THE ANALYSIS OF S.C. CROPS FOR THE PREVALENCE OF MOLD AND THE REDUCTION OF AFLATOXINS BY OZONE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1016394
• Project Start Date: Aug 1, 2018
• Project End Date: Jul 31, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH CAROLINA STATE UNIVERSITY
(N/A)
ORANGEBURG,SC 29117

Performing Department
Engineering Technologies

Non Technical Summary
This proposal will investigate the effects of ozone on aflatoxin in wheat, soybeans and cottonseeds grow in South Carolina. There are 2 objectives for the proposed research project: to determine the prevalence of aflatoxins produced by the molds Aspergillus flavis and Aspergillus parasiticus are found in the wheat, cottonseed, and soybean crops in S.C. and to develop an innovative approach to effectively inhibit the levels of aflatoxins in wheat, cottonseed, and soybean crops using various ozone treatments. Mycotoxins contaminate many types of food crops throughout the food chain. Among these toxins, aflatoxins are highly toxic secondary metabolites predominantly produced by the filamentous fungi A. flavus and A. parasiticus. Aflatoxins can contaminate agricultural commodities including corn, wheat, rice, peanut, and many other crops. Aflatoxin B1 is the most important and toxic to human beings from the public health point of view. It is the most toxic and potent carcinogen, teratogen and mutagen to humans and animals, causing damage such as toxic hepatitis, hemorrhage, edema, immunosuppression and hepatic carcinoma. In the US, it was reported that income losses due to aflatoxins contamination cost an average of more than $100 million per year to US producers. To perform this research study, $500,000 is requested for 3 years to purchase the necessary equipment and supplies. In addition, funds are needed to support the PI, Co-PI and 2 undergraduate students. A major goal of this proposal is to strengthen the research capabilities of the students and faculty of South Carolina State University. Students involved in the project will develop research skills promoting acceptance into graduate school. Moreover, further building the capacity of food safety laboratory at SCSU will place the university in a more competitive stance to submit grant applications for future funding from governmental agencies i.e., USDA.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4020	1150	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1150 - Toxicology;

Keywords

aflatoxin

aspergillus flavis

aspergillus parasiticus

ozone

Goals / Objectives
The hypothesis is that the prevalence of mold and aflatoxin contamination will vary among the farms and crops tested in S.C. The goal of the study is to analyze the incidence of mold and aflatoxin contamination on wheat, soybean, and cottonseed crops obtained from S.C. farms. Moreover, the effectiveness of ozone for contamination purposes will be tested.

Project Methods
Samples of wheat, cottonseed and soybeans will be collected from farmers in Hampton Cluster, Orangeburg Cluster, Williamsburg Cluster, and Charleston County during the Spring and Fall of each year. The extension agent will coordinate with farmers. The samples will labeled, packaged in sterile polyethylene bags, transferred to the laboratory and kept in a cool place (3-5°C) until aflatoxins analysis and ozone studies are performed. Sample preparation (extraction of sample): Fifty grams of each homogenized and pulverized sample will be extracted in 100 mL MeOH : H2O (80:20 v/v). The sample suspensions will be blended and the blended extracts will be filtered through Whatman Number 1 filter paper and clear supernatants will be collected in separate airtight amber vials. Samples will be analyzed for aflatoxins using HPLC system consisting of a degasser, auto sampler, and quaternary pump, and fluorescence detector. The chromatographic separation will be performed with a reversed-phase column (Extend-C18, Zorbax column, 4.6 mm i.d., 250 mm, 5 μm, Agilent Co.). The sample will be analyzed isocratically using 60:20:20 water/methanol/acetonitrile mixture as the mobile phase. The column temperature will be adjusted at 30°C at a flow rate of 1.0 mL/min to achieve the optimum resolution of the aflatoxins. The injection volume will be maintained at 20 μL for both the sample and standard solutions. Ozone will be generated with a commercial ozone generator model TS 10 (10g/h) (Ozone solutions Inc., Ohio, USA) using atmospheric air as the oxygen source. The ozonized air produced at a constant flow rate by the apparatus was passed by a silicone tube and the concentration will be measured by ozone analyzer (0-20%) (Ozone solutions Inc., Ohio, USA). Fifty grams of ground sample will be weighed and placed in blender for homogenization. One sample (control) will be mixed with 100 ml of 80% MeOH, the other one will be treated with ozone gas (5% ozone was flushed for 10 min) and mixed with 100 ml of 80% MeOH, and the third one will be mixed with sterile distill water. The blended extracts will be filtered, passed through immunoafinity column and analyzed by HPLC to detect any structural changes. In addition, the Ames test will be used to detect decreased levels of mutagenic properties of the aflatoxins following ozone treatment.

