Aflatoxin B1 degradation by liquid cultures and lysates of three bacterial strains

International Journal of Food Microbiology 233 (2016) 11–19

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Aflatoxin B1 degradation by liquid cultures and lysates of three bacterial strains Oluwafemi Ayodeji Adebo a,⁎, Patrick Berka Njobeh a, Sibusiso Sidu b, Matsobane Godfrey Tlou c, Vuyo Mavumengwana a a b c

Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein 2028, Gauteng, South Africa Gold One International Limited, Corner Cloverfield Ave & Auteniqua Road, Eastvale, Springs, South Africa Department of Biochemistry, Faculty of Science, University of Johannesburg, P.O. Box 254, Auckland Park 2006, Gauteng, South Africa

a r t i c l e

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Article history: Received 25 February 2016 Received in revised form 24 May 2016 Accepted 6 June 2016 Available online 07 June 2016 Keywords: Aflatoxin B1 Biodegradation Bacterial lysate Cytotoxicity

a b s t r a c t Aflatoxin contamination remains a daunting issue to address in food safety. In spite of the efforts geared towards prevention and elimination of this toxin, it still persists in agricultural commodities. This has necessitated the search for other measures such as microbial degradation to combat this hazard. In this study, we investigated the biodegradation of aflatoxin B1 (AFB1), using lysates of three bacterial strains (Pseudomonas anguilliseptica VGF1, Pseudomonas fluorescens and Staphylococcus sp. VGF2) isolated from a gold mine aquifer. The bacterial cells were intermittently lysed in the presence and absence of protease inhibitors to obtain protease free lysates, subsequently incubated with AFB1 for 3, 6, 12, 24, and 48 h to investigate whether any possible AFB1 degradation occurred using high performance liquid chromatography (HPLC) for detection. Results obtained revealed that after 6 h of incubation, protease inhibited lysates of Staphylococcus sp. VGF2 demonstrated the highest degradation capacity of 100%, whereas P. anguilliseptica VGF1 and P. fluorescens lysates degraded AFB1 by 66.5 and 63%, respectively. After further incubation to 12 h, no residual AFB1 was detected for all the lysates. Lower degrading ability was however observed for liquid cultures and uninhibited lysates. Data on cytotoxicity studies against human lymphocytes showed that the degraded products were less toxic than the parent AFB1. From this study, it can thus be deduced that the mechanism of degradation by these bacterial lysates is enzymatic. This study shows the efficacy of crude bacterial lysates for detoxifying AFB1 indicating potential for application in the food and feed industry. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Mycotoxins are secondary metabolites produced by a variety of widespread toxigenic strains of fungi. These compounds contaminate agricultural commodities during pre- and/or post-harvest conditions (Rocha et al., 2014). Although several mycotoxins have been detected in various commodities worldwide, aflatoxins (AFs) are considered the most important mycotoxins in human foods and animal feeds (Makun et al., 2012; Strosnider et al., 2006). Aflatoxins are secondary metabolites (toxins) primarily produced by Aspergillus flavus and Aspergillus parasiticus. They were first brought to prominence in the early 1960s when about 100,000 turkeys died after consuming feeds contaminated with the toxin (Makun et al., 2012). Since then, over 18 analogues of AFs have been identified and characterized, but those of agricultural and health significance are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 ⁎ Corresponding author. E-mail address: [email protected] (O.A. Adebo).

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.06.007 0168-1605/© 2016 Elsevier B.V. All rights reserved.

(AFG2) (Dors et al., 2011; Marin et al., 2013). Aflatoxins M1 (AFM1) and M2 (AFM2) are also considered because they are hydroxylated metabolites of AFB1 and AFB2 respectively. Of all the AFs, the most important member is AFB1 (Makun et al., 2012), as it has been recognized as the most potent naturally occurring carcinogen known (IARC, 2002). AFs are recognised as mutagenic, carcinogenic, hepatotoxic, teratogenic and immunosuppressive (Dors et al., 2011). Due to its continued presence along the food chain and their impact on health and the economy, AFB1 has attracted worldwide attention (Adebo et al., 2015; Makun et al., 2012). The ongoing contamination of agricultural produce by AFs has triggered the need to mitigate their formation or at best reduce or deactivate them in these commodities (Adebo et al., 2015). Several approaches and strategies for their removal and elimination have been extensively studied, including physical or chemical measures (Bailey et al., 1994; Mishra and Das, 2003; Park, 2002). None of these approaches can however, completely fulfil the required efficacy, safety and nutrient retention (Zhao et al., 2011). Microbial degradation offers a more promising alternative in decontaminating AFs as it provides a

