Progress 01/23/14 to 09/15/15
Outputs
Target Audience:These projects target the agrigultural and farming community, scientific researchers, food and energy consumers, as well as regulatory laboratories. Efforts: We maintained outreach services to the community by hosting agricultural and food/feed safety experiences within our laboratory. Overall, 104 individuals from the community participated in following outreach activities: "An Agricultural Crime Case-Focusing on Mycotoxin Analysis" activity as a forensic/feed safety experience for Winston Academy; Facility tours were provided to 4-H Participants during Summer 2015 highlight food and feed safety; We partnered with MSU, City of Starkville Police Department, MEMA, and 47th Civil Support Team for an emergency response exercise pertaining to an agricultural terrorism event. We also offered a demonstration of the practical application of analytical testing for petroleum products for environmental and mechanical engineers (47 individuals). Additionally, we have published our findings in peer-reviewed journals and presented our research at scientific conferences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?We maintained outreach services to the community by hosting agricultural and food/feed safety experiences within our laboratory. Overall, 104 individuals from the community participated in following outreach activities: "An Agricultural Crime Case-Focusing on Mycotoxin Analysis" activity as a forensic/feed safety experience for Winston Academy; Facility tours were provided to 4-H Participants during Summer 2015 highlight food and feed safety; We partnered with MSU, City of Starkville Police Department, MEMA, and 47th Civil Support Team for an emergency response exercise pertaining to an agricultural terrorism event. We also offered a demonstration of the practical application of analytical testing for petroleum products for environmental and mechanical engineers (47 individuals). Additionally, we have published our findings in peer-reviewed journals and presented our research at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This describes an effective sample preparation method for the extraction of aflatoxin B1 from Aspergillus flavus infected maize kernels. The method involves liquid extraction with a methanol/water solution. The samples were analyzed by liquid chromatography coupled to an electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in positive ion multiple reaction monitoring (MRM) mode. The spiked levels for the recovery experiment were 4 and 20 ng/g. Recoveries ranged between 72% and 113% (90.8% average), with RSD below 15% (10.6% on average). Key Words: Aspergillus flavus, aflatoxin B1, LC/MS, extraction Introduction A. flavus is a fungus that commonly grows in the soil and is known to infect a variety of crops including cotton, peanuts, and corn. A. flavus can cause ear rot in corn, aflaroot in peanuts, and yellow spot disease in cotton. Environmental conditions such as high temperatures, high humidity, drought stress, and poor crop storage can cause A. flavus to start producing aflatoxins. Aflatoxins are a secondary metabolite of the fungus and are extremely carcinogenic [1]. The four major types of aflatoxins are aflatoxin B1, B2, G1, and G2. Aflatoxin B1 and B2 fluoresce blue under UV light while aflatoxin G1 and G2 fluoresce green in the presence of UV light. Aflatoxin B1 is the most common and potent of these compounds (Figure 1). Aflatoxin B1 is converted in vivo to an aflatoxin B1-exo-8, 9-epoxide by the liver enzyme cytochrome p450 oxidase. The epoxide that is formed is a highly reactive electrophile and has an extremely high regiospecificity for the N7 position of the guanine residue in DNA. It specifically binds to the p53 encoding region to form a DNA adduct. This results in nonfunctioning p53 proteins which are important tumor suppressors in humans [2]. In 1988 the International Agency for Research on Cancer classified aflatoxin B1 as a Class 1 human carcinogen. Therefore, the Food and Drug Administration (FDA) has restricted the amount of aflatoxin in food for human consumption to 20 parts per billion (ppb) in the United States. The European Commission (EC) limits aflatoxins in food for human consumption to 4 ppb in the European Union [3, 4]. The Council of Agricultural Science and Technology has estimated that the US has an annual loss of almost a billion dollars due to crop damage from mycotoxins. An estimated $225 million of that are due to aflatoxin contamination in maize crops [4]. Agriculture is a vital part of Mississippi's economy, and corn is one of its largest crops. In order to better track the progression of the A flavus in an infected maize cob and the production of aflatoxin during infection, we have developed a single maize kernel aflatoxin extraction method. Reagents and Chemicals All solvents and reagents were Optima LC/MS grade. Methanol, water, formic acid, and ammonium acetate were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Purified aflatoxin B1 and M1 standards were acquired from Sigma Aldrich (St. Louis, MO, USA). Sample Preparation Maize kernels were flash frozen in liquid nitrogen. The kernels were ground into a fine powder with a mortar and pestle. 