Aflatoxin B1 inhibition in Aspergillus flavus by

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International Journal of Food Microbiology 256 (2017) 1–10

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Aflatoxin B1 inhibition in Aspergillus flavus by Aspergillus niger through downregulating expression of major biosynthetic genes and AFB1 degradation by atoxigenic A. flavus

MARK

Fuguo Xing⁎,1, Limin Wang1, Xiao Liu, Jonathan Nimal Selvaraj, Yan Wang, Yueju Zhao, Yang Liu⁎ Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Aspergillus flavus Aspergillus niger Aflatoxin Inhibition Degradation

Twenty Aspergillus niger strains were isolated from peanuts and 14 strains were able to completely inhibit AFB1 production with co-cultivation. By using a Spin-X centrifuge system, it was confirmed that there are some soluble signal molecules or antibiotics involved in the inhibition by A. niger, although they are absent during the initial 24 h of A. flavus growth when it is sensitive to inhibition. In A. flavus, 19 of 20 aflatoxin biosynthetic genes were down-regulated by A. niger. Importantly, the expression of aflS was significantly down-regulated, resulting in a reduction of AflS/AflR ratio. The results suggest that A. niger could directly inhibit AFB1 biosynthesis through reducing the abundance of aflS to aflR mRNAs. Interestingly, atoxigenic A. flavus JZ2 and GZ15 effectively degrade AFB1. Two new metabolites were identified and the key toxic lactone and furofuran rings both were destroyed and hydrogenated, meaning that lactonase and reductase might be involved in the degradation process.

1. Introduction Aspergillus flavus is one of the most frequently isolated mold species in agriculture and medicine and a saprophytic filamentous fungus that is distributed all over the world especially in warm and moist fields (Cleveland et al., 2009). As a contaminant of stored grains, other crops and feeds, A. flavus produces an abundance of diverse secondary metabolites, such as aflatoxins and cyclopiazonic acid (CPA). Of them, the best known group of metabolites is aflatoxin, the most potent naturally occurring toxic and hepatocarcinogenic compounds (Squire, 1981). Aflatoxin is estimated to cause up to 28% of the total worldwide cases of hepatocellular carcinoma (HCC), the most common form of liver cancer (Wu, 2014). In addition to liver cancer, consumption of aflatoxin-contaminated foods and feeds can cause acute poisoning, immune-system dysfunction and stunted growth in children. People whose livers are already compromised by infection with hepatitis B virus (HBV) are particularly susceptible to aflatoxin-induced liver cancer (Groopman et al., 2008). Studies by Wu and her team (Wu, 2014) suggest that up to 172,000 cases of HCC per year can be attributed to exposure to aflatoxin in the diet, and most of these individuals are infected with HBV. The majority of cases occur in sub-



1

Corresponding authors. E-mail addresses: [email protected] (F. Xing), [email protected] (Y. Liu). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ijfoodmicro.2017.05.013 Received 8 January 2017; Received in revised form 11 May 2017; Accepted 21 May 2017 Available online 22 May 2017 0168-1605/ © 2017 Elsevier B.V. All rights reserved.

Saharan Africa, Southeast Asia and the Western Pacific region (including China), as well as in parts of Central America (Wu, 2014). A. flavus also can cause direct infection and systematic disease in humans. After Aspergillus fumigatus, A. flavus is the second leading cause of invasive and non-invasive aspergillosis in immunocompromised patients (Cleveland et al., 2009). So, A. flavus and its metabolite aflatoxin not only are one of the important threats to human health, but also cause significant economic losses in many countries. To prevent, control and eliminate harmful aflatoxin in foods for human consumption, numerous strategies have been utilized to control fungal growth and aflatoxin production (Amaike and Keller, 2011), and remove or degrade aflatoxin in products (Shcherbakova et al., 2015). These include the prevention of fungal infection on crops by applying atoxigenic biocompetitive A. flavus and/or Aspergillus parasiticus strains or yeast (Chang et al., 2012), enhancing host resistance, postharvest control of fungal growth, and prevention of aflatoxin production by using microorganisms and natural products (Ding et al., 2015; Liang et al., 2015). Due to efficiently eliminating toxins and safe-guarding the quality of food and feed, biological control of aflatoxin provides an attractive alternative. Except for atoxigenic A. flavus and A. parasiticus, many other filamentous fungi such as Aspergillus chevalieri, Aspergillus

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1 kDa). Then the concentrated culture filtrate was sterilized using 0.2 μm disposable syringe filters (Millipore, Bedford, MA, USA) for the following experiments.