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possible means of degrading or removing these toxic substances under mild conditions, limiting significant losses in aesthetic and nutritional value of food commodities (Alberts et al., 2009; Samuel et al., 2014). There have been various studies investigating AF degradation by bacteria including Corynebacterium rubrum (Mann and Rehm, 1977; Wu et al., 2009), Flavobacterium auranticum (Doyle et al., 1982; Smiley and Draughon, 2000), Mycobacterium fluoranthenivorans (Hormisch et al., 2004), Brevundimonas sp. (Guan et al., 2008), Nocardia corynebacteroides (Tejada et al., 2008), Rhodococcus erythropolis (Alberts et al., 2006; Cserhati et al., 2013; Teniola et al., 2005), Myxococcus fulvus (Zhao et al., 2011) and Pseudomonas spp. (Krifaton et al., 2011; Samuel et al., 2014; Sangare et al., 2014). Likewise, a number of fungal strains such as Armillariella tabescens (Liu et al., 2001), Phanerochaete sordida (Wang et al., 2011), Pleurotus ostreatus (Yehia, 2014) and a host of others (Adebo et al., 2015; Alberts et al., 2009; Wu et al., 2009) have been found to transform AFB1 to less toxic products. Shortcomings associated with the use of fungi or products from them have however, made them less desirable for the biodegradation of AFs (Teniola et al., 2005). Additionally, lengthy degradation time and the possibility of fungal strains producing toxins under certain conditions limit their application (Eshelli et al., 2015). Although bacteria are more desirable for AF degradation, the use of whole cultures has less potential for large scale utilization in the industry (Kolosova and Stroka, 2011). Therefore, the aim of this study was to investigate the degradation of AFB1 by bacterial lysates and to determine toxicity of products after degradation. 2. Materials and methods 2.1. Bacterial strain isolation and identification Water samples were collected from a gold mine aquifer in Springs (26°15′17″S 28°26′34″E), East Rand in the Gauteng province of South Africa. All the samples were collected in sterile bottles and subsequent serial dilutions were done. 1 mL of this sample was aseptically transferred into 9 mL of sterile phosphate buffered saline (PBS), vortexed, enriched with Nutrient Agar (M012, HIMEDIA) and incubated for 24 h at 37 °C. Colonies growing on each plate were isolated and identified based on certain morphological features, considering pigmentation and size. Each isolate was subjected to successive streak plating for 24 h at 37 °C to ensure clone purity. The isolates were preserved in 70% glycerol at − 80 °C. DNA was subsequently extracted using a ZR fungal /Bacterial DNA Kit (Zymo Research, California, USA) and PCR performed using universal primers 27F: GAGTTTGATCCTGGCTCAG and 1492R: GTTACCTTGTTACGACT. A GeneAmp PCR system was used in amplifying genes at 94 °C, using annealing temperature for 90 s at 53 ° C and extension time 2 min. PCR products were purified using ExoSAP (Affymetrix, Inc., California, USA) and sequenced on the ABI 3500 XL Genetic analyzer (Applied Biosystems, California, USA). The purified PCR products were sequenced through a nucleotide sequencer and sequencing results further studied by using Geneious software (Biomatters Ltd., New Zealand). The 16S rRNA gene sequences were compared with those of the GenBank and European molecular biology laboratory (EMBL) databases by advanced BLAST searches from the National Center for Biotechnology (NCBI). These sequences were then utilized in finding and constructing a phylogenetic tree using MEGA-6 (Tamura et al., 2013).

2.5 μg/mL stock solution of AFB1 standard (Sigma Aldrich, Germany) dissolved in dimethyl sulphoxide (DMSO) to a final AFB1 concentration of 0.5 μg/mL. These were incubated at 37 °C for 3, 6, 12, 24 and 48 h. As further described by Teniola et al. (2005) and Farzaneh et al. (2012), the following were included (i) sterile NB containing AFB1, (ii) sterile NB inoculated with 400 μL of the respective bacterial strains and (iii) Proteinase K treated liquid cultures as controls. To investigate the kinetics of AFB1 degradation, the respective bacterial cell absorbances were also measured at 600 nm at 3, 6, 12, 24 and 48 h. 2.3. AFB1 extraction and quantification After degradation, AFB1 was extracted using 1 mL of chloroform (3 ×), from an aliquot of the degradation mix (Teniola et al., 2005). The chloroform fractions were pooled in a screw capped vial and evaporated under nitrogen gas. The residue was then re-dissolved in HPLC grade methanol, filtered (0.22 μm pore size, Gema Medical S.L, Spain) and preserved for HPLC analysis. Firstly and in triplicate, the recovery of AFB1 in terms of extraction yield and matrix effects was determined by spiking 2.5 μg/mL of the mycotoxin to a known volume of extract which was thoroughly mixed and allowed to stand in a fume hood. Recovery of AFB1 was calculated as: % Recovery = [(A − B) / C] × 100, where A, B and C were are respectively, concentrations of AFB1 found in spiked and non-spiked extracts and the toxin spiked. Thereafter, determination of AFB1 degradation was analyzed on a Shimadzu LC-20AB HPLC system (Shimadzu Corporation, Kyoto, Japan) that consisted of a RP-C18 symmetry column [250 × 4.6 mm i.d., 5 μm (particle size), Waters, Milford, USA]. Presence of AF fluorescence was detected on a photo diode array (PDA) detector (SPDM20A, Shimadzu, Japan) at excitation and emission wavelengths of 333 and 500 nm, respectively. Elution was carried out under isocratic conditions using acetonitrile/methanol/water (60:20:20, v/v/v) as the mobile phase at an injection volume of 5 μL at a constant flow rate of 1 mL/min. The oven temperature was maintained at 30 °C using a column heater (CTO-20A, Shimadzu, Japan). Working standard levels were 10, 20 and 40 μg/mL. Percentage degradation of AFB1 was calculated as [A − B] / A × 100, where A and B are the initial and final AFB1 concentrations, respectively. 2.4. Lysate preparation Bacterial strains were cultured in sterile NB. Subsequently, 40 mL of a 24 h broth culture was inoculated into 1000 mL sterile NB and incubated for 24 h at 37 °C with agitation at 200 rpm using a shaking incubator (Labcon 3081U, Labcon, South Africa). After incubation, the bacterial cells were harvested and pelleted using a centrifuge (Centrofriger-BL II, Labex, South Africa) at an operating speed of 12,000 rpm for 10 min at 4 °C. The pellets were then washed using sterile phosphate buffer saline (PBS) (pH 7.4) and resuspended in the same buffer, before cell lysis. The respective suspensions were then lysed intermittently using a sonicator (Bandelin Sonopuls HD 2070, Bandelin Electronic GmbH & Co. Germany) at 4 °C, 4 pulse cycles with each lasting for 10 min. After the sonication process, the lysed cells were centrifuged at 20,000 rpm for 1 h at 4 °C (Beckman Coulter Avanti™ J-301, Beckman Coulter, USA). Each lysate was each filtered using a 0.22 μm pore size syringe filter (Gema Medical S.L, Spain) and stored at −20 °C until further use.