200 mg of each ground sample was placed into a 1.5 mL micro-centrifuge tube. Aflatoxin free ground maize was spiked with aflatoxin B1 to yield two levels of spiked samples (4 ppb and 20 ppb) of aflatoxin B1 for recovery. These concentrations were chosen because they are the limit for aflatoxin in food for human consumption in the European Union and the United States, respectively. A solution of 1 mL methanol: water (70/30, v/v) was added to each 1.5 mL microcentrifuge tube. The samples were vortex for 1 minute and then centrifuged for 5 minutes at 14,000 rpm. Polytetrafluoroethylene (PTFE) syringe filters (0.45 µm) were used to purify the samples after centrifugation (Figure 2). The liquid extractions were transferred to auto-sampler vials and analyzed using an Agilent 6460 LC/MS/MS Triple Quadruple with electrospray ionization. Aflatoxin M1 was added as an internal standard at a concentration of 10 ng/g. The calibration curve was matrix matched with extracted aflatoxin free maize solution. Experimental The samples were analyzed using an Agilent 6460 LC/MS/MS Triple Quadruple ESI, using an Agilent 1200 Series HPLC. The HPLC system consists of a binary pump (G1312B), infinity high performance degasser (G1379B), high performance autosampler (G1367E), thermostatted column compartment (G1316B), sampler thermostat (G1330B), and MassHunter data software. Aflatoxin B1 and M1 were optimized using the Agilent Optimization software. The Agilent Optimization software produces the ideal fragmentor voltage and collision energy for each MRM transition of aflatoxin B1 and M1 (Table 1). Instrumentation HPLC Method Conditions: Column: Zorbax Eclipse Plus-C18 Narrow Bore 2.1 x 50mm, 5µm Column Temperature: 40°C Injection Volume: 10 µL Autosampler Temp: 4°C Mobile Phase: A = 5 mM NH4 acetate and 0.1% formic acid in water B = 5 mM NH4 acetate and 0.1% formic acid in methanol Flow Rate: 0.6 mL/min Gradient: Time (min) %B 0 5 5 100 6 100 Analysis Time: 6 min Equilibration Time: 3 min Total Run Time: 9 min MS Method Conditions: Ion Mode: ESI/Agilent Jet Stream, Positive Ionization Capillary Voltage: 4000 V Drying Gas: 10 L/min Drying Gas Temperature: 325°C Nebulizer Gas: 50 psi Sheath Gas Flow Rate: 11 L/min Sheath Gas Temperature: 350°C Delta EMV: 300 V Results and Discussion Table 2 demonstrates the average percent recoveries and relative standard deviation values (RSD) values obtained from the spiked corn samples after performing the single maize kernel aflatoxin extraction. The analysis was performed in replicates of five at each of the two levels. The average percent recoveries for the 4 ng/g and the 20 ng/g aflatoxin B1 spiked maize were 90.83% with a RSD of 4.11% and 90.72% with a RSD value of 14.45% respectively. Calibration standards were matrix matched with a range from 1 ppb to 100 ppb with a linear correlation (R2) of 0.996 (Figure 4). The limit of detection (LOD) and limit of quantification (LOQ) were estimated from the concentration of aflatoxin B1 required to give a signal to noise ratio of 3:1 and 10:1 respectively. The LOD was determined to be 0.344 ng/g and the LOQ was estimated to be 1.042 ng/g. The internal standard, aflatoxin M1, injected at 10 ppb accounts for variation due to the instrument. Aflatoxin M1 was chosen to be the internal standard because it has a structure similar to aflatoxin B1 but is not produced by A. flavus. Conclusion This application note demonstrates a fast, simple, and effective analytical method for determining aflatoxin concentrations in a single maize kernel using an Agilent 6460 Triple Quadruple Mass Spectrometer. The detection levels for aflatoxin B1 were below both the limit set by the FDA in the United States and the limit set by the EC in the European Union. The recovery percentages for aflatoxin B1 were 90.83% for 4 ng/g and 90.72% for 20 ng/g with a satisfactory average RSD less than 15%. The single kernel extraction method will be a useful technique in determining how aflatoxin producing Aspergillus flavus affects infected maize.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Womack, E.D.; *Sparks, D.L.; Reid, C.X.; Brown, A.E.; DuBien, J.L.; Ward, S. (2015) Validation of Aflatoxin M1 in Raw Milk Using QuEChERS as an Extraction Method. Journal of Chromatography A. In Review.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Williams, C.L.; Dill-McFarland, K.A.; Sparks, D.L.; Kouba, A.J.; Willard, S.T.; Suen, G.; *Brown, A.E (2015) Dietary Changes During Weaning Shape the Gut Microbiota of Red Pandas (Ailurus Fulgens). Microbiome, In Review.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Womack, E.; *Sparks, D.L.; Brown, A.E. (2015) Aflatoxin M1 in Milk and Milk Products: A Short Review. World Mycotoxin Journal, Accepted for Publication.