candidus, Aspergillus oryzae and Aspergillus niger could inhibit aflatoxin accumulation (Cvetnic and Pepeljnjak, 2007). Of these filamentous fungi, non-toxigenic A. niger isolated from food has the best prospective due to its safety (Xu et al., 2013). Nevertheless, to date, little is known about the mechanism of inhibition of aflatoxin B1 (AFB1) production and degradation of AFB1 by A. niger. The genes encoding the pathway for aflatoxin biosynthesis are within a 75 kb cluster in A. flavus. Up to now, 34 genes have been identified as members of the aflatoxin pathway gene cluster (Yu et al., 2004). Most of their functions in the pathway have been elucidated (Cleveland et al., 2009). Certain microorganisms and natural compounds can inhibit aflatoxin production by reducing the expression of the aflatoxin gene pathway. For example, co-cultivation of A. flavus with Bacillus megaterium down-regulates the expression of aflF, aflT, aflJ, aflL and aflX (Kong et al., 2014); 2-phenylethanol from Pichia anomala down-regulates the structural genes (aflC, aflD, aflO and alfM) involved in aflatoxin biosynthesis (Hua et al., 2014); curcumin inhibits AFB1 production in A. parasiticus by down-regulating the aflatoxin pathway genes (aflM, aflD, aflC, aflP and aflR) (Jahanshiri et al., 2012); Zataria multiflora Boiss. essential oil down-regulates the gene expression of aflD, aflM and aflP in A. parasiticus (Yahyaraeyat et al., 2013); and cinnamaldehyde, citral and eugenol down-regulate the expression of aflR, aflT, aflD, aflM and aflP (Liang et al., 2015). The aim of the present work was to investigate the mechanism behind the inhibitory role of A. niger on aflatoxin production by A. flavus, molecular expression profile of two key regulatory genes aflR and aflS, and 18 biosynthetic structural genes in pathway cluster, and the key regulatory gene of secondary metabolite laeA, and transcriptional activator genes of asexual sporulation brlA, were analyzed using real-time PCR. In addition, the effect of A. niger filtrates on aflatoxin production was also characterized by using the Spin-X centrifuge system. The AFB1 degradation activity of atoxigenic GZ15 and JZ2 was investigated. And the degradation products and pathway were characterized.

2.3. Effect of A. niger on the growth of A. flavus and aflatoxin production 2.3.1. Effect of co-cultivation of A. niger on the growth of A. flavus and AFB1 production The conidial suspensions of A. niger were adjusted to 1 × 106 conidia/mL with sterile 0.1% tween-20 solution, respectively. YES broth (50 mL) containing 1 mL of 1 × 106 conidia/mL suspensions of A. flavus was respectively inoculated with 1 mL of suspensions of A. niger conidia at different concentration and incubated in the dark at 28 ± 2 °C for 15 days with shaking at 150 rpm. Control without the conidia of A. niger was carried out under the same conditions. All treatments were tested in triplicate. The fungal growth and AFB1 production in the culture medium were assayed every three days of incubation. 2.3.2. Effect of the culture filtrate of A. niger on radial growth of A. flavus Influence of the culture filtrate of A. niger on radial growth of A. flavus mycelium was assayed according to the method described by Gandomi et al. (2009) with minor modification. The concentrated culture filtrate was added to molten PDA medium with the concentration of 2% (v/v). A 5 mm sterile diameter Whatman No. 1 filter paper disc was placed at the center of each plate and inoculated with 10 μL of A. flavus conidia suspension. Plates were incubated for 7 days at 28 ± 2 °C in the darkness. For the control, only PDA medium was used. All treatments were tested in triplicate. The diameter of colony was measured in two directions at right angles to each other to obtain the mean diameter. 2.3.3. Effect of culture filtrate on growth of A. flavus and AFB1 production YES (50 mL) containing 2% (v/v) of the concentrated culture filtrate was inoculated with 1 mL of conidia suspensions of A. flavus and incubated in the dark at 28 ± 2 °C on a rotary shaker (150 rpm) for 15 days. YES without the culture filtrate was incubated under the same conditions as the control. The fungal growth and AFB1 in the culture medium were assayed every three days of incubation.

2. Materials and methods 2.1. Fungal strains and culture conditions The aflatoxigenic strain of A. flavus YC15 (high AFB1 producer) was used as the pathogenic fungus. A. flavus JZ2 and GZ15 were isolated from peanut fields and proved in our lab to be atoxigenic strains without some key aflatoxin biosynthetic genes. Twenty strains of A. niger were isolated from peanut kernels in our lab (Ding et al., 2015). These strains were maintained on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potato, 20 g glucose and 20 g agar in 1 L of distilled water) at 4 °C. Conidial suspensions were harvested from sporulated cultures (7-day-old) of fungi on PDA plates by surface washing with a 0.01% Tween-20 solution in sterile deionized water. Conidia were counted with a hemocytometer and adjusted to 1 × 106 conidia/mL with 0.01% Tween-20 solution. For studying the aflatoxin biosynthetic gene expression level, fungal conidia were inoculated into 150 mL flasks containing 50 mL Yeast Extract Sucrose broth (YES, 20 g yeast extract, 150 g sucrose, and 0.5 g MgSO4·7H2O in 1 L of deionized water) and grown at 28 °C on a rotary incubator at 150 rpm for 5 d.

2.3.4. Effect of culture filtrate of A. niger on AFB1 production by A. flavus using Spin-X centrifuge filter system Conidial suspensions were diluted to 1 × 106 conidia/mL mixed with YES medium. The effect of A. niger on the aflatoxin production was assayed according to the method described by Huang et al. (2011) with minor modifications. Conidial-medium suspensions (500 μL of 1 × 106 conidia/mL) were placed in the filter tube insert above a 0.45 μm cellulose acetate filter in the Spin-X tube. As shown in Fig. 2, four treatments with four replications were included: 1) A. niger to exchange, 2) A. flavus to exchange, 3) A. niger and A. flavus together, 4) A. flavus alone. The tubes were incubated in an Eppendorf microfuge at 28 °C and centrifuged at 5000 rpm (2000 g) for 1 min every 3 h for 6 days. At the end of each centrifugation the insert, now free of liquid but containing growing fungus, was removed from the tube. In the last two treatments the medium contained in the tube was poured back into the insert from which it came, placed back in the tube, capped and mixed on a vortex before placing back in the centrifuge to incubate another 3 h. The first two treatments were done in the same way except the filtrate in the tubes from A. niger isolate was placed into the inserts containing A. flavus and vice versa. Every 3 h the first two treatments were exchanged so the A. flavus isolate grew for 3 days of the whole duration of the experiment in broth medium in which A. niger isolate had grown. Then, they were again centrifuged, and 600 μL methanol was added, the tube capped and vortexed before pouring over an alumina column into a vial for HPLC analysis of AFB1. All of them were filtered using 0.22 μm disposable syringe filters for HPLC detection.

