Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of

Iran
ABSTRACT
Pistachio nuts are among the commodities with the highest risk of aflatoxin
comtamination in Iran. Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for
humans and livestock. In nature, there are microorganisms which are capable of reducing
aflatoxins contamination in food and feed products. In this study, Bacillus subtilis strain
UTBSP1 was isolated from pistachio nuts and studied for the degradation of AFB1. The
AFB1 contents were determined by the use of HPTLC and HPLC as well as multiple
reactions monitoring (MRM) method in LC-MS/MS. The results indicated Bacillus
subtilis UTBSP1 could considerably remendiate AFB1 from nutrient broth culture and
pistachio nut by 85.66% and 95% respectively. Cell free supernatant fluid caused an
apperent 78.39% decrease in AFB1 content. The optimal conditions for AFB1
degradation by cell free supernatant appeared at 35-400C, during 24h. Furthermore, the
results indicated that AFB1 degradation is enzymes are extracellular and constitutively
produced. The destructive AFB1 differed from standard AFB1 chemically, and lost a
fluorescence property.
1. Introduction
Nuts, in gereral, and pistachio nuts, in specific, are very sensitive to Aspergillus flavus
and Aspergiluus parasiticus, weak opportunistic plant pathogenic fungi ( Mojtahedi,
Rabie, Lubben, Steyn, & Danesh, 1979; Sinha & Bhatnagar, 1998). Both A.flavus and A.
parasiticus have a wide host ranges and by producing aflatoxins (AFs) cause to
contaminate agriculture crops, feed and food products ( Shephard, 2005). AFs, especially
AFB1, result in carcinogenic, teratogenic, hepatotoxic and immunosuppressive effects on
human and animals.
Pistachio nuts are among the commodities with the highest risk of AFs contamination.
Iran is the largest pistachio nut producer in the world and AFs contamination has been
reported in Iran pistachio nut especially during harvest/postharvest period and storage
stage. In a survey in Iran, 7.5% of the analyzed pistachio nuts contained total AF (AFT)
higher than maximum tolerated level (15ng/g). Consequently, in recent years, Iranian
health and agriculture authorities have implemented strict regulations to manage of
contamination by promoting good agriculture practice principles in the orchards and
hazard analysis and critical control point principles in storage and processing plants.

Hence, preventive action cannot be achieved, several strategies have been proposed to
inactivate and detoxify AFs in crops, including physical, biological and chemical
methods. Most physical and chemical detoxification methods have many limitations, such
as loss of product nutritional value, organoleptic qualities, undesirable health effects, and
high cost of equipment. Among biological methods, beneficial microorganisms such as
bacteria and fungi substantially contribute to reducing AFs in contaminated media.
However, bacteria have more application for the aflatoxin remediation because of some
advantages such as more elimination within shorter time as well as producing non
pigments. Several reports have been published about AFB1 reduction by some bacterial
isolates. Lactic acid bacteria such as Lactobacillus, Bifidobacterium, Propionibacterium
and Lactococcus were found to be active in removing AFB1 primarily by the adhesion
method. In addition, some bacteria such as Rhodococcus erythopolis, Bacillus sp.,
Stenotrophomonas maltophilia, Mycobacterium fluoranthenivorans and Nocardia
corynebacterioides were reported to degrade AFB1.
The objectives of this study were to find a native bacterial isolate, from the bacterial
population of Rafsanjan pistachio phyllosphere, with strong ability to reduce AFB1, to
identify the promising strain based on physical and chemical characterization as well as
16S rRNA gene sequencing, to find initial mechanism, optimal temperature and time
kinetics of degradation of AFB1 by the strain.
2. Materials & methods
2.1. Bacterial isolate and AFB1 preparation
Bacterium UTBSP1 was isolated from mature pistachio nut fruits of Rafsanjan orchards
in September 2008. Briefly, 10 mature pistachio nuts were placed in 100-ml flasks
containing 20ml of sterile 0.85% NaCl and were vigorously shaken at 350 rpm for 90
min. the resulting suspensions (100l) were plated on NA medium and inoculated for 72h
at 300C. This strain could considerably inhibit the mycelia growth of A.flavus.
AFB1 was purchased from Arcos Organics Ltd, USA. Stock standard solution of AFB1 at
the 500g/ml concentrations was prepared in methanol, kept at -200C. Working solutions
at the concentrations of 200, 100 and 50g/ml. were diluted in methanol and stored at
-200C.
2.2. Identification of the UTBSP1 Strain
To identify isolate UTBSP1, physiological and biochemical tests were carried out
followed by Schaad, Jones, and Chun. In addition, 16s Gene Sequence analysis was