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
Tabler, T.; Brown, A.; Hagood, G.; Farnell, M. (2015) Nutrient Content in Mississippi Broiler Litter. Mississippi State University Extension Service, MSUcares.com. Pub 2878:POD-02-15.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Candace L. Williams (2015) The Gastrointestinal Tract Microbioto of the Obligate Foragers, the Giant Panda (Aliuropoda melanoleuca) and the Red Panda (Ailurus fulgens). MSU Published Dissertation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Ashli Brown, Cedric Reid, Curtis Atkinson, Darrell Sparks (2015) Using FT-IR as a Tool for Identification of Toxin Producing Agricultural Phytopathogens. AOAC 129th Annual Meeting, September 27-30, Los Angeles, CA
|
Progress 01/23/14 to 09/30/14
Outputs
Target Audience: These projects target scientific researchers, the farming community, food and energy consumers, as well as regulatory food and feed safety laboratories. Efforts: We maintained outreach services to the community by hosting agricultural and food/feed safety experiences within our laboratory. Individuals for the community (74) participated in the following activities: “What killed my bees and contaminated my honey?” activity as a forensic experience for Bug Camp; “An Agricultural Crime Case-Focusing on Mycotoxin Analysis” activity as a forensic/feed safety experience for Winston Academy; Facility tours were provided to 4-H Participants during Summer 2014 highlight food and feed safety. We also offered a demonstration of the practical application of analytical testing for petroleum products for environmental and mechanical engineers (50 individuals). Additionally, we have published our findings in peer-reviewed journals and presented our research at scientific conferences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The two graduate students working on these projects attended and presented material at national research conferences. Both of these students provided extened absttacts and they were awarded ACS National Travel Grants. How have the results been disseminated to communities of interest? These poject methodologies where invovled in the mentoring of 2 graduate students and 2 undergraduate students. The students and PI attended national scientific symposia and presented published abstracts. The data generated by these projects resulted in the submission of 3 peer-reviewed publication. : We also maintained outreach services to the community by hosting agricultural and food/feed safety experiences within our laboratory. What do you plan to do during the next reporting period to accomplish the goals? We will continue to develop data for use in the assement of mycotoxins in human and animal health. Additionally, we will outline strategies to mitigate mycotoxin contamination in food and feed products.
Impacts
What was accomplished under these goals?