2.2. Preparation of the culture filtrate of A. niger The culture filtrate of A. niger was prepared according to the method described by Xu et al. (2013) with minor modifications. Ten milliliters of a conidial suspension from a strain of A. niger was added to 500 mL of potato dextrose broth (PDB) and incubated at 28 ± 2 °C for 5 days with shaking at 150 rpm. The mycelia were separated from the substrate by filtration with four layers of cheese cloth and the culture filtrates were concentrated 10-fold by ultrafiltration (membrane cut off 2

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2.4. Effect of A. niger culture filtrate on the expression of aflatoxin biosynthesis-related genes

Table 1 Primers used for real-time PCR.

A. flavus was inoculated into 50 mL of YES broth containing 2% (v/ v) of the concentrated culture filtrate to provide the final concentrations of 106 conidia/mL. YES broth with only 106 conidia/mL of A. flavus was the control. They were incubated in the dark at 28 °C on a rotary shaker (ZWY-2102C, Shanghai Zhicheng, China) at 150 rpm in triplicate. On the 5th day, fungal mycelia were collected by filtering. The concentration of aflatoxins in YES filtrate was detected by HPLC. Total RNA was isolated from fungal mycelia by using the Plant Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. RNA samples were treated with DNA-free DNase. The purity and concentrations of RNA were determined by measuring the absorbance of samples at 260 and 280 nm using spectrophotometric quantification in a Beckman DU800 (Beckman, USA). The cDNA was obtained from 5 μg of total RNA by reverse transcription using a Takara RNA PCR Kit (AMV) ver. 3.0 (Takara, Japan). A. flavus NRRL 3357 genome database sequence was used to design primers for specific genes in the 75 kb aflatoxin biosynthesis gene cluster. Primers were designed with Primer Premier 5.0 software (Premier Biosoft International, USA) and Oligo 6.71 software (Molecular Biology Insights, Cascade, Co., USA). The nucleotide sequences were submitted to a BLASTn sequence similarity search to confirm the specificity of the primers. The primers were synthesized by Sangon Biotech (Shanghai, China). Primers used in this study were listed in Table 1. Real-time PCR reactions were performed in an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, Calif., USA). SYBR Green Real-time PCR Master Mix (Applied Biosystems) with fluorescent tag was used as the amplification detector. The amplification of 18S rRNA sequence was used as an internal control. Each reaction was prepared in 20 μL containing 100 ng cDNA (1 μL), SYBR Premix Ex Taq 10 μL, Nuclease-Free water 8 μL, and 10 mmol/L primers 0.5 μL (forward and reverse). The PCR program included an initial denaturation step at 94 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, and a final annealing step at 57 °C for 30 s. The fluorescence signals obtained were continuously measured once per cycle after the annealing and extension step. A melting curve was generated at the end of every run to ensure product uniformity. Plates and quantification assay documents were created in the sequence detection system software 1.9.1 (Applied Biosystems). The relative quantification of gene expression changes was computed by using the 2−ΔΔCt method (Livak and Schmittgen, 2001). The gene expression of each gene without treatment was considered as control and treated as 1.0. Each sample was run in triplicate in each experiment for all genes tested. Each experiment was repeated 3 times. 2.5. Degradation of aflatoxin B1 by atoxigenic A. flavus

Genes

Genes

Length (bp)