Progress 08/01/18 to 07/31/21

Outputs
Target Audience: SCSU Faculty SCSU Students 1890 Extension agent Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Eight undergraduate students were trained in food safety. These students were trained with aflatoxin extraction method from soybean, and corn. Students were also trained with the HPLC and ozone generator. A literature review of aflatoxin research was conducted and discussed virtually every week with students and researchers. The project has allowed for the preliminary experiments for testing the presence of aflatoxin associated with wheat, soybean and cottonseed collected from local farms. This project also allowed determining the amounts of aflatoxins in wheat and soybean products from local grocery store. Moreover, the results were presented at The IDEA Networks of Biomedical Research Excellence (INBRE) conference. How have the results been disseminated to communities of interest?Poster presentations entitled i) Quantitative Determination Of Aflatoxins In Corn Starch, Peanut Butter And Almond From Local Grocery Stores Using HPLC ii) Determining The Quantity Of Aflatoxins In Breakfast Cereals From Local Grocery Stores and iii) Rapid Analysis Of Aflatoxins Found In Pasta Products And Vicam Corn" at the LS-SCAMP conference held at South Carolina State University on July 25, 2019. Poster presentation entitled "A Comparison of Aflatoxins Levels in Corn Oil Obtained from Local Grocery Stores." Association of Research Directors Conference,March 29, 2019 - April 3, 2019.Jacksonville, Florida. Poster presentations entitled "The Effects of Ozone Treatment On The Reduction Aflatoxins In Corn" at the 77th PAWC hosted by Tuskegee University on December 7-9, 2019. Oral presentations entitled "The Analysis of South Carolina Crops for the Prevalence of Mold and the Reduction of Aflatoxins by Ozone" at the MANNRS meeting held at 1890 Research and Extension, South Carolina State University on December 2, 2019. A Journal club was arranged with students and researchers to study aflatoxin related research articles every week. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Eight undergraduate students were hired to work on the project. These students were exposed to cutting edge research techniques to analyze the presence of aflatoxins in farm wheat, corn, soybean, and cottonseed were analyzed by HPLC. Samples of soybean and corn were collected from farmers in South Carolina. The extension agent coordinated with farmers and samples were labeled, packaged in sterile polyethylene bags, transferred to the laboratory, and kept in a cool place (3-5°C) until aflatoxins analysis by HPLC. Equipment such as High-Performance Liquid Chromatography (HPLC) and evaporator were installed. Chemicals and supplies were ordered. Aflatoxin amounts in farm wheat, soybean and cottonseed were analyzed by HPLC. All samples collected from South Carolina local farms had very small amounts of aflatoxins (less than 1 ppb) which are safe for human consumption. About 33% and 40% of aflatoxin B1 was reduced from aflatoxin contaminated Vicam corn by purging ozone gas for 10 min at 2% and 5% levels, respectively. Aflatoxins G2 (0.01 ppb), B2 (0.12 ppb) and B1 (1.29 ppb) were determined in farm soybean while aflatoxin G1 (0.009 ppb), B2 (0.372 ppb) and B1 (5.575 ppb) were determined in corn by HPLC. All samples collected from South Carolina local farms have very small amounts of aflatoxins which are safe for human consumption. A virtual journal club was arranged every week to study aflatoxin related research paper with students and researchers. A new protocol was developed testing the effects of ozone on farm corn. This protocol sought to explore the efficacy of using ozone as a deterrent for aflatoxin contamination in corn. About 56% and 39% of aflatoxin B2 and B1 was reduced from farm corn by purging 5% ozone gas for 10 min. A manuscript was prepared and submitted to the Journal of Food Science and Nutrition in June.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: James B. Stukes, Nazimuddin Mohammed, Deasia Gathers, David Bottenberg (2021). Analysis of aflatoxins in South Carolina Farms Corn, peanut, wheat, soybean and cotton seed was submitted to the Journal of Food Science and Nutrition on June 28, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jazmyne Cortese, James B. Stukes, Nazimuddin Mohammed and Myrandi Roper, Determination of Aflatoxins In Corn Oil. Proceedings at the South Carolina State University on November 9, 2018 as well as the Annual Biomedical Research Conference for Minority Students (ABRCMS) conference held on November 14, 2018 in Indianapolis, Indiana.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J.L. Cortese*, J.B. Stukes, N. Mohammed, and M. Roper, A Comparison of Aflatoxins Levels in Corn Oil Obtained from Local Grocery Stores. 1890 Research, Department of Biological and Physical Sciences, South Carolina State University, Orangeburg, South Carolina. Association of Research Directors Conference, March 29, 2019 - April 3, 2019. Jacksonville, Florida.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Myrandi Roper, Dr. James B. Stukes, Dr. Nazimuddin Mohammed, Determining The Quantity of Aflatoxins In Breakfast Cereals From Local Grocery Stores. Proceedings at the INBRE conference held at the South Carolina State University on July 25, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: DeAsia Stuckey, Dr. James B. Stukes, Dr. Nazimuddin Mohammed, Quantitative Determination of Aflatoxins In Corn Starch, Peanut Butter And Almond From Local Grocery Stores Using HPLC. Proceedings at the INBRE conference held at the South Carolina State University on July 25, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Deasia Gathers, Dr. N. Mohammed and Dr. J.B. Stukes, The Effects of Ozone Treatment On The Reduction Aflatoxins In Corn. Proceedings at the 77th PAWC conference held at Tuskegee University on December 6, 2019.