Material and Methods: A fresh batch of raw bovine milk was obtained from Mississippi State University’s Department of Animal and Dairy Sciences (Mississippi State University, MS). The adsorbents used included powdered activated carbon (PAC) at concentrations of 0.1, 0.25, and 0.4% and 3 commercial binders: Biomin Mycofix (bentonite), Bios Agricorp Biofix, and Mycosorb Alltech binder (glucomannan yeast) at concentrations of 2, 3, and 4%. A total of 36 artificially spiked (0.5 ppb AFM1) raw milk samples (r=3) and/or an adsorbent were homogenized with a Burrell Wrist Shaker for 60 min and extracted using a modified version of the QuEChERS extractions method. A volume of 15 mL of acetonitrile (ACN) was added to each sample, which was then homogenized in a SPEX Sample Prep Geno-Grinder for 1 min at 1000 strokes min-1. Agilent QuEChERS extraction salts (6 g MgSO4, 1.5 g NaOAc) were added to each sample, vigorously shaken in the Geno-Grinder, and centrifuged for 15 min at 3200 rpm for separation. The supernatant was collected and final clean-up was performed using 0.45 µm PTFE syringe filters. Measurements were performed on an Agilent 1260 Infinity high performance liquid chromatography (HPLC) system coupled to a Triple Quadrupole Mass Spectrometer (MS). Samples were injected onto a Zorbax Eclipse Plus-C18 Narrow Bore 2.1 x 50 mm, 5 µm column from Agilent (Santa Clara, CA) with a column temperature of 40°C. Mobile phase A consisted of water, 5 mM ammonium acetate, and 0.1% formic acid. Mobile phase B was composed of methanol, 5 mM ammonium acetate, and 0.1% formic acid. The total injection volume was 5 μL comprised of 4 μL from the sample and 1 μL from the internal standard (AFB1). The mobile phase was pumped at a flow rate of 0.6 mL min-1 under gradient elution. The MS was operated in positive ion mode, the drying gas (N2) temperature was 325°C, and the drying gas flow rate was set to 10 L min-1. Protein, lactose, and fat from the milk samples were analyzed to determine if the most effective adsorbent was binding the AFM1 from the milk, as well as these milk constituents. Analysis of protein in milk was performed by the Mississippi State Chemical Laboratory using the Leco FP-628 (Dumas N2 analyzer). A weight of 1.0 g of milk sample was combusted at a high temperature in the presence of oxygen leading to the release of CO2, H2O, and N2. The N2 content was measured by passing the remaining gas through a column with a thermal conductivity detector and then converted to protein value. Lactose was measured using an Agilent 1260 Infinity HPLC-evaporative light scattering detector (HPLC-ELSD). All samples were diluted by a 100-fold dilution using 70% ACN and filtered using nylon syringe filters. An injection volume of 10 μL was injected on an amino column with a column temperature of 35°C. The mobile phase of the HPLC-ELSD consisted of 65% ACN at a flow rate of 1 mL min-1. Soxhlet extraction was used to determine the amount of total fat in the milk samples before and after the addition of the PAC. A mass of 2 g of the milk samples was mixed with 80 g of anhydrous sodium sulfate, placed in a desiccator overnight, and extracted using hexane. After extraction, the samples were evaporated and re-weighed to calculate percent fat. Discussion: Physical removal or separation of aflatoxin-contaminated crops is an important strategy for reducing aflatoxin levels. The practice of adding binders or sequestering agents to feed has been studied to reduce toxicity by acting as an enterosorbent and reducing the bioavailability in intestinal absorption . These dietary additives (inorganic silica-based compound, organic charcoal-based polymer, or an indigestible carbohydrate) offer one of the greatest potential solutions for preventing toxicity in a stable digestive tract where the bound aflatoxin can be excreted via urine or fecal material. Research studies have demonstrated the utilization of AC, bentonite, and hydrated sodium calcium aluminosilicate (HSCAS) that effectively reduced AFB1 toxicity in feed . However, not all AFB1 is bound and maybe metabolized to AFM1 and excreted in the milk. Instead of discarding an entire batch of milk that has exceeded the maximum limit of AFM1, a reasonable approach for reducing aflatoxin contamination would be the physical removal of AFM1 from milk. This study was performed to evaluate the effectiveness of PAC, Alltech, Biofix, and Mycofix adsorbents to obtain artificially spiked AFM1 from bovine raw milk. The commercial binders that consisted of bentonite and glucomannan yeast had no significant effect on the reduction of 0.5 ppb AFM1 from raw