar well diffusion assay was performed. The inhibition rates of the cell-free supernatant against A.ochraceus
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www.soci.org normal (Figs 2A and 2C). Similar results could be seen in the study of Islam.19 According to the above results, CW 14 has potential as a biological agent to control OTA levels in crops.
Figure 4. TLC of cell-free supernatant and intracellular supernatant of Bacillus subtilis CW 14 tested for OTA degradation. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 24 h under aerobic conditions in the dark. Lanes: CK, OTA in LB-nutrient broth; A and B, OTA in cell-free supernatant; C and D, OTA in intracellular supernatant. No detectable degradation productions were produced.
3.4412 and A. carbonarius were 28.8 and 44.4% respectively in comparison with the above results, indicating the presence of diffusible inhibitory substances. In the optical microscope and scanning electron microscope, CW 14 induced curling (Figs 2B and 2D) in the confronting hyphae; moreover, there was excessive branching (ca one- to twofold higher than the control). In contrast, the hyphae in control samples were straight and branching was
OTA degradation by B. subtilis CW 14 The ability of B. subtilis CW 14 to bind OTA in vitro is shown in Fig. 3. Viable cells bound 66.6% while autoclaved cells bound 87.9% of 6 µg mL−1 OTA after 24 h. OTA adsorption by B. subtilis CW 14 was similar to that by Saccharomyces and Lactobacillus, as all are based on physical adsorption to the cell wall of the microorganism. However, the absorption rate of B. subtilis CW 14 was less than that of Lactobacillus (≥95%)7 and similar to that of Saccharomyces (∼75%).20 In our study, autoclaved (121 ◦ C, 20 min) cells bound 21.3% more OTA than viable cells. This phenomenon may due to the enhancement of adsorption sites by heat treatment.20 The results agreed with previous reports.20,21 Next, to further study OTA degradation by B. subtilis CW 14, different cell fractions were tested for their OTA degradation ability. As shown in Fig. 4, no degradation was observed in the intracellular supernatant (lanes C and D), while the cell-free supernatant had very high OTA-degrading ability (lanes A and B). For quantification of OTA degradation by the cell-free supernatant, HPLC was used. The OTA was 97.6% degraded (Fig. 5B) and no degradation products were observed on the chromatogram. There have been reports that OTA could be metabolized by microorganisms and/or enzymes through hydrolysis of the amide bond (formation of OTα),8,11,12 dechlorination of the isocoumarin moiety (formation
A
B
Figure 5. HPLC of Bacillus subtilis CW 14 cell-free supernatant tested for OTA degradation: A, OTA in LB-nutrient broth; B, OTA in cell-free supernatant. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 24 h under aerobic conditions in the dark. The retention time of OTA was 8.34 min. No detectable degradation products were produced.
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and incubated for 24 h at 30 ◦ C. The B. subtilis CW 14 grew well, indicating that CW 14 could be grown in autoclaved and contaminated maize.
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100 80
CONCLUSION
60 40 20 0 0
2
4
8
12
24
Time (h) Figure 6. Time course of OTA degradation by Bacillus subtilis CW 14 cell-free supernatant. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 0, 2, 4, 8, 12 and 24 h under aerobic conditions in the dark.