performed according to Chen et al. Briefly, total DNA was extracted by using Ultrapure
TM DNA extraction kit and the gene, encoding 16s rRNA, was amplified by PCR using
universal primers. The amplified nucleotide product was sequenced and similar
sequences were identified using online BLAST in NCBI nucleotide database. A multiple
alignment and a phylogenetic tree were obtained using CLUSTAL X 2.0 software and
MEGA 4 software.
2.3.2.Reduction of AFB1 from aqueous solution by bacterial cells and cell free
supernatant fluid of strain UTBSP1
The effects of bacterial cells and cell free supernatant fluid (extracellular fraction) on
reduction of AFB1 were studied according to Teniola et al. and El-Nezamirespectively,
within 1, 2, 4, 6, 8, 12, 24, 48, and 72h. The influence of temperature on AFB1 reduction
efficacy of extracellular fraction was studied at 20, 25, 30, 35 and 400C, for 72h. To
determine the AFB1 degradation ability, extracellular fraction was treated with 1mg/ml
proteinase K and 1% SDS at 300C, in the dark condition for 6h, according to Alberts,
Engelbrecht, .The effect of heat treatment was determined by dipping the cell free
supernatant in boiling water bath for 10min. The sterile NB culture and untreated cell free
supernatant culture (crude) were used as control. Each experiment was terminated by the
addition of an equal volume of chloroform to extract the remaining AFB1.
2.3.3. degradation of AFB1 by strain UTBSP1 in pistachio nut
Hulls and shells of undamaged mature pistachio nuts were removed and then kernels
were dried in oven at 600C for 5h. The dried kernels were ground with a blender and
placed at Petri plates. The ground kernels (10g ) were thoroughly contamination of 2g
AFB1 per gram kernel, and then inoculated with 1ml of the isolate UTBSP1 (10 8cfu/ml).
The Petri plates were incubated in dark conditions at 300C, and their AFB1 contents were
determined during 1, 3 and 5 days.The following controls were ground kernel and AFB1
contaminated ground kernel without bacterial inoculation.
2.4. Extraction of residual AFB1
AFB1 content in liquid culture was extracted three times by chloroform. The chloroform
was evaporated with a gentle stream of nitrogen gas at 500C and the samples were
dissolved in 1 ml of methanol, filtered (0.22m) and were then analyzed for AFB1.
AFB1 was extracted from pistachio nut according to the method reported by.The
immunoaffinity column clean-up was used prior to analysis.
2.5. Quantification and qualification of residual AFB1

Analyses of AFB1 were performed by high performance thin layer chromatography

(HPTLC), reversed- phase high performance liquid chromatography mass spectrometry
(LCMS).
TLC analysis was performed according to Alberts et al. (2006) on TLC silica gel 60 F254
plates by CAMAG TLC system at 365nm. RP-HPLC analysis was performed using the
C18 column on Agilent 1100 series liquid chromatography according to Yazdanpanah
The LC-MS/MS system was used according to Takinoby Agilent HP1200 series liquid
chromatography coupled with triple-quadrupole mass spectrometer. The symmetry
ZORBAX SB-C18 column, and the mobile phase, methanol and water with 10 nM
ammonium acetate (60:40), at the flow rate of 0.2 mL/min were applied. LC-ESI/CID
mass spectrometry was used to create product ions of the AFB1 precursor ion. For the
CID experiment, helium was used as the collision gas and the collision energy (CE) was
set at 20-50% to find the best CE for three abundant product ions. Quantitative analysis
was carried out using multiple reactions monitoring (MRM) mode.