2.2. Degradation of AFB1 by bacterial cultures 2.5. Protein determination The bacterial strains were cultured in sterile Nutrient Broth (NB) (HG000C24 Merck, South Africa) for 24 h in a 250 mL flask. Aliquots of the grown cultures were inoculated in 50 mL sterile NB and incubated at 37 °C with agitation at 200 rpm (Labcon 3081U, Labcon, South Africa). Degradation studies used a modified method of Teniola et al. (2005), by supplementing the respective bacterial cultures (400 μL) with 100 μL of

Protein concentration of the lysates was determined using the Bradford method (Bradford, 1976). Standard curves were prepared using known concentrations of bovine serum albumin. Total protein concentrations of the crude extracts were subsequently extrapolated from the standard curve.

O.A. Adebo et al. / International Journal of Food Microbiology 233 (2016) 11–19 Table 1 Mean protein concentration (mg/mL) of differently treated lysates. Lysate condition

Pseudomonas fluorescens

Pseudomonas anguilliseptica VGF1

Staphylococcus sp. VGF2

PIǂ PK§ Lysates only HIϕ

0.82a ± 0.04 0.36a ± 0.03 0.77a ± 0.04 0.29a ± 0.02

1.10b ± 0.01 0.79b ± 0.01 1.04b ± 0.02 0.65b ± 0.01

1.32c ± 0.08 0.84c ± 0.02 1.23c ± 0.06 0.73c ± 0.01

Each value is a mean ± SEM of triplicate values. Means with no common letters within a row significantly differ (p b 0.05). ǂ PI — protease inhibited. § PK — proteinase K treated. ϕ HI — heat inactivated.

2.6. Lysate degradation of AFB1 Degradation experiments were performed in 1.5 mL Eppendorf tube using a modified method of Teniola et al. (2005). Briefly, 100 μL of 2.5 μg/mL AFB1 (Sigma Aldrich, Germany) dissolved in DMSO was added to 400 μL of the lysate. The mixture was incubated (Labcon 3081U, Labcon, South Africa) at 37 °C for 3, 6, 12, 24 and 48 h. Control experiments were done using sterile PBS. Extraction, detection and quantification of AFB1 was done as earlier described in Section 2.3. 2.7. Heat inactivation, protease inhibitor and proteinase K treatments of the lysates Protease inhibitor cocktail (Sigma Aldrich, Germany) was added to the bacterial cells before cell lysis [1 mL of the cocktail solution to 20 mL of cells (containing 4 g wet weight of the cells)], to inhibit protease activity (upon cell lysis). Subsequent cell lysis was done as previously described in Section 2.3. Heat inactivation of the lysates was done following the method described by Guan et al. (2010), and the effect of proteinase K treatment on the lysates was investigated using the method of Smiley and Draughon (2000) by exposing them to 1 mg/ mL proteinase K (Thermo Scientific, South Africa). The obtained lysates were incubated with AFB1 at 37 °C for 3, 6, 12, 24 and 48 h. Extraction, detection and quantification of AFB1 were done as earlier described in Section 2.3. 2.8. Cytotoxicity studies 2.8.1. Lymphocyte isolation and culture Five (5) mL of human blood was collected from a healthy volunteer and lymphocytes were extracted using the method described by Meky et al. (2001). Using the method described by Meky et al. (2001), the lymphocyte cells were grown in culture flasks consisting of a complete culture medium [Fetal Bovine Serum (FBS), supplemented with 100 U/ mL penicillin and 100 mg/mL streptomycin and RPMI 1640 medium (with L-glutamine) (Gibco)]. The cells were incubated at 37 °C in a 5% CO2 buffered and humidified incubator (Forma Scientific 3111, Thermo Scientific, USA). Cell concentrations were examined on a Neubauer hemocytometer and those having 100% viability were transferred into a cell culture flask containing 100 mL complete culture medium and incubated at 37 °C for 24 h in a 5% CO2 buffered and humidified incubator (Meky et al., 2001). One-hundred and eighty microliter of cultured cells were in triplicate, pipetted into 96-well plates and cells stimulated with 20 μL of 10 μg/mL phytohaemagglutunin-p (PHA-p) in preparation for MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 2.8.2. MTT assay MTT assay was done as described by Meky et al. (2001) with the modifications of Njobeh et al. (2009). Viable cells were exposed to 20, 40 and 80 μL of AFB1 standard solution, extracts containing the AFB1