of OTB) or opening of the lactone ring (formation of OP-OTA).22 Some Aspergillus spp.11 and Bacillus licheniformis8 could hydrolyze OTA to OTα; moreover, their degradation rate (>90%) was close to that of B. subtilis CW 14. However, in our study, no OTα or other degradation products were produced. Xiao23 reported that Aspergillus fumigatus, Aspergillus japonicus and Aspergillus niger degraded 2 mg L−1 OTA after 10 days; OTα was detected later and degraded into unknown compounds. To establish the optimal incubation time for OTA degradation by the cell-free supernatant, an experiment on the incubation time dependence of OTA degradation was conducted. The results are shown in Fig. 6. The fastest degradation of OTA occurred in the first 2 h, and after 12 h virtually no further degradation had occurred; moreover, no degradation products were detected at these time points. Our research indicated that neither OTα nor other degradation products were detectable, even in the first hour of degradation. Thus it is unlikely that OTA is hydrolyzed by cell-free supernatants via the same mechanism as it is hydrolyzed by carboxypeptidase A (CPA) (EC 3.4.17.1) from bovine pancreas12 or by other hydrolases.24 We propose that another degradation pathway is in operation or that more than one enzyme is involved in the hydrolysis of OTA, as with aflatoxin degradation.25 Effects of B. subtilis CW 14 on OTA production in contaminated maize Autoclaved (121 ◦ C, 20 min) and contaminated maize (3 g, with ca 0.3 mg of OTA) was incubated with B. subtilis CW 14 for 72 h at 30 ◦ C and extracted for OTA. OTA was assessed by HPLC. The OTA was 47.1% degraded, without any detectable degradation products, which was a lower degradation rate than that of pure OTA. Varga et al.10 reported that Rhizopus stolonifer could degrade 96.5% of OTA on wheat at 30 ◦ C in 10 days. In our study the concentration of OTA in the contaminated maize (0.1 mg g−1 ) was approximately 17 times greater than in pure OTA (6 µg mL−1 ). The environmental conditions in the contaminated maize, such as humidity, pH, nutrients and metabolites produced by A. ochraceus 3.4412, likely had effects on the degradation by B. subtilis CW 14. The observation that B. subtilis CW 14 is able to degrade OTA in contaminated maize is promising. In the future, B. subtilis CW 14 might be used as a food additive for degradation of animal feed and crops. Before extraction, B. subtilis CW 14 from the autoclaved and contaminated maize was inoculated onto LB-nutrient agar plates
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Bacillus subtilis CW 14 and its cell-free supernatant inhibited the growth of the toxin-producing species A. ochraceus 3.4412 and A. carbonarius and disrupted their hyphae. Bacillus subtilis CW 14 could also adsorb and degrade OTA, and the degradation was due to metabolites secreted into the culture medium. Thus B. subtilis CW 14 has potential for use as a biocontrol agent to prevent OTA production by Aspergillus spp. and as an additive agent to degrade OTA in crops. It remains to be determined whether inhibition and degradation are both accomplished by the same substance(s). Further studies are in progress to isolate and identify the active substances.
ACKNOWLEDGEMENT This work was funded by Fundamental Research Funds for the Central Universities (grants 2011 K0805 and 2013QJ036). The funders have no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Revised: 14 November 2013
Accepted article published: 30 November 2013
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6507
Ochratoxin A biocontrol and biodegradation by Bacillus subtilis CW 14 Lei Shi,a,b Zhihong Liang,a∗ Junxia Li,a Junran Hao,a Yuancong Xu,a Kunlun Huang,a,b,c Jingjing Tian,a Xiaoyun Hea and Wentao Xua,b,c∗ Abstract BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species. In this study a strain of Bacillus subtilis was tested for its effects on OTA-producing Aspergillus and OTA degradation. The mechanisms of the effects were also investigated. RESULTS: A strain of Bacillus spp. isolated from fresh elk droppings was screened out using the methods described by Guan et al. (Int J Mol Sci 9:1489–1503 (2008)). The 16S rRNA gene sequence suggested that it was B. subtilis CW 14. It could inhibit the growth of the OTA-producing species Aspergillus ochraceus 3.4412 and Aspergillus carbonarius, with inhibition rates of 33.0 and 33.3% respectively. At 6 µg mL−1 OTA, both viable and autoclaved (121 ◦ C, 20 min) cells of CW 14 bound more than 60% of OTA. In addition, OTA was degraded by the cell-free supernatant of CW 14. By high-performance liquid chromatography, the cell-free supernatant degraded 97.6% of OTA after 24 h of incubation at 30 ◦ C, and no degradation products were produced. The fastest degradation occurred during the first 2 h. In 3 g samples of contaminated maize, 47.1% of OTA was degraded by 50 mL inocula of overnight cultures of CW 14. CONCLUSION: These findings indicated that B. subtilis CW 14 could both prevent OTA contamination and degrade OTA in crops. c 2013 Society of Chemical Industry Keywords: biocontrol; biodegradation; ochratoxin A; Bacillius subtilis CW 14