Primers

18s

18s

110

aflR

aflR

88

aflS

aflS

109

aflA

fans-2

111

aflC

pksA

61

aflD

nor-1

106

aflE

norA

134

aflF

norB

119

aflG

avnA

134

aflH

adhA

112

aflI

avfA

112

aflJ

estA

146

aflK

vbs

114

aflL

verB

127

aflM

ver-1

100

aflN

verA

114

aflP

omtA

109

aflO

omtB

115

aflQ

ordA

134

aflU

cypA

112

aflX

ordB

112

aflT

aflT

105

nadA

nadA

105

laeA

leaA

121

abaA

abaA

120

brlA

brlA

131

F:GCTCTTTTGGGTCTCGTAATTGG R:CGCTATTGGAGCTGGAATTACC F:CCTTTCTCACTACTCGGGTTT R:GCAGGTAATCAATAATGTCGG F:CTCGATGCGGCAGTGTATCT R:ACACCTCCACATGAGCCTTG F:CATGCTGTTAACCCCCGACT R:AATTGGGCTAGGAAACCGGG F:TGCATGGCGATGTGGTAGTT R:GTAAGGCCGCGGAAGAAAG F:ATGCTCCCGTCCTACTGTTT R:ATGTTGGTGATGGTGCTGAT F: GTGTGGAGGAAGTGATGCGA R: CGGGGTAAGTCCGTTAGCTC F: GGTTCGATGTTTGCTGAGGG R: GGGTGAGGACGAATTGGCTT F:GCACCAACAATTCGGCTCTG R:TGTGGAAGGGTGGAAGATGC F:ACCAGGTTGACCACGTCTTG R:CACGAGGTGTAGTAGACGCC F:GTGAGTGCCTCAGGAATGCT R:TGGAGCACGGATATGATGGC F: GCGTGATCAGTCGTCAATGC R: CAGGATGAGCGGTTGGTTCT F: GCTGGGCATTCCAGTACGAT R: CCCATCAACTGACTGTGGCT F:CTCTGATATACACCTACTACCT R:TGAACATCTTCCTTGATACG F:GAGCCAAAGTCGTGGTGAAC R:GCCTGGATTGCGATAGCGTC F:CAAGGCGAGGTGTTTCCTCT R:GGCAAGTGGGTGATCCTTGA F:CACGCTTTCAGAGCAGGTAA R:TTCGGTGGAGGAGGGAGTT F: GTCCCGTTTCCTGGGTTGAT R: GCTTTCGATTGCTGCCCAAA F:GCACCAACAATTCGGCTCTG R:TGTGGAAGGGTGGAAGATGC F:GTGAGTGCCTCAGGAATGCT R:TGGAGCACGGATATGATGGC F: AGTCCTCAACATAGCCGCTG R: TAGTCCCCCAGGTTTGACGA F: GCGTCCGCCTATCTACTGAC R: GAAAATACCCAGGGCGACGA F:GAAACCCGATAATCCCGCCT R:TGGTATCCAGTGGCACATCG F: AAAGGTTGCTCGCTGGTACA R: GACTTCTGACGAAATGCGCC F:ACTGGCAAAAGGAGGTCGAG R:ATTCGAACGGTCTGCTGGTT F:TCTAGCGGGGATGACCTCAA R:CCGAAGGAAGCCAAAAGTGC

et al. (2015). AFB1 was extracted from 10 mL of culture medium. The extracts were cleaned using an immunoaffinity column and analyzed using a Water 2695 HPLC coupled to a Water 2475 fluorescence detector and a post-column derivation system, and an Agilent TC-C18 column. The mean recovery was calculated by spiking YES broth at different levels ranging from 1 to 100 ng/g of AFB1 and estimated at 95.2 ± 8.4%. The lowest detection limit was 1 ng/g.

The degradation experiment of AFB1 by atoxigenic A. flavus was performed in YES broth medium. Atoxigenic A. flavus was inoculated into YES broth to provide the final concentrations of 106 conidia/mL. Fifty microliters of AFB1 stock (500 μg/mL) was added to 50 mL of YES broth in 150 mL conical flasks to a final concentration of 500 ng/mL. In total, 54 flasks were used for each experiment in three sets: I-control, without atoxigenic A. flavus inoculation; II-with atoxigenic A. flavus JZ2; III-with atoxigenic A. flavus GZ15. All flasks were incubated at 28 °C in a shaker incubator at 180 rpm for 9 d. Every 3 d, three flasks were removed from each set for analysis of AFB1 and its metabolites. The remaining AFB1 in the filtrate was analyzed by HPLC and LCQTOF/MS.

2.7. Determination of AFB1 metabolites by LC-qTOF/MS Operational procedures for LC-qTOF/MS were as follows: degradation products of AFB1 were first separated by the separation column, and the separated components were ionized by the MS ion source. Through the first quadruple, the TOF was reached by a mass analyzer without CID. The CID mode was used for MS/MS analysis. The parent ions were fragmented in the cell of CID, and the fragmentation pathways were obtained.

2.6. Determination of AFB1 by HPLC Aflatoxins levels were determined according to the method by Liang 3

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LC was performed on Agilent 1200 series HPLC (Agilent, Palo Alto, CA) equipped with an autoinjector and a quaternary HPLC pump. Chromatography was performed on a 2.1 × 150 mm inner diameter, 5 μm, Agilent Plus C18 column. The injection volume was 2 μL. The mobile phase was acetonitrile and an aqueous solution containing 0.1% formic acid in 70:30 (v/v) solution. The total run time was 12 min, with a flow rate of 0.4 mL/min. MS was performed with Agilent 6520 accurate-mass qTOF LC/MS (Agilent, Palo Alto, CA). The optimized conditions were as follows: compounds were analyzed in positive-ion mode. Capillary and fragmentor voltages were 3500 and 175 V, respectively, and the skimmer voltage was 65.0 V. The flow rate of drying gas was 10.0 L/min, and nebulizer was 40 psi. Nitrogen was used as the collision gas. Mass spectra were acquired in a full-scan analysis within the range of m/z 100–1000 using extend dynamic range and a scan rate of 1.4 spectra/s and varying the collision energy with mass. The data station operating software used was the Mass Hunter Workstation software (version B. 04.00). A reference mass solution containing reference ions 121.0508 and 922.0097 was used to maintain mass accuracy during the run time.

Table 2 Production of aflatoxin B1 by A. flavus YC15 in mixed cultures with A. niger strains grown in YES broth.

2.8. Statistical analysis of the data All experiments were carried out in triplicate. Data were analyzed by one way analysis of variance (ANOVA) in the statistical software SAS v. 8.0 (SAS, Cary, NC, USA). Mean separations were performed by Turkey's multiple range tests. Difference was considered significant as P < 0.05.