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degradation products (previously evaporated to dryness and redissolved in DMSO) as well as the bacterial cultures and their respective lysates. Exactly 160 μL of culture media were then added to each well and the plates incubated at 37 °C in a 5% CO2 buffered and humidified incubator for 24, 48 and 72 h. After each incubation period, 30 μL of MTT solution (5 mg/mL in 0.14 M PBS) was added, the plates incubated for 3 h in the same incubator, then 50 μL of DMSO was added to each well to dissolve the formed crystals and the plates incubated for a further 2 h. At the end of incubation, absorbance was read using a microplate reader (iMARK, Biorad South Africa) at a wavelength of 560 nm. Percentage cell viability was expressed as [AS / AC] × 100, where AS is the absorbance of the sample and AC the absorbance of the untreated samples (control). 2.8.3. Statistical analysis The protein concentration, AFB1 percentage degradation and cytotoxicity data obtained were analyzed by analysis of variance (ANOVA) using IBM SPSS Statistics 22 (SPSS/IBM, Chicago, Illinois) (Bricker et al., 2014). Significant F-tests at p ≤ 0.05 level of probability were then reported. Duncan's multiple range test (DMRT) was used to determine significant differences among the means, when a significant F-value was established. Microsoft Excel, 2013 (Microsoft Corp., USA) was used to plot various graphs. A multivariate analysis, principal component analysis (PCA) was performed using JMP 12.1.0 statistical software (SAS Institute, Cary, North Carolina) to further summarize and describe the variations in the descriptive experimental factors (Eshelli et al., 2015). The results represent the average of triplicate determinations and are expressed as mean ± standard error of mean (SEM). 3. Results and discussion 3.1. Characterization of bacterial strains The gold mines of South Africa are among the deepest excavations in the world (Takai et al., 2001). These mines and environments around them present favorable conditions under which microorganisms reside and flourish. Based on 16S rRNA and phylogenetic evolution, the bacterial isolates in this study were found to represent new strains of Staphylococcus and Pseudomonas species. The nucleotide sequences of Staphylococcus sp. VGF2, Pseudomonas anguilliseptica VGF1 and Pseudomonas fluorescens were subsequently deposited in GenBank with accession numbers KX008390, KX008391 and KP192770.1, respectively, generated. The strains are part of a bacterial culture collection belonging to the Department of Biotechnology and Food Technology, University of Johannesburg, South Africa. Similar occurrence of bacterial strains in South African gold mines belonging to these genera has also been reported (DeFlaun et al., 2007; Onsott et al., 1997) and a few of these reported strains have degraded other polyaromatic hydrocarbons and mycotoxins (Booyjzen, 2007; DeFlaun et al., 2007; Krifaton et al., 2011; Onsott et al., 1997; Mujahid et al., 2015; Samuel et al., 2014;

Fig. 1. Mean AFB1 degradation kinetics by bacterial liquid cultures over 48 h.

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Fig. 2. Monitoring optical density during the AFB1 degradation by the bacterial cultures over 48 h. Fig. 4. Mean AFB1 degradation kinetics by uninhibited bacterial lysates over 48 h.

Shapira and Paster, 2004; Shekhar et al., 2014). Microorganisms thriving under such conditions are of great interest as their naturally developed resistance to harsh conditions makes them suitable for a wide array of biocatalytic reactions (Deive et al., 2012). Furthermore, we have earlier confirmed the potential of such microorganisms to degrade AFB1 (Adebo et al., 2016).

3.2. Protein concentration The degradation of AFs to safe limits is vital to ensure the safety of foods throughout the food and feed chain. Cells of the three bacterial strains were intermittently lysed using a probe sonicator in the presence and absence of protease inhibitors. The total protein concentrations of the resulting lysates, according to their respective treatments were determined using the Bradford assay and data presented in Table 1. From all the bacterial strains examined, lysates of Staphylococcus sp. VGF2 had a total protein concentration of 1.2 mg/mL, coinciding with a higher AFB1 degradation. For all the lysates, protein concentration and AFB1 degradation were enhanced by protease inhibitor treatments. The protease inhibited (PI) lysates of Staphylococcus sp. VGF2 exhibited a higher protein concentration of 1.3 mg/mL whereas the PI lysates of P. fluorescens had the lowest protein concentration of 0.8 mg/mL. Compared to both the protease inhibited and uninhibited lysates, heat inactivation significantly (p ≤ 0.05) decreased protein concentration (Table 1). Heat inactivated lysates of Staphylococcus sp. VGF2 had a decreased protein concentration of 0.7 mg/mL, and P. fluorescens had the least protein concentration of 0.3 mg/mL corresponding to a reduced level of AFB1 degradation. A similar trend was also observed for the proteinase K treated lysates (Table 1).

Fig. 3. Mean AFB1 degradation kinetics by PI bacterial lysates over 48 h.

As stated by Teniola et al. (2005), total protein concentration can be a viable marker for establishing the enzymatic basis for AFB1 degradation among microbial strains. The protein concentrations obtained in this study, correlated well with the degradation patterns of the respective bacterial lysates. Due to the presence of endogenous enzymes in crude cells, proteases can degrade valuable enzymes. It is therefore necessary to protect the integrity of the enzymes of interest during cell lysis (disruption). The slight increase in total protein concentration as observed for the protease inhibited lysates compared to the uninhibited fractions suggests that degradation of enzymes occurred during cell lysis and the protease inhibitor cocktail used in this study shielded the enzymes. Conversely, a decrease in protein contents observed in the heat inactivated and proteinase K treated lysates indicates the partial denaturation of the proteins (enzymes) present. 3.3. AFB1 degradation by liquid cultures and lysates Liquid cultures of the bacterial strains were investigated for their ability to degrade AFB1. As observed by HPLC, the bacterial strains were able to effectively degrade AFB1 to varying extents over a period of 48 h. As illustrated in Fig. 1, in 6 h, a 27.8% AFB1 degradation was observed in the presence of the liquid cultures of P. fluorescens as compared to 37.5 and 42.1% observed for P. anguilliseptica VGF1 and Staphylococcus sp. VGF2 respectively. After 48 h of incubation, a 56.8% AFB1 degradation was observed for liquid cultures of Staphylococcus sp. VGF2. Under similar conditions, a significant (p ≤ 0.05) lower AFB1 degradation was observed for liquid cultures of P. anguilliseptica VGF1

Fig. 5. Mean AFB1 degradation by lysates, heat inactivated lysates, protease inhibited lysates, proteinase K treated lysates, liquid cultures and proteinase K treated liquid cultures after 48 h of incubation.

O.A. Adebo et al. / International Journal of Food Microbiology 233 (2016) 11–19

(51.7%) and P. fluorescens (47.7%) (Fig. 1) and at longer incubation periods, there was no significant difference in AFB1 decrease for all the bacterial cultures. In contrast and in tandem to similar reports of

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Teniola et al. (2005), no significant reduction occurred when untreated sample (AFB1 + sterile NB) as well as heat inactivated cultures were tested. A significant (p ≤ 0.05) reduction in AFB1 degradation observed

Fig. 6. Mean % cell viability of the bio-transformed extracts after (a) 24 h, (b) 48 h and (c) 72 h.

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Fig. 7. Mean % cell viability of the bacterial cultures and lysates after (a) 24 h, (b) 48 h and (c) 72 h.

O.A. Adebo et al. / International Journal of Food Microbiology 233 (2016) 11–19

when cultures were treated with proteinase K further suggests that the bacterial strains investigated herein, were involved in the AFB1-degradation process (Farzaneh et al., 2012; Teniola et al., 2005) and not via binding/adhesion. Further monitoring of the optical density of the bacterial strain and their corresponding decrease in AFB1 content, revealed that an increasing bacterial population resulted in a reduced AFB1 content (Fig. 2). Comparable results previously obtained in other similar studies also suggest that such degradation is a constitutive activity of the tested bacteria (Eshelli et al., 2015; Farzaneh et al., 2012; Guan et al., 2008; Samuel et al., 2014; Teniola et al., 2005). Different reports have been presented on AF degradation by different bacterial isolates (Adebo et al., 2015; Ciegler et al., 1966; Doyle et al., 1982; Samuel et al., 2014; Smiley and Draughon, 2000), but a few on bacterial lysates and fractions (Guan et al., 2008; Teniola et al., 2005). Ciegler et al. (1966) investigated the ability of about 1000 different microorganisms and only Nocardia corynebacteroides exhibited up to 70% AF reduction in some food commodities. Further studies revealed that this bacterium transformed AFB1 into aflatoxicol (AFL) (Doyle et al., 1982) and the degradation mechanism was proven to be enzymatic (Smiley and Draughon, 2000). Liquid cultures and cell free extracts of Rhodococcus erythropolis have also been reported to degrade AFB1 with a residual amount of 3–6% left after 72 h of incubation (Teniola et al., 2005). Recent studies by Cserhati et al. (2013) have also demonstrated that strains of Rhodococcus possess high degradation ability for other mycotoxins such as zearalenone, fumonisin B1 and T2 toxin. This study thus investigated the ability of bacterial lysates to degrade effectively AFB1 over time. Different levels of AFB1 degradation were recorded and within 3 h of incubation, ˃60% AFB1 degradation for all the protease inhibited lysates was noted. In 6 h, a 100% AFB1 elimination by Staphylococcus sp. VGF2 lysates was observed (Fig. 3). Among all protease inhibited extracts tested, no residual AFB1 was detected when incubation time was extended to 12 h. It was found that uninhibited bacterial lysates had a much lower degradation capacity. Accordingly, only 44.7% AFB1 degradation was observed for lysates of the Staphylococcus sp. VGF2 after 48 h (Fig. 4), which corresponds to a reduced protein concentration (Table 1). This can be explained by the fact that the absence of protease inhibitors decreases protein concentration as found in this study. Similar trends were also observed for uninhibited lysates with those of P. fluorescens giving the least AFB1 degradation of 35.8% after 48 h of incubation. To further investigate the mode of AFB1 degradation, lysates were heat inactivated and incubated together with a known concentration of AFB1. It was found that after 48 h of incubation, heat inactivated lysates demonstrated a much significantly (p ≤ 0.05) lower degradation ability with P. fluorescens, P. anguilliseptica VGF1 and Staphylococcus sp.

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VGF2 recording a percentage AFB1 degradation of 21.9, 25.8 and 27.4%, respectively (Fig. 5). According to Smiley and Draughon (2000), heat treatment of protein extracts does not conclusively prove that AFB1 degradation is enzymatic. Hence, proteinase K was used to treat the lysates prior to incubation with AFB1, which then resulted in a significant (p ≤ 0.05) reduction in % AFB1 degradation (Fig. 5). Conversely, AFB1 was found to be stable in the presence of PBS and protease inhibitor cocktail. However, a 100% degradation of AF was observed by Teniola et al. (2005) for intracellular extracts of R. erythropolis and Mycobacterium fluoranthenivorans after 8 h as well as a 74% degradation by cells of Flavobacterium aurantiacum in 68 h (Ciegler et al., 1966), meanwhile a degradation of ˂20% was observed for cell extracts of Myxococcus fulvus ANSM068 (Guan et al., 2010). A much reduced AFB1 degradation of less than 50% in 48 h for the tested lysates (protease uninhibited) was recorded, which directly correlated to a reduced protein content as seen in these extracts, thus indicating that protease inhibition of cells before lysis protected the proteins (enzymes), yielding significantly improved AFB1 degradation. These results demonstrate that cells treated with protease inhibitors had the integrity of their proteins safeguarded, resulting in an increased AFB1 degradation potential. A much lower AFB1 degradation ability was recorded for heat inactivated lysates. Similar results have previously been reported attributing this reduction to heat inactivation and proteinase K treatment of cell free and crude protein extracts (Teniola et al., 2005; Smiley and Draughon, 2000). These findings also corroborated earlier assertions reported in the literature (Farzaneh et al., 2012; Guan et al., 2008; Smiley and Draughon, 2000), suggesting that an enzyme or protein is responsible for AFB1 degradation. Enzyme activity takes place over time, hence the continuous increase in degradation over time also posits that enzymes are responsible (Guan et al., 2008). HPLC chromatograms for all bio-transformed extracts (liquid cultures and the lysates degraded AFB1) tested did not show the appearance of any new peaks. It is most likely that AFB1 was hydrolyzed and bio-transformed to other compounds structurally different from the parent molecule. Therefore, it could only be speculative that the degradation products are such that they could not be detected by PDA. 3.4. Cytotoxicity In this study, it was necessary to ascertain the toxicity of AF degradation products (extracts) in comparison to the AF parent molecule. This is because degradation of AF could possibly result in a partial or complete hydrolysis of the parent AF to other toxic, less toxic or nontoxic compounds. It therefore becomes extremely important to investigate the toxicity of the degraded compound. Cytotoxicity of the lysate degraded

Fig. 8. PCA multivariate data analysis of AFB1 degradation by different bacterial strains at different time (a) score plot (b) loadings plot.

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AFB1 was determined on human lymphocytes following the MTT assay for 24, 48 and 72 h. As generally observed, percentage cell viability was inversely proportional to AFB1 concentration and time of exposure (Fig. 6a-c). Results obtained also indicated that the cell viability was reduced to varying extents when cell were exposed to the bio-transformed extracts, compared with AFB1 (2.5 μg/mL) which significantly induced (p˂0.05) lymphocyte mortality of 56.3% after 72 h (Fig. 6c). Bio-transformed extracts of all the uninhibited bacterial lysates of Staphylococcus sp. VGF2, P. anguilliseptica VGF1 and P. fluorescens induced similar significant (p˂0.05) cell death of 24.9, 29.3, 31.6 and 34.1%, respectively. A significant (p˂0.05) reduction in cell mortality, ranging between 5 and 9% and corresponding higher percentage cell viability of more than 90% were observed for all the protease inhibited lysates after 72 h (Fig. 6c). In overall, percentage AFB1 degradation by the bacterial lysates coincided with a reduced cytotoxic effect on human lymphocytes. The corresponding reduction in the cytotoxic effect of AFB1 and decreased fluorescence property of the degraded AFB1 molecule suggest that the bacterial lysates cleaved and modified the lactone ring of the AFB1 (Lee et al., 1981; Motomura et al., 2003). The cytotoxicity of bacterial cultures and lysates on human lymphocytes were further studied to ascertain the toxicity and safety. This is a vital prerequisite for the use of bacterial strains to degrade or inactivate mycotoxins in foods as they are expected not to leave or subsequently produce toxic residues thereafter and to ascertain they are generally recognized as safe (GRAS) (Shapira and Paster, 2004). Furthermore, some bacterial cultures are not only toxic, but also possesses the ability to release endotoxins after lysis as the case may be for fungi in producing mycotoxins, which could result in higher cytotoxicity compared to the parent AFB1 molecule. Reports have documented endotoxin production from the bacterial species utilized in this study (Bjork et al., 1992; Evora et al., 1998; Nya and Austin, 2010; Williams, 2007; Zodrovenko et al., 1990), thus necessitating the investigation of the potential GRAS status of these strains. Percentage cell viability after exposure to the bacterial cultures and lysates is presented in Fig. 7a-c. Percentage cell viability from both the cultures and lysates was ˃90% throughout the study. It was found that after 72 h of incubation, cell viability ranged between 94 and 98%, demonstrating that the tested extracts were non-toxic to human lymphocyte cells and have the potential to be considered as GRAS bacterial strains. 3.5. Principal component analysis Multivariate analysis was utilized to analyze the data and to determine the group similarity of samples according to principle components, where each group is described as a cluster. Within a cluster, samples have closely related properties and behaviors, while different clusters have factors with strong variations based on the Principal Component Analysis (PCA) model of the JMP 12.1.0 statistical software. The loadings plot show the relationship between the variables and how they influence the distribution pattern on the coordinate system of the PCA model. As shown in Fig. 8a, the clusters of the bacterial strains are distinct from each other and their components are quite different. This suggests that the degradation of AFB1 by these bacterial lysates is independent of each other. The loadings plot (Fig. 8b) of this study also revealed that the markers are more pulled towards Staphylococcus sp. VGF2 suggesting that the lysates of this bacterial strain are better AFB1 degraders. 4. Conclusion The findings from this study demonstrate the ability of three bacterial cultures and their lysates to effectively degrade AFB1 to less toxic products. The AFB1 degradation phenomenon was found to be time dependent and was influenced by addition of protease inhibitors before cell lysis. This affords promising prospects for the application of these bacterial lysates for the sequestration and the decontamination of AFs.

These lysates are also possible practical choices for the detoxification of AFB1 in food commodities, enhancing the safety of foods and feeds. Further research on the purification and identification of enzymes responsible for AFB1 degradation herein is required to fully understand the mechanisms involved during the degradation. Acknowledgements This work was financially supported via the Global Excellence and Stature (GES) Fellowship of the University of Johannesburg (UJ) granted to the main author (Adebo, O.A.). This work was also partly supported by the National Research Foundation (NRF) Center of Excellence (CoE) in Food Security co-hosted by the University of Pretoria (UP) and the University of Western Cape (UWC), South Africa. References Adebo, O.A., Njobeh, P.B., Gbashi, S., Nwinyi, O.C., Mavumengwana, V., 2015. Review on microbial degradation of aflatoxins. Crit. Rev. Food Sci. Nutr. http://dx.doi.org/10. 1080/10408398.2015.1106440. Adebo, O.A., Njobeh, P.B., Mavumengwana, V., 2016. Degradation and detoxification of AFB1 by Staphylocococcus warneri, Sporosarcina sp. and Lysinibacillus fusiformis. Food Control 68, 92–96. Alberts, J.F., Engelbrecht, Y., Steyn, P.S., Holzapfel, W.H., Van, Z.W.H., 2006. Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures. Int. J. Food Microbiol. 109, 121–126. Alberts, J.F., Gelderblom, W.C.A., Botha, A., van Zyl, W.H., 2009. Degradation of aflatoxin B1 by fungal laccase enzymes. Int. J. Food Microbiol. 135, 47–52. Bailey, G.S., Price, R.L., Park, D.L., Hendricks, J.D., 1994. Effect of ammoniation of aflatoxin B1-contaminated cottonseed feedstock on the aflatoxin M1 contents of cows' milk and hepatocarcinogenicity in the trout bioassay. Food Chem. Toxicol. 32 (8), 707–715. Bjork, L., Andersson, J., Ceska, M., Andersson, U., 1992. Endotoxin and Staphylococcus aureus enterotoxin a induce different patterns of cytokines. Cytokine 4 (6), 513–519. Booyjzen, C., 2007. Bioremediation of hydrocarbon water pollution by bioaugmentation using Southern African bacterial isolates. An MSc Dissertation Submitted to the Faculty of Science. University of Witwatersrand, South Africa. Bradford, M.M., 1976. Rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Bricker, G.V., Riedl, K.M., Ralston, R.A., Tober, K.L., Oberyszyn, T.M., Schwartz, B., 2014. Isothiocynate metabolism, distribution, and interconversion in mice following consumption of thermally processed broccoli sprouts or purified sulforaphane. Mol. Nutr. Food Res. 58 (10), 1991–2000. Ciegler, A., Lillehoj, B., Peterson, R.E., Hall, H., 1966. Microbial detoxification of aflatoxins. Appl. Microbiol. 14, 934–939. Cserhati, M., Kriszt, B., Krifaton, C., Szoboszlay, S., Hahn, J., Toth, S., Nagy, I., Kukolya, J., 2013. Mycotoxin-degradation profile of Rhodococcus strains. Int. J. Food Microbiol. 166, 176–185. DeFlaun, M.F., Fredrickson, J.K., Dong, H., Pfiffner, S.M., Onstoot, T.C., Balkwill, D.L., Streger, S.H., Stackebrandt, E., Knoessen, S., van Herdeen, E., 2007. Isolation and characterization of a Geobacillus thermoleovorans strain from an ultra-deep South African gold mine. Syst. Appl. Microbiol. 30, 152–164. Deive, F.J., Lopez, E., Rodriguez, A., Longo, M.A., Sanroman, M.A., 2012. Targeting the production of biomolecules by extremophiles at bioreactor scale. Chem. Eng. Technol. 35, 1565–1575. Dors, G.C., Caldas, S.S., Feddern, V., Bemvenuti, R.H., Hackbart, H.C.S., de Souza, M.M., Olivera, M., Garda-Buffon, J., Primel, E.G., Badiale-Furlong, E., 2011. Aflatoxins: contamination, analysis and control. In: Guevara-Gonzalez, R.G. (Ed.), Aflatoxins—Biochemistry and Molecular Biology. InTech, Croatia, pp. 415–438. Doyle, P.A., Applebaum, R.S., Brackett, R.E., Marth, E.H., 1982. Physical, chemical and biological degradation of mycotoxins in foods and agricultural commodities. J. Food Prot. 45, 964–971. Eshelli, M., Harvey, L., Edrada-Ebel, R., McNeil, B., 2015. Metabolomics of the biodegradation process of aflatoxin B1 by Actinomycetes at an initial pH of 6.0. Toxins 7, 439–456. Evora, P.R.B., Ekin, S., Pearson, P.J., Shaff, H.J., 1998. Endothelium-dependent vasolidation in response to Pseudomonas aeruginosa lipopolysaccharide: an in vitro study on canine arteries. Braz. J. Med. Biol. Res. 31 (10), 1329–1334. Farzaneh, M., Shi, Z.Q., Ghassempour, A., Sedaghat, N., Ahmadzadeh, M., Mirabolfathy, M., Javan-Nikkhah, M., 2012. Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran. Food Control 23, 100–106. Guan, S., Ji, C., Zhou, T., Li, J.X., Ma, Q., Niu, T., 2008. Aflatoxin B degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium. Int. J. Mol. Sci. 9, 1489–1503. Guan, S., Zhao, L., Ma, Q., Zhou, T., Wang, N., Hu, X., Ji, C., 2010. In vitro efficacy of Myxococcus fulvus ANSM068 to biotransform aflatoxin B1. Int. J. Mol. Sci. 11, 4063–4079. Hormisch, D., Brost, I., Kohring, G.W., Giffhorn, F., Krippenstedt, R.M., Stackebrandt, E., Farber, P., Holtzapfel, W.H., 2004. Mycobacterium fluoranthenivorans sp. Nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant. System. Appl. Environ. Microbiol. 27, 553–660.

O.A. Adebo et al. / International Journal of Food Microbiology 233 (2016) 11–19 IARC (International Agency for Research on Cancer), 2002. Summaries and Evaluations: Aflatoxins. IARC Press, Lyon, France. Kolosova, A., Stroka, J., 2011. Substances for reduction of the contamination of feed by mycotoxins: a review. World Mycotoxin J. 4 (3), 225–256. Krifaton, C., Kriszt, B., Szoboszlay, S., Cserhati, M., Szucs, A., Kukolya, J., 2011. Analysis of aflatoxin-B1-degrading microbes by use of a combined toxicity profiling method. Mutat. Res. 726, 1–7. Lee, L.S., Dunn, J.J., De Lucca, A.J., Ciegler, A., 1981. Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity. Experientia 37, 16–17. Liu, D.L., Yao, D.S., Liang, Y.Q., Zhou, T.H., Song, Y.P., Zhao, L., Ma, L., 2001. Production, purification and characterization of an intracellular aflatoxin-detoxifizyme from Armillariella tabescens (E-20). Food Chem. Toxicol. 39, 461–466. Makun, H.A., Dutton, M.F., Njobeh, P.B., Gbodi, T.M., Ogbadu, G.H., 2012. Aflatoxin contamination in foods and feeds: A special focus on Africa. In: Ayman-Amer, E. (Ed.), Trends in Vital Food and Control Engineering. InTech, Croatia, pp. 187–234. Mann, R., Rehm, H.J., 1977. Degradation of aflatoxin B1 by various microorganisms. Z. Lebensm.Unters. Forsch. 163, 39–43. Marin, S., Ramos, A.J., Cano-Sancho, G., Sanchis, 2013. Mycotoxins: Occurrence, toxicology, and exposure assessment. Food Chem. Toxicol. 60, 218–237. Meky, F.A., Hardie, L.J., Evans, S.W., Wild, C.P., 2001. Deoxynivalenol induced immunomodulation of human lymphocyte proliferation and cytokine production. Food Chem. Toxicol. 39, 827–836. Mishra, H.N., Das, C., 2003. A review on biological control and metabolism of aflatoxin. Crit. Rev. Food Sci. Nutr. 43, 245–264. Motomura, M., Toyomasu, T., Mizuno, K., Shinozawa, T., 2003. Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus. Microbiol. Res. 158 (3), 237–242. Mujahid, T.Y., Wahab, A., Padhiar, S.H., Subhan, S.A., Baloch, M.N., Pirzada, Z.A., 2015. Isolation and characterization of hydrocarbon degrading bacteris from petrol contaminated soil. J. Basic Appl. Sci. 11, 223–231. Njobeh, P.B., Dutton, M.F., Chuturgoon, A.A., Koch, S.H., Steenkamp, P.A., Stoev, S.D., 2009. Identification of novel metabolite and its cytotoxic effect on human lymphocyte cells in comparison to other mycotoxins. Int. J. Biol. Chem. Sci. 3 (3), 524–531. Nya, E.J., Austin, B., 2010. Use of bacterial lipopolysaccharide (LPS) as an immunostimulant for the control of Aeromonas hydrophila infections in rainbow trout Oncorhynchus mykiss (walbaum). J. Appl. Microbiol. 108 (2), 686–694. Onsott, T.C., Tobin, K., Dong, H., DeFlaun, M.F., Fredrickson, J.K., Bailey, T., Brockman, F., Kieft, T., Peacock, A., White, D.C., Balkwill, D., Phelps, T.J., Boone, D.R., 1997. The deep gold mines of South Africa: windows into the subsurface biosphere. SPIE 42nd Annual Meeting Proceedings 3111, pp. 344–357. Park, D.L., 2002. Effect of processing on aflatoxin. Advanced Experimental and Medical Biology 504, 173–179. Rocha, M.E., Freire, F.C., Maia, F.E., Guedes, M.I., Rodina, D., 2014. Mycotoxins and their effects on human and animal health. Food Control 36, 159–165.

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Samuel, M.S., Sivaramakrishna, A., Mehta, A., 2014. Degradation and detoxification of aflatoxin B1 by Pseudomonas putida. Int. Biodeterior. Biodegrad. 86, 202–209. Sangare, L., Zhao, Y., Folly, Y.M.E., Chang, J., Li, J., Selvaraj, J.N., Xing, F., Zhou, L., Wang, Y., Liu, Y., 2014. Aflatoxin B1 degradation by a Pseudomonas strain. Toxins 6, 3028–3030. Shapira, R., Paster, N., 2004. Control of mycotoxins in storage and techniques for their decontamination. In: Magan, N., Olsen, M. (Eds.), Mycotoxins in Food: Detection and Control. Woodhead Publishing, Cambridge, UK, pp. 190–223. Shekhar, S.K., Godheja, J., Modi, D.R., 2014. Hydrocarbon bioremediation efficiency by five indigenous bacterial strains isolated from contaminated soils. Int. J. Curr. Microbiol. Appl. Sci. 4 (3), 892–905. Smiley, R.D., Draughon, F.A., 2000. Preliminary evidence that degradation of aflatoxin B1 by Flavobacterium aurantiacum is enzymatic. J. Food Prot. 63 (3), 415–418. Strosnider, H.E., Azziz-Baumgartner, M., Banziger, M., Bhat, R.V., Breiman, R., et al., 2006. Workgroup report: public health strategies for reducing aflatoxin exposure in developing countries. Environ. Health Perspect. 114, 1898–1903. Takai, K., Moser, D.P., Onstott, T.C., Spoelstra, N., Pfiffner, S.M., Dohnalkova, A., Fredrickson, J.K., 2001. Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine. Int. J. Syst. Evol. Microbiol. 51, 1245–1256. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tejada, C.Z.I., Avila, G.E., Casaubon, H.M.T., Cervantes, O.R.A., Vasquez, P.C., Hernandez, B.E.M., Moreno, M.E., 2008. Biodetoxification of aflatoxin contaminated chick feed metabolism and nutrition. Poult. Sci. 87, 15691576. Teniola, O.D., Addo, P.A., Brost, I.M., Farber, P., Jany, K.D., Alberts, J.F., van Zyl, W.H., Steyn, P.S., Holzapfel, W.H., 2005. Degradation of aflatoxin B1 by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. Nov. DSM44556 (T). Int. J. Food Microbiol. 105 (2), 111–117. Wang, J., Ogata, M., Hirai, H., Kawagishi, H., 2011. Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624. FEMS Microbiol. Lett. 314, 164–169. Williams, K.L., 2007. Endotoxin structure, function and activity. In: Williams, K.L. (Ed.), Endotoxins: Pyrogens. LAL Testing and Depyrogenation. CRC Press, US, pp. 67–90. Wu, Q., Jezkova, A., Yuan, Z., Pavlikova, L., Dohnal, V., Kuca, K., 2009. Biological degradation of aflatoxins. Drug Metab. Rev. 41, 1–7. Yehia, R.S., 2014. Aflatoxin detoxification by manganese peroxidase purified from Pleutorus ostreatus. Braz. J. Microbiol. 45 (1), 127–133. Zhao, L.H., Guan, S., Gao, X., Ma, Q.G., Lei, Y., Bai, X.M., Ji, C., 2011. Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068. J. Appl. Microbiol. 110 (1), 147–155. Zodrovenko, G.M., Veremeychenko, S.N., Zakharova, I.Y., Knirel, Y.A., 1990. Endotoxins of Pseudomonas fluorescens. Endotoxin of Pseudomonas fluorescens. Endotoxin. Springer, US, pp. 131–135.