INTRODUCTION Each year, approximately 25% of the world’s commodities are contaminated by mycotoxins according to the Food and Agricultural Organization (FAO). Among the more than 300 known mycotoxins, ochratoxin A (OTA) is one of the most important. It is nephrotoxic, hepatotoxic, teratogenic and carcinogenic in animals and is also involved in the pathogenesis of Balkan endemic nephropathy. In 1993 the International Agency for Research on Cancer (IARC) classified OTA as a possible human carcinogen (group 2B). OTA is produced by several Aspergillus and Penicillium species. Aspergillus ochraceus was the first OTA producer discovered by Van Der Merwe et al.,1 and it affects crops in the tropics and subtropics. Aspergillus carbonarius is another major source of OTA contamination in fruits, especially in grapes and grape products.2 Penicillium verrucosum has better OTA production ability than A. carbonarius and mainly contaminates crops in cold regions.3 In the present experiments, A. ochraceus 3.4412 and A. carbonarius were chosen for inhibition assays. The best approach to reduce OTA levels in crops is to prevent fungal growth and OTA production in crops both before and after harvest. Biological control appears to be a more promising method than physical and chemical methods. Munimbazi and Bullerman4 reported that antifungal metabolites produced by Bacillus pumilus inhibited the mycelial growth of many species of Aspergillus, Pencillium and Fusarium, as well as inhibiting the production of aflatoxins, cyclopiazonic acid, OTA and patulin. Some non-toxigenic Aspergillus strains have been used to reduce J Sci Food Agric (2013)
aflatoxin contamination by 70–90%,5,6 and this inhibition is based on competitively excluding naturally toxigenic strains in the same niche and competing for crop substrates. Although the ideal solution for management of mycotoxins is to prevent them from contaminating foods, contamination cannot be completely avoided, so degradation approaches must also be taken. Biological degradation is the method of choice to deactivate mycotoxins. Such degradation comprises toxin binding by adsorptive materials and microbial inactivation by specific microorganisms or enzymes. Various organisms have been employed for this purpose, including lactic acid bacteria,7 Bacillus spp.,8 Phaffia rhodozyma9 and mycetes.10,11 Moreover, some enzymes were also reported to hydrolyze OTA. Carboxypeptidase A (CPA) (EC 3.4.17.1) from bovine pancreas was the first enzyme
∗
Correspondence to: Zhihong Liang; Wentao Xu, Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China. E-mail: [email protected], xuwentaoboy@ sina.com
a Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China b The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing, 100083, China c Beijing Key Laboratory of Nutrition, Health and Food Safety, Beijing, 100083, China
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www.soci.org found to hydrolyze the amide bond of OTA to L-β-phenylalanine and ochratoxin α (OTα), which was less toxic than OTA.12 Bacillus subtilis is a Gram-positive, rod-shaped and endosporeforming aerobic bacterium. It is found in soil and rotting plant material and is non-pathogenic. The formed spores can survive in adverse conditions and are resistant to many physicochemical factors. Bacillus subtilis also produces many inhibitory metabolites.13 Several strains related to B. subtilis are used in the commercial production of extracellular enzymes such as α-amylase. Other strains produce insect toxins, peptide antibiotics and antifungals, some of which have been used in agricultural crop protection. In this study a strain of B. subtilis CW 14 isolated from fresh elk droppings was tested for its ability to remove OTA, and a preliminary investigation of the mechanisms of OTA biocontrol and degradation was conducted.
MATERIALS AND METHODS Materials Standard OTA was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Aspergillus ochraceus 3.4412 was purchased from the Institute of Microbiology, Chinese Academy of Sciences. Aspergillus carbonarius was isolated from grapes in our laboratory. Methanol and acetonitrile of high-performance liquid chromatography (HPLC) grade were purchased from Fisher Scientific (Pittsburgh, PA, USA). Isolation, screening and identification of microorganisms Fresh elk droppings were collected from Beijing Zoo. A 1 g sample was suspended in 9 mL of phosphate-buffered saline (PBS, pH 7.4), mixed well by vortex and then agitated for 30 min. Appropriate dilutions (up to 107 colony-forming units (CFU) mL−1 ) were made with PBS. Each dilution (100 µL) was spread on Luria–Bertani (LB)nutrient agar (10 g L−1 pancreatic peptone, 5 g L−1 yeast extract, 10 g L−1 NaCl, 15 g L−1 agar, pH 7) and incubated at 37 ◦ C for 24 h. Colonies appearing on the plates were harvested and maintained on LB-nutrient agar at 4 ◦ C. Culture stocks were prepared by cultivation on LB-nutrient broth (10 g L−1 pancreatic peptone, 5 g L−1 yeast extract, 10 g L−1 NaCl, pH 7) and stored at −80 ◦ C after addition of sterile glycerol to 200 g L−1 . Two hundred colonies were first characterized by Gram’s staining technique and morphological observation. The Grampositive bacteria grown well were inoculated in LB-nutrient agar with 0.6 and 3 µg mL−1 OTA at 37 ◦ C for 24 h and stored at −80 ◦ C after addition of sterile glycerol to 200 g L−1 . Four Bacillus spp. were selected as the most effective using isocoumarin as the sole carbon source.14 To identify the bacterial strains, the 16S rRNA gene sequences of one interesting colony were amplified using the methods described by Upadhaya et al.15 Growth inhibition of A. ochraceus 3.4412 and A. carbonarius by B. subtilis CW 14 and its cell-free supernatant Bacillus subtilis CW 14 was initially studied for antagonism against A. ochraceus 3.4412 and A. carbonarius using the dual culture technique. The culture media used were LB-nutrient agar and potato dextrose agar (PDA, 200 g L−1 potato, 20 g L−1 glucose, 15 g L−1 agar).8 The media were poured into 90 mm Petri dishes. A single colony of CW 14 was inoculated at one-third of the diameter, and A. ochraceus 3.4412 or A. carbonarius was inoculated at two-thirds of the diameter. The plates were incubated at 28 ◦ C
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for 7 days. Triplicates were used throughout for each combination. Radial growth reduction was calculated according to the formula8 mycelial inhibition (%) =
r − r /r × 100
where r (mm) is the growth of the fungus from the center of the colony towards the edge of the Petri dish and r (mm) is the growth of the fungus from the center of the colony towards the center of the bacteria tested. The antifungal activity of the cell-free supernatant of B. subtilis was studied using an agar well assay. Bacillus subtilis CW 14 was incubated in LB-nutrient broth at 37 ◦ C overnight. The freshly grown culture was then centrifuged at 6000 × g for 5 min at 4 ◦ C and the supernatant was transferred to a new sterile centrifuge tube. A plug of fungi was plated at the center of a Petri dish containing 25 mL of PDA and incubated at 28 ◦ C for 2 days. After this incubation, three wells with a diameter of 6 mm were cut into the agar using a sterile cork-borer. Cell-free supernatant (100 µL) was added to the wells, and the plate was incubated for a further 5 days. Each test was conducted in triplicate and the mycelial inhibition was calculated as above. Next, the hyphae of A. ochraceus 3.4412 and A. carbonarius were harvested for microscopic examination. Briefly, the hyphae facing the B. subtilis CW 14 colony were sectioned uniformly (∼0.25 mm thickness) with a sterilized stainless blade. The hyphae were stained with methyl blue and observed with an optical microscope (DM2500, Leica, Wetzlar, Germany) and further assessed using a scanning electron microscope (S-3400 N, Hitachi, Tokyo, Japan). OTA degradation by B. subtilis CW 14 Bacillus subtilis CW 14 was cultured in LB-nutrient broth at 37 ◦ C in an orbital shaker at 200 rpm for 24 h. The cells were harvested by centrifugation at 6000 × g for 10 min at 4 ◦ C and divided into two portions for use as viable (untreated) or autoclaved (121 ◦ C, 20 min) cells. Cell pellets were washed twice with 3 mL of sterile PBS and resuspended in 2.9 mL of sterile PBS to which 0.1 mL of OTA was added. The final concentration of OTA was 6 µg mL−1 . The cell suspension was incubated at 30 ◦ C and 200 rpm for 24 h in the dark. Bacillus subtilis CW 14 was grown in LB-nutrient broth at 37 ◦ C and 200 rpm for 24 h. The cell-free supernatant was harvested by centrifugation at 6000 × g for 10 min at 4 ◦ C. The cell pellets were washed twice with 3 mL of PBS, resuspended in 4 mL of PBS and sonicated for 10 min with an ultrasonicator (JY92-I2N, SCIENTZ, Ningbo, China). The resuspended solution was centrifuged at 11 000 × g for 20 min at 4 ◦ C. The intracellular supernatant and the cell-free supernatant were aseptically filtered using 0.22 µm sterile cellulose pyrogen-free disposable filters. Aliquots (3 mL) of the cell-free supernatant or intracellular supernatant containing 6 µg mL−1 of OTA were incubated at 30 ◦ C and 200 rpm for 24 h. The extraction of OTA was performed according to Abrunhosa et al.11 Briefly, the suspensions were acidified with 2 mol L−1 HCl and extracted twice with 1 mL of chloroform. The extracts were centrifuged at 6000 × g for 5 min at 4 ◦ C to recover OTA. Determination of OTA Thin layer chromatography (TLC) analysis was performed according to Liang.16 Briefly, chloroform extracts (30 µL) were spotted on silica gel plates and developed in toluene/ethyl acetate/formic acid (6:3:1 v/v/v). OTA was examined under UV light.