A. niger strains

Concentration of AFB1 (ng/mL)

Inhibition rate (%)

A. flavus YC15 AN1 AN2 AN3 AN4 AN5 AN6 AN7 AN8 AN9 AN10 AN11 AN12 AN13 AN14 AN15 AN16 AN17 AN18 AN19 AN20

1733.58 ± 24.55 nd nd nd 1745.32 ± 28.92 1734.23 ± 27.36 nd nd 1753.84 ± 34.57 525.28 ± 18.85⁎⁎ 12.83 ± 1.02⁎⁎ nd nd nd nd nd 15.85 ± 1.24⁎⁎ nd nd nd nd

– 100 100 100 0 0 100 100 0 69.7 99.3 100 100 100 100 100 99.1 100 100 100 100

nd: AFB1 was not detected. ⁎⁎ Statistically significant when compared to control, P < 0.01.

day 15. In the control, the AFB1 concentration in YES filtrate was 1503.72 ng/mL. When YES medium contained 2% of the concentrated culture filtrates of A. niger, AFB1 production was inhibited with the inhibition rate of 0–92.3% (Fig. 1B). Of the 20 strains, A. niger No. 2 and No. 6 were found to be highly effective in inhibiting AFB1 production with inhibition rate of 92.3% and 78.7%, respectively.

3. Results 3.1. Identification of A. niger and confirmation of atoxigenic strains Of the Aspergillus isolated from peanut kernels, 20 strains were identified as A. niger according to the results of morphological characterization and ITS sequence analysis. These 20 A. niger strains were numbered No. 1 to No. 20, respectively. They all were proved to be non-producer of AFB1, ochratoxin A and fumonisin.

3.4. Effect of the culture filtrate of A. niger No. 2 and No. 6 on AFB1 accumulation at different times To further evaluate the ability of the culture filtrate of A. niger No. 2 and No. 6 to inhibit AFB1 production, AFB1 concentrations in the filtrate were determined at different time. As shown in Fig. 1C, in the control, the level of AFB1 achieved maximum concentration at the 6th day and sharply decreased at 9th and 12th day. After the period, the concentration of AFB1 increased up to 15 days and then decreased at 20 days. When YES medium contained 2% of the concentrated filtrate of A. niger No. 6, the level of AFB1 was significantly lower than the control (P < 0.05) with the similar variation trend. However, AFB1 concentrations of A. niger No. 2 group decreased from day 3 to day 20 and always remained significantly lower level than detected in the control (P < 0.01). Generally, the strong inhibitory efficacy was observed from 6 to 20 days during incubation with the reduction of AFB1 ranging from 81.7% to 93.5%.

3.2. Effect of A. niger by co-cultivation with A. flavus on aflatoxin accumulation The effects of all 20 A. niger strains on the production of AFB1 by A. flavus YC15 after 7 days of incubation were summarized in Table 2. Obviously, A. niger strains grown as mixed cultures do not support growth, sporulation, and toxin production, and their anti-toxigenic potential is very strong. Of 20 A. niger strains, 14 were shown to completely inhibit the production of AFB1 by A. flavus in co-cultivation, 3 strains No. 9, 10 and 16 significantly reduced the production of AFB1 with inhibition rate 69.7%, 99.35% and 99.1%, respectively. Other three strains No. 4, 5 and 8 could not reduce AFB1 accumulation. Macroscopically, inhibition of AFB1 production in co-cultivation may have been caused by competition (e. g. nutritional) or by metabolites produced by these strains which specifically inhibit aflatoxin biosynthesis.

3.5. Determining if soluble signal molecules are involved in the inhibition of A. niger No. 2 on the aflatoxin accumulation by A. flavus

3.3. Effect of the culture filtrate of A. niger on A. flavus growth and AFB1 accumulation

A. niger No. 2 significantly inhibited AFB1 production by A. flavus but the mechanism of this function is unclear. One possible reason could be that there were metabolites or soluble signal molecules which down-regulate aflatoxin biosynthesis. To confirm this postulate, the Spin-X centrifuge filter system was used. As shown in Fig. 2, the amount of aflatoxin in AF exchange group was same with AN2 exchange and was similar with AN2 + AF group (P > 0.05). They all were obviously lower than AF self group (P < 0.01). Thus there appeared to be some soluble signal molecules or antibiotics which play a role in this phenomenon.

A. flavus colonies grown on untreated PDA and PDA treated with the culture filtrate of A. niger strains were observed. In the control, an obvious halo and well-developed mycelia could be observed after 7 days, and colony diameter was 6.85 ± 0.05 cm. The inhibitory effect of the culture filtrate of A. niger on A. flavus growth on PDA was not obvious, and the inhibition rate was only 0.4%–11.5% (Fig. 1A). To evaluate the ability of the culture filtrate of 20 A. niger strains to inhibit AFB1 production, AFB1 levels in the filtrate were determined at 4

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Fig. 1. Effect of the culture filtrate of A. niger on A. flavus growth and AFB1 production. (A) A. flavus; (B) AFB1 production; (C) AFB1 inhibition by the culture filtrate of A. niger strain No.2 and No.6. CK: control; * statistically significant when compared to control, P < 0.05; ** statistically significant when compared to control, P < 0.01.

metabolite laeA was up-regulated with an average of 2.1-fold increase, while the transcriptional activator gene of asexual sporulation brlA was slightly down-regulated with an average of 1.1-fold decrease. As shown in Fig. 3B, for A. niger No. 6, of 20 aflatoxin pathway genes, 19 genes also were down-regulated in the range from nearly to 11-fold decrease, while aflF was up-regulated with an average of 2.0fold increase. Among the 19 down-regulated genes, aflJ was the most strongly down-regulated gene, followed by aflL and aflK. The laeA and brlA genes were up-regulated with an average of 3.1 and 1.7-fold increases, respectively.

3.6. Effect of A. niger on the expression of aflatoxin biosynthetic and regulatory genes To reveal the molecular mechanism behind the inhibitory role of A. niger in aflatoxin production, molecular expression profile of 22 aflatoxin biosynthetic and regulatory genes was analyzed using realtime PCR. After 5 days of cultivation, the AFB1 concentration of the control was 463.49 ng/mL. However, in the treatment with the A. niger No. 2 and No. 6 culture filtrates, the AFB1 concentrations were 2.33 and 2.59 ng/mL with the inhibition rate of 99.5% and 99.4%, respectively. As shown in Fig. 3A, for A. niger No.2, of 20 aflatoxin biosynthetic pathway genes, 19 genes were down-regulated in the range from nearly to 90-fold decrease, while the key regulatory gene aflR was upregulated with an average of 1.4-fold increase. Among the 19 downregulated genes, aflL was the most highly down-regulated gene, followed by aflO and aflN. The key regulatory gene of secondary

3.7. Degradation of AFB1 in the culture of atoxigenic A. flavus GZ15 and JZ2 To reveal the fluctuation of AFB1 concentration in the culture during the incubation of A. flavus (Fig. 1C), biological degradation of AFB1 in 5

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Fig. 2. Determining if soluble signal molecules are involved. AFB1 production in Spin-X centrifuge filter units. Tubes spun every 3 h for 6 days and filtrates from tubes changed (AN2 exchange group and AF exchange group); or filtrates added back to itself (AN2 + AF self group and AF self group). Toxigenic A. flavus and atoxigenic A. niger No.2 were grown for total of 6 days. AN2: A. niger No.2; AF: A. flavus.

the culture of atoxigenic A. flavus GZ15 and JZ2 was investigated. As shown in Table 3, AFB1 was stable over the 9 d incubation period in PDB broth as no significant difference was observed between AFB1 control samples after 3 and 9 d. However, a significant (P < 0.01) reduction of AFB1 was observed from 3 to 9 d when treated with atoxigenic A. flavus GZ15 and JZ2. After 3 days, the concentration of AFB1 was reduced from 389.3 ng/mL to 294.3 and 204.1 ng/mL by A. flavus GZ15 and JZ12, respectively. After 6 days, the concentration of AFB1 was reduced from 372.5 ng/mL to 206.7 and 90.6 ng/mL by GZ15 and JZ2 strains, respectively. After 9 days, the concentration of AFB1 was reduced from 354.3 ng/mL to 164.1 and 67.8 ng/mL by GZ15 and JZ2, with degradation rate of 53.7% and 80.9%, respectively. Furthermore, the biodegradation products of AFB1 by atoxigenic A. flavus were investigated. Fig. 4 shows the total ion chromatograms of AFB1 and its biodegradation products at different incubation times. Compared with the control, one new peak (product 1) was observed for AFB1 treated with both atoxigenic A. flavus GZ15 and JZ2 after 3 days incubation, which indicates that AFB1 was decomposed through the biodegradation. After 6 days incubation, the second new peak (product 2) was observed for AFB1 treated with GZ15 strain, while no new peak was observed for AFB1 treated with JZ2. Molecular formulas of biodegradation products are calculated using the formula calculator based on m/z, a function of Agilent Mass Hunter Qualitative Analysis software. To confirm the structures of biodegradation products, it is important to further analyze the fragmentation patterns and their accurate masses. TOF/MS/MS of biodegradation products as precursor ions was preformed to pass to the collision cell using the ion-filtering function of QTOF. MS/MS spectra and fragmentation of six main precursor compounds are shown in Fig. 4. On the basis of the accurate masses of the parent ions and the fragments obtained from TOF/MS/MS experiments, the structures of two products are deduced as follows

(Fig. 4). 4. Discussion The interaction of A. niger with A. flavus causing a reduction in AFB1 production has been reported in several publications (Horn and Wicklow, 1983; Shantha and Rati, 1990; Shantha et al., 1990; Xu et al., 2013). Nevertheless, to date, the mechanism of the reduction of AFB1 production by A. flavus remained undefined. Horn and Wicklow (1983) found that A. niger could interfere with the production of aflatoxin when grown with A. flavus on autoclaved corn. They concluded that A. niger reduced substrate pH below 2.8–3.0 which is sufficient to suppress aflatoxin production. They found also that a water extract of corn kernels fermented with A. niger caused an additional inhibition of AFB1 production. Further studies performed by Shantha and Rati (1990) reported that oxalic acid was one of major inhibitory factors produced by A. niger which inhibited biosynthesis of aflatoxin by A. flavus in liquid synthetic medium without inhibiting fungal growth. However, they thought the inhibition by oxalic acid was not due to the lowering of pH. Meanwhile, the same research group found that gluconic acid secreted by A. niger partly prevented biosynthesis of aflatoxin, and the changes in pH of the medium had no role in inhibiting aflatoxin production by A. flavus (Shantha et al., 1990). Xu et al. (2013) found that the culture filtrate of A. niger strain FS10 did not lower the pH value of substrate and confirmed that lowering of pH does not play a role in reducing AFB1 contamination by A. niger. Lee et al. (1999) reported that an antifungal peptide (Anafp) secreted by A. niger, which is single polypeptide chain with 58 amino acids including 6 cysteine residues, could inhibit the growth of A. flavus. The cysteinespacing pattern of Anafp was similar to that of the antifungal peptide (PAF) from Penicillium chrysogenum. Antifungal proteins from filamen6

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Fig. 3. Effect of A. niger No. 2 (A) and No. 6 (B) on the expressions of aflatoxin biosynthetic and regulatory genes.