milk contrary to the results obtain in other studies [18-20]. Soha et al. (2006) found that Bentonite B1 at concentrations of 0.5% and 2% significantly reduced the 0.087 ppb AFM1-naturally contaminated milk both by 91% and Bentonite X reduced the AFM1 levels by 49% (0.5% Bentonite X) and 71% (2% Bentonite X) when the binders were added to the milk [17]. The effectiveness of activated carbon (AC) to bind aflatoxin is variable [21-22]. Diaz et al. (2004) evaluated AC (SA-20), along with other binders, to sequester dietary AFB1 from an animal’s gastrointestinal tract and consequently, reduce the transfer of AFB1 to AFM1 in milk. Contaminated corn of 55 ppb of AFB1 were given to lactating cows. A concentration of 0.25% of AC had no significant effect on the reduction of AFB1 to AFM1 in cow’s milk (5.4% reduction) [23]. However, findings have shown AC to be more effective in aqueous solutions due to its high surface area. Di Natale and Nigro (2009) performed a study that examined the adsorbents selection for aflatoxins removal in bovine milks. It was concluded that the highest removal of AFM1 was obtained by AC due to the high surface area, wide micropore size, and high affinity between AFM1 molecules and the aromatic structure of the carbons (>93% removal of 0.5 µg kg-1). However, there was an effect on nutritional factors removing as high as 80% on organic acids, chlorides, lactose, total protein and pH [24]. A concentration of 0.4% PAC in the current study successfully bound the AFM1 from the milk removing 52% without significantly eliminating milk constituents, milk fat, lactose, and protein. Monitoring the concentrations of AFM1 in milk can be challenging because of very low levels. Therefore, extraction is also important in the quantification of AFM1. QuEChERS has been proven to be a highly beneficial approach that vastly simplifies extraction, cutting costs and time. The QuEChERS method, introduced by USDA scientists in early 2003 for the analysis of pesticide residues in foods, has been extended in the analysis of veterinary drug residues, antibiotics, acrylamide, and mycotoxins in different matrices [25]. Because QuEChERS sample extraction is flexible and has many options of analysis for a diverse range of organic chemicals, modifications of the original method have been made for the extraction of AFM1 from milk. To our knowledge this is the first study describing a QuEChERS method for the quantitative determination of AFM1 in raw milk using HPLC-MS. The effectiveness of the binders to separate AFM1 from the milk using QuEChERS as an extraction method was possible as QuEChERS produced 3 layers separating the AFM1 in the organic layer, the middle layer contained the binder from the sample (and AFM1 if the binder was efficient), and the lower aqueous layer and the excess salts. Recoveries ranged from 92-111% with 0.5 ppb AFM1 artificially spiked raw milk.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Womack, E.D.; *Sparks, D.L.; Reid, C.X.; Brown, A.E.; DuBien, J.L.; Ward, S. (2014) Validation of Aflatoxin M1 in Raw Milk Using QuEChERS as an Extraction Method. Journal of Chromatography A. In Review.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Atkinson, C.; Pechanova, O.; Sparks, D.L.; *Brown, A.; *Rodriquez, J.M. (2014) Differentiation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergilli by FT-IR Spectroscopy. Applied Spectroscopy. 68(8) DOI:10.1002/PMIC.201100659.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Womack, E.D.; *Brown, A.E.; Sparks, D.L. (2014) A Recent Review of Non-biological Remediation of Aflatoxin-Contaminated Crops. Journal of Science, Food and Agriculture. 94:1706-1714.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Erika Womack, Darrell Sparks, Cedric Reid, Ashli Brown, Jan DuBien, Stephanie Hill (2014) Validation of alfatoxin M1 in raw milk using QuEChERS as an extraction method. American Chemical Society National Meeting, San Francisco, CA, August 10-14.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Cedric Reid, Erik Mylorie, Ashli Brown, Darrell Sparks, W. Paul Williams (2014) Correlating aflatoxin accumulation and fungal biomass in Aspergillus flavus inoculated maize. American Chemical Society National Meeting, San Francisco, CA, August 10-14.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Erika Womack, Darrell Sparks, Ashli Brown, Stephanie Hill (2014) Evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk. The American Dairy Science Association, The American Socienty of Animal Science and The Canadian Society of Animal Science Joint Annual Meeting, Kansas City, MO, July 20-24.
|