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Figure 1. Sequence comparison between CW 14 and Bacillus subtilis (Genbank No. GU191901).
For HPLC analysis the chloroform extracts were filtered using 0.22 µm cellulose pyrogen-free disposable filters, evaporated, redissolved and diluted with methanol. Aliquots of 20 µL were used for HPLC analyses. Kinetic studies were repeated three times. The HPLC equipment consisted of a solvent delivery system with a fluorescence detector (λex = 334 nm, λem = 460 nm). BST Rutin C18 BD HPLC columns (250 mm × 4 mm, particle size 10 µm; BioSeparation Techniques, Budapest, Hungary) were used. OTA was eluted with acetonitrile/water/acetic acid (100:100:1 v/v/v) as the mobile phase at a flow rate of 1 mL min−1 .11 The relative standard deviation of this analysis method is 4–5%. The rate of OTA degradation was calculated using the formula OTA degradation (%) = (1 − OTA peak area in treatment /OTA peak area in control) × 100 Effects of B. subtilis CW 14 on OTA production in contaminated maize Aspergillus ochraceus 3.4412 was chosen as the toxin-producing fungus. A spore suspension of A. ochraceus 3.4412 was prepared as follows. Aspergillus ochraceus 3.4412 was induced to sporulate on a PDA plate at 28 ◦ C for 7 days. Spores were harvested by adding 10 mL of sterile water to the plate and gently joggling and dislodging spores into a 50 mL Erlenmeyer flask with glass beads. The Erlenmeyer flask was shaken for 12 h and the spore suspension was filtered through four layers of sterile cheesecloth to remove mycelial debris. The concentration of spores was determined using a hemocytometer and adjusted to 107 spores mL−1 . The maize used in this study was purchased from local supermarket outlets. Autoclaved maize (30 g) was inoculated with 0.5 mL of the spore suspension (107 spores mL−1 ) described above. The final moisture content of the maize was adjusted to 180 g kg−1 J Sci Food Agric (2013)
with sterile water. After incubation in the dark at 28 ◦ C and 200 rpm for 21 days, the maize was sterilized at 121 ◦ C for 20 min and then 3 g of maize was mixed with 50 mL of the overnight culture of B. subtilis. The mixture was incubated at 30 ◦ C and 200 rpm for 72 h and extracted for OTA according to the method described by Liang.16 The extract was diluted with an appropriate volume of methanol and stored at −20 ◦ C for HPLC analysis. As a control, maize without B. subtilis was prepared. All tests were performed in triplicate. Statistics Statistical analyses were performed on repeated measurements by one-way analysis of variance (ANOVA) using Prism Version 5.0 (GraphPad Software, Inc., San Diego, CA, USA), followed by Bonferroni’s multiple comparison test. P values less than 0.05 were considered statistically significant.
RESULTS AND DISCUSSION Bacillus isolation, screening and identification Two hundred colonies were isolated from the sample of fresh elk droppings on LB-nutrient agar. The results of Gram’s staining technique showed that about two-thirds of the strains were Gram-positive bacteria. About 60 Gram-positive bacteria were cultivated in LB-nutrient agar with OTA, of which 12 could grow well at 0.6 µg mL−1 and four at 3 µg mL−1 . To obtain the most effective strain, the latter four strains were cultivated again using isocoumarin as the sole carbon source, and only one strain, named CW 14, was able to use isocoumarin and grow well. OTA was a derivative composed of isocoumarin, phenylalanine and different structure groups, mainly methyl and ethyl groups. It was viable that isocoumarin as a selective agent was used for screening instead of
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A
B
C
D
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Figure 2. In vitro interactions between Aspergillus ochraceus 3.4412 and Bacillus subtilis CW 14 in dual culture at day 7: A and C, normal hyphal growth in a control (arrows); B and D, curly growth of A. ochraceus 3.4412 hyphae facing a B. subtilis CW 14 colony (arrows). A and B were imaged with a digital camera (Leica DFC295) connected to an optical microscope (Leica DM2500); C and D were imaged with a scanning electron microscope (Hitachi S-3400N). 120
Mycelial inhibition (%) OTA-producing species
B. subtilis CW 14
A. ochraceus 3.4412 A. carbonarius a
30.0 33.3
Cell-free supernatant 28.8 44.4
Samples were incubated at 28 ◦ C for 7 days under aerobic conditions.
OTA. Guan et al.14 used coumarin as the sole carbon and energy source to screen bacteria degrading aflatoxin B1 (AFB1), and 25 bacterial isolates reducing AFB1 were obtained from 65 samples. CW 14 was confirmed by 16S rRNA gene sequences and showed 99% sequence similarity to B. subtilis (Fig. 1). Bacillus subtilis CW 14 and its cell-free supernatant inhibit growth of A. ochraceus 3.4412 and A. carbonarius Bacillus subtilis CW 14 clearly inhibited the growth of A. ochraceus 3.4412 and A. carbonarius in dual test plates at rates of 30.0 and 33.3% respectively (Table 1). The inhibition rate was close to that of Bacillus spp. for Aspergillus westerdijkiae NRRL 3174 (34%)8 and less than that of Bacillus spp. for Aspergillus flavus (52%).8
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OTA adsorption (%)
Table 1. Effects of Bacillus subtilis CW 14 and its cell-free supernatant on growth of Aspergillus ochraceus 3.4412 and Aspergillus carbonariusa
80
40
0 Viable cells
Autoclaved cells
Figure 3. OTA adsorption by viable and autoclaved (121 ◦ C, 20 min) cells of Bacillus subtilis CW 14 (107 CFU mL−1 ) for 24 h. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 24 h under aerobic conditions in the dark.
However, the inhibitory effect might be due to competition for nutrients and/or space between B. subtilis CW 14 and the mycete17 or to diffusible inhibitory substances produced by B. subtilis CW 14 that suppressed the growth of the mycete.4,18 To further study the inhibition mechanism, an agar well diffusion assay was performed. The inhibition rates of the cell-free supernatant against A.ochraceus
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www.soci.org normal (Figs 2A and 2C). Similar results could be seen in the study of Islam.19 According to the above results, CW 14 has potential as a biological agent to control OTA levels in crops.
Figure 4. TLC of cell-free supernatant and intracellular supernatant of Bacillus subtilis CW 14 tested for OTA degradation. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 24 h under aerobic conditions in the dark. Lanes: CK, OTA in LB-nutrient broth; A and B, OTA in cell-free supernatant; C and D, OTA in intracellular supernatant. No detectable degradation productions were produced.
3.4412 and A. carbonarius were 28.8 and 44.4% respectively in comparison with the above results, indicating the presence of diffusible inhibitory substances. In the optical microscope and scanning electron microscope, CW 14 induced curling (Figs 2B and 2D) in the confronting hyphae; moreover, there was excessive branching (ca one- to twofold higher than the control). In contrast, the hyphae in control samples were straight and branching was
OTA degradation by B. subtilis CW 14 The ability of B. subtilis CW 14 to bind OTA in vitro is shown in Fig. 3. Viable cells bound 66.6% while autoclaved cells bound 87.9% of 6 µg mL−1 OTA after 24 h. OTA adsorption by B. subtilis CW 14 was similar to that by Saccharomyces and Lactobacillus, as all are based on physical adsorption to the cell wall of the microorganism. However, the absorption rate of B. subtilis CW 14 was less than that of Lactobacillus (≥95%)7 and similar to that of Saccharomyces (∼75%).20 In our study, autoclaved (121 ◦ C, 20 min) cells bound 21.3% more OTA than viable cells. This phenomenon may due to the enhancement of adsorption sites by heat treatment.20 The results agreed with previous reports.20,21 Next, to further study OTA degradation by B. subtilis CW 14, different cell fractions were tested for their OTA degradation ability. As shown in Fig. 4, no degradation was observed in the intracellular supernatant (lanes C and D), while the cell-free supernatant had very high OTA-degrading ability (lanes A and B). For quantification of OTA degradation by the cell-free supernatant, HPLC was used. The OTA was 97.6% degraded (Fig. 5B) and no degradation products were observed on the chromatogram. There have been reports that OTA could be metabolized by microorganisms and/or enzymes through hydrolysis of the amide bond (formation of OTα),8,11,12 dechlorination of the isocoumarin moiety (formation
A
B
Figure 5. HPLC of Bacillus subtilis CW 14 cell-free supernatant tested for OTA degradation: A, OTA in LB-nutrient broth; B, OTA in cell-free supernatant. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 24 h under aerobic conditions in the dark. The retention time of OTA was 8.34 min. No detectable degradation products were produced.
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and incubated for 24 h at 30 ◦ C. The B. subtilis CW 14 grew well, indicating that CW 14 could be grown in autoclaved and contaminated maize.
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CONCLUSION
60 40 20 0 0
2
4
8
12
24
Time (h) Figure 6. Time course of OTA degradation by Bacillus subtilis CW 14 cell-free supernatant. The final concentration of OTA was 6 µg mL−1 . Samples were incubated at 30 ◦ C for 0, 2, 4, 8, 12 and 24 h under aerobic conditions in the dark.
of OTB) or opening of the lactone ring (formation of OP-OTA).22 Some Aspergillus spp.11 and Bacillus licheniformis8 could hydrolyze OTA to OTα; moreover, their degradation rate (>90%) was close to that of B. subtilis CW 14. However, in our study, no OTα or other degradation products were produced. Xiao23 reported that Aspergillus fumigatus, Aspergillus japonicus and Aspergillus niger degraded 2 mg L−1 OTA after 10 days; OTα was detected later and degraded into unknown compounds. To establish the optimal incubation time for OTA degradation by the cell-free supernatant, an experiment on the incubation time dependence of OTA degradation was conducted. The results are shown in Fig. 6. The fastest degradation of OTA occurred in the first 2 h, and after 12 h virtually no further degradation had occurred; moreover, no degradation products were detected at these time points. Our research indicated that neither OTα nor other degradation products were detectable, even in the first hour of degradation. Thus it is unlikely that OTA is hydrolyzed by cell-free supernatants via the same mechanism as it is hydrolyzed by carboxypeptidase A (CPA) (EC 3.4.17.1) from bovine pancreas12 or by other hydrolases.24 We propose that another degradation pathway is in operation or that more than one enzyme is involved in the hydrolysis of OTA, as with aflatoxin degradation.25 Effects of B. subtilis CW 14 on OTA production in contaminated maize Autoclaved (121 ◦ C, 20 min) and contaminated maize (3 g, with ca 0.3 mg of OTA) was incubated with B. subtilis CW 14 for 72 h at 30 ◦ C and extracted for OTA. OTA was assessed by HPLC. The OTA was 47.1% degraded, without any detectable degradation products, which was a lower degradation rate than that of pure OTA. Varga et al.10 reported that Rhizopus stolonifer could degrade 96.5% of OTA on wheat at 30 ◦ C in 10 days. In our study the concentration of OTA in the contaminated maize (0.1 mg g−1 ) was approximately 17 times greater than in pure OTA (6 µg mL−1 ). The environmental conditions in the contaminated maize, such as humidity, pH, nutrients and metabolites produced by A. ochraceus 3.4412, likely had effects on the degradation by B. subtilis CW 14. The observation that B. subtilis CW 14 is able to degrade OTA in contaminated maize is promising. In the future, B. subtilis CW 14 might be used as a food additive for degradation of animal feed and crops. Before extraction, B. subtilis CW 14 from the autoclaved and contaminated maize was inoculated onto LB-nutrient agar plates
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Bacillus subtilis CW 14 and its cell-free supernatant inhibited the growth of the toxin-producing species A. ochraceus 3.4412 and A. carbonarius and disrupted their hyphae. Bacillus subtilis CW 14 could also adsorb and degrade OTA, and the degradation was due to metabolites secreted into the culture medium. Thus B. subtilis CW 14 has potential for use as a biocontrol agent to prevent OTA production by Aspergillus spp. and as an additive agent to degrade OTA in crops. It remains to be determined whether inhibition and degradation are both accomplished by the same substance(s). Further studies are in progress to isolate and identify the active substances.
ACKNOWLEDGEMENT This work was funded by Fundamental Research Funds for the Central Universities (grants 2011 K0805 and 2013QJ036). The funders have no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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