However, the signal molecules were absent during the critical initial 24 h of the aflatoxigenic A. flavus growth when it is sensitive to inhibition. A similar result was reported by Huang et al. (2011) using the same Spin-X centrifuge system. They found that growing toxigenic isolate 53 for 12 of the first 24 h in media in which atoxigenic isolate 51 had grown did not cause intraspecific aflatoxin inhibition in the toxigenic isolate. To confirm the inhibitory effect of A. niger on AFB1 biosynthesis and reveal the molecular mechanism behind the inhibitory role of A. niger, the molecular expression profile of 22 aflatoxin biosynthetic and regulatory genes was analyzed (Fig. 3). With both A. niger No.2 and No.6, 19 of 20 aflatoxin biosynthetic pathway genes were downregulated by the culture filtrate of A. niger. Importantly, the expression of the regulatory gene aflS was down-regulated by both A. niger strains, which subsequently caused a significant decrease of the ratio of aflS/ aflR gene transcript abundance. The decrease of aflS/aflR ratio then inactivated the expression of aflatoxin biosynthetic structural genes. AflR, the aflatoxin regulatory gene, encodes a positive regulator named AFLR, which activates the structural gene transcription in the aflatoxin pathway (Chang et al., 1995). The aflS is involved in regulation of aflatoxin biosynthesis in close association with the aflR gene. Disruption or deletion of aflS gene resulted in loss of the ability to convert aflatoxin intermediates to aflatoxins (Chang, 2003; Kiyota et al., 2011). The results of yeast two-hybrid experiment showed that AflS and AflR are co-activators of transcription of aflatoxin pathway cluster structural genes (Chang, 2003). AflS binds to AflR to form a

Table 3 Degradation of AFB1 in the culture of non-aflatoxigenic A. flavus GZ15 and JZ2. Time (days)

Group

AFB1 concentration (ng/ mL)

3

Control GZ15 JZ2 Control GZ15 JZ2 Control GZ15 JZ2

389.3 ± 40.7 294.3 ± 34.6⁎⁎ 204.1 ± 10.9⁎⁎ 372.5 ± 10.3 206.7 ± 35.9⁎⁎ 90.6 ± 20.4⁎⁎ 354.3 ± 38.6 164.1 ± 15.0⁎⁎ 67.8 ± 11.3⁎⁎

6

9

⁎⁎

Degradation ratio of AFB1 (%)

24.4 47.6 44.5 75.7 53.7 80.9

Statistically significant when compared to control, P < 0.01.

tous fungi such as PAF and AFP are low molecular weight, cysteine-rich and cationic proteins that inhibit the growth of opportunistic zoo- and plant-pathogenic fungi by interfering with PCK/MPK and cAMP/PKA signals (Binder et al., 2010). These results suggested that some soluble signal molecules secreted by A. niger, such as Anafp, oxalic acid, gluconic acid, could inhibit AFB1 biosynthesis. A similar result was obtained in the present study. The experiment using the Spin-X centrifuge system to force metabolites and media through the filter every 3 h for 6 days and alternately exchange these solutions between A. niger No. 2 and A. flavus (Fig. 2) confirms strongly the presence of signal molecules in the culture filtrate of A. niger. 7

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Fig. 4. LC-QTOF/MS profile of AFB1 degradation and proposed fragmentation of biodegradation products of AFB1 in the process of atoxigenic A. flavus treatment. (A) Electrospray ionization (ESI) total ion chromatogram (TIC) scan of positive control (500 ng/mL in YES broth, after incubation in the dark at 37 °C for 3 d, the samples were extracted with acetonitrile). (B) ESI TIC scan of sample (500 ng/mL in YES broth, after incubation with A. flavus GZ15 in the dark at 37 °C for 3 d, the samples were extracted with acetonitrile). (C) ESI TIC scan of sample (500 ng/mL in YES broth, after incubation with A. flavus JZ2 in the dark at 37 °C for 3 d, the samples were extracted with acetonitrile). (D) ESI TIC scan of sample (500 ng/mL in YES broth, after incubation with A. flavus GZ15 in the dark at 37 °C for 6 d, the samples were extracted with acetonitrile). (E) ESI TIC scan of sample (500 ng/mL in YES broth, after incubation with A. flavus JZ2 in the dark at 37 °C for 6 d, the samples were extracted with acetonitrile). (F) Product 1; (G) product 2; (H) the deduced biodegradation pathway of AFB1.

findings were achieved by Kong et al. (2014), who found Bacillus megaterium inhibited aflatoxin biosynthesis by down-regulating the aflS gene expression and changing the ratio of AflS/AflR. Yu et al. (2011) found that high temperature (37 °C) negatively affected aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster. In the present study, a similar result was obtained. The ratio of aflS/alfR gene transcript abundance with the culture filtrates of A. niger No. 2 and No. 6 was significantly reduced, which played a key role

complex, then both together bind to the promoter region called AflR binding site of each of the structure genes in the aflatoxin gene cluster. AflS is a pathway regulator, while AflR is a transcription activator. When aflS and alfR genes are expressed normally, there are enough AflS proteins bounded with AflR to form a functional activation complex in a ratio of roughly 4 AflS to 1 AflR (Kong et al., 2014). Down-regulation of aflS resulted in less AflS production, which could subsequently provide an opportunity for potential inhibitors as AflS substitutes to bind to AflR. As a consequence, the AflS-AflR-dependent aflatoxin gene transcription would be reduced and little aflatoxin is produced. Similar

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process. In conclusion, our results showed that A. niger has potential biocontrol activity against the decay and aflatoxin biosynthesis caused by A. flavus. This potential may provide the antagonist and its metabolites which are safe for human consumption. Through qRTPCR analysis profiling of the aflatoxin biosynthetic pathway genes with the culture filtrate of A. niger, most of these genes were shown to be highly down-regulated. Importantly, the expression of aflS was significantly down-regulated compared to that of aflR, resulting in a significant reduction of AflS/AflR ratio. In addition, this study confirmed that atoxigenic A. flavus could effectively degrade AFB1. Two new degradation products were identified. The structures showed that the furofuran and lactone rings were both destroyed and hydrogenated, meaning that enzymes belonging to lactonase and reductase are involved in the degradation process.

in the inhibition of aflatoxin biosynthesis. LaeA has been shown to encode a putative methyltransferase that affects the expression of genes in many different secondary metabolite gene clusters, among which are clusters involved in production of aflatoxins, sterigmatocystin, penicillin, emericellamide, terrequinone, gliotoxin, and lovastatin (Brakhage and Schroeckh, 2011). Furthermore, LaeA was identified as a protein that was thought to be necessary for expression of aflR, the gene that encodes the transcriptional regulator of genes in the sterigmatocystin cluster in Aspergillus nidulans and the aflatoxin cluster in A. flavus (Kale et al., 2008). In the present study, the laeA gene was up-regulated by both A. niger No. 2 and No. 6. Based on the transcriptional level of laeA, the expression of aflR should be significantly increased. However, the expression of aflR gene was not significantly affected by the culture filtrate of A. niger. The result confirmed that the culture filtrate of A. niger directly reduced aflatoxin biosynthesis through reducing the abundance of aflS and aflR mRNAs in A. flavus. Although aflatoxins are produced by fungi, certain species such as A. flavus, A. parasiticus, A. niger, Alternaria spp., Absidia repens, Armillariella tabescens, Candida utilis, Dactylium dendroides, Mucor spp., Paecilomyces lilacinus, Penicillium spp., Peniophora spp., Phoma spp., Pleurotus ostreatus, Rhizopus spp. and Trichoderma spp. have been reported to degrade them as well (Adebo et al., 2015). Wu et al. (2009) thought that some fungal metabolites can reduce the pH of the medium and the subsequent acidic condition could decrease aflatoxins levels. Some fungi have been shown to possess genes coding for aflatoxin-degrading enzymes such as oxidases, peroxidases and laccases (Shcherbakova et al., 2015). Interestingly, both A. flavus and A. parasiticus, which are the predominant aflatoxin-producing fungi, can degrade aflatoxins. The production and degradation of AFB1 and AFG1 by A. parasiticus strains (NRRL 2999, 3000, A-13570 and A-13367; also called M001) in liquid medium during cultivation were investigated (Ciegler et al., 1966). For the first 72 h of growth, the aflatoxin production was observed and depended on mycelia dispersion and aeration. After this time, reduction of aflatoxin concentrations was observed while the degradation was not dependent on carbohydrate concentration. Moreover, the fermentation of mycelia of previously aflatoxin non-degrading fungi (A. parasiticus NRRL 2999 and M 001) induced their ability to degrade aflatoxins. The degradation can be prevented by fermentation at low temperatures and a low agitation rate. Similarly, the degradation of AFB1, AFB2, AFG1 and AFG2 the four major aflatoxins by the mycelia and filtrates of A. parasiticus (such as NRRL 3353 and NRRL 3000) after 24 h of incubation has been reported in several studies (Doyle and Marth, 1978; Shih and Marth, 1975). Peroxidase was later confirmed to be the enzyme involved in the aflatoxin degradation by this fungus (Doyle and Marth, 1979). Hamid and Smith (1987) also reported the aflatoxin detoxifying activity of cell free extracts (CFE) and mycelium of A. flavus 102,566. Enzymes belonging to the cytochrome P-450 monooxygenase system were considered to be involved in the degradation process. Nakazato et al. (1990) found that atoxigenic A. flavus could convert AFB1 to aflatoxicol-A (AFL-A) by reducing the cyclopentenone carbonyl of AFB1, then AFL-A was converted to aflatoxicol-B (AFL-B) by the actions of medium components or organic acids produced from the fungi. In addition, the interconversion between AFB1 and AFL was ascertained to occur during proliferation of the fungi and was suggested to be mediated by intracellular enzymes of A. flavus. Furthermore, the sum of AFL and AFB1 was found to be decreased with time, which suggested both AFB1 and AFL were further metabolized to unknown substances by the fungus (Wu et al., 2009). In the present study, we confirmed that atoxigenic A. flavus (GZ15 and JZ2) could effectively degrade AFB1. Interestingly, two new metabolites (Product 1 and Product 2) were observed and identified using TOF/MS/MS. The two key toxic groups, the furofuran and lactone ring, were destroyed and hydrogenated at the same time. The results suggest that enzymes belonging to lactonase and reductase are involved in the degradation

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