• Type: Other Status: Published Year Published: 2019 Citation: Student researcher, DeAsia Gathers received the Highest scoring undergraduate student poster recognition at The Gamma Sigma Delta The Honor Society of Agriculture Tuskegee University Chapter on December 10, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sasha Prioleau, Dr. James B. Stukes, Dr. Nazimuddin Mohammed, Rapid Analysis of Aflatoxins Found In Pasta Products And Vicam Corn. Proceedings at the INBRE Conference held at the South Carolina State University on July 25, 2019.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:1.SCSU faculty 2. SCSU Students 3. 1890 Extension agent Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students were trained in food safety. These students were trained with aflatoxin extraction method from soybean, and corn. Students were also trained to use the HPLC and ozone generator. A literature review of aflatoxin research was conducted and discussed virtually every week with students and researchers. How have the results been disseminated to communities of interest?A Journal club was arranged with students and researchers to study aflatoxin related research articles every week. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Two undergraduate students were hired to work on the project. These students were exposed to cutting edge research techniques to analyze the presence of aflatoxins in farm wheat, corn, soybean, and cottonseed were analyzed by HPLC. Samples of soybean and corn were collected from farmers in South Carolina. The extension agent was coordinated with farmers and samples were labeled, packaged in sterile polyethylene bags, transferred to the laboratory, and kept in a cool place (3-5°C) until aflatoxins analysis by HPLC. Aflatoxins G2 (0.01 ppb), B2 (0.12 ppb) and B1 (1.29 ppb) were determined in farm soybean while aflatoxin G1 (0.009 ppb), B2 (0.372 ppb) and B1 (5.575 ppb) were determined in corn by HPLC. All samples collected from South Carolina local farms have very small amount of aflatoxins which are safe for human consumption. Virtual Journal club was arranged every week to study aflatoxin related research paper with students and researchers. A new protocol was developed testing the effects of ozone on farm corn. This protocol sought to explore the efficacy of using ozone as a deterrent for aflatoxin contamination in corn. About 56% and 39% of aflatoxin B2 and B1 was reduced from farm corn by purging 5% ozone gas for 10 min.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: SCSU faculty SCSU Students 1890 Extension agent Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has allowed for the preliminary experiments for testing the presence of aflatoxin associated with wheat, soybean and cottonseed collected from local farms. This project also allowed determining the amount of aflatoxins in wheat and soybean products from local grocery store. Moreover, the results were presented at The IDEA Networks of Biomedical Research Excellence (INBRE) conference, MANNRS meeting, 77th Professional Agricultural Workers Conference (PAWC) and ARD symposium. How have the results been disseminated to communities of interest? Poster presentations entitled i) Quantitative Determination Of Aflatoxins In Corn Starch, Peanut Butter And Almond From Local Grocery Stores Using HPLC ii) Determining The Quantity Of Aflatoxins In Breakfast Cereals From Local Grocery Stores and iii) Rapid Analysis Of Aflatoxins Found In Pasta Products And Vicam Corn" at the INBRE conference held at South Carolina State University on July 25, 2019. Oral presentations entitled "The Analysis of South Carolina Crops for the Prevalence of Mold and the Reduction of Aflatoxins by Ozone" at the MANNRS meeting held at 1890 Research and Extension, South Carolina State University on December 2, 2019. Poster presentations entitled "The Effects Of Ozone Treatment On The Reduction Aflatoxins In Corn" at the 77th PAWC hosted by Tuskegee University on December 7-9, 2019. Poster presentations entitled "A Comparison of Aflatoxins Levels in Corn Oil Obtained from Local Grocery Stores". March 29-April 3, 2019 ARD symposium, Jacksonville, Fl. Oral presentations entitled "The Effects of Ozone Treatment on the Reduction Aflatoxins in Corn and Peanuts". March 29-April 3, 2019 ARD symposium, Jacksonville, Fl. What do you plan to do during the next reporting period to accomplish the goals? More farm samples such as wheat, soybean and cottonseed will be will be collected from local farmers and analyzed aflatoxin levels by HPLC. Farm samples will be contaminated with aflatoxins.

Impacts
What was accomplished under these goals? Two undergraduate students were hired to work on the project. Equipment such as High Performance Liquid Chromatography (HPLC) and evaporator were installed. Chemicals and supplies were ordered. Aflatoxin amounts in farm wheat, soybean and cottonseed were analyzed by HPLC. All samples collected from South Carolina local farms have very small amount of aflatoxins (less than 1 ppb) which are safe for human consumption. About 33% and 40% of aflatoxin B1 was reduced from aflatoxin contaminated Vicam corn by purging ozone gas for 10 min at 2% and 5% levels, respectively

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Myrandi Roper, Dr. James B. Stukes, Dr. Nazimuddin Mohammed, Determining The Quantity Of Aflatoxins In Breakfast Cereals From Local Grocery Stores. Proceedings at the INBRE conference held at the South Carolina State University on July 25, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: DeAsia Stuckey,?Dr. James?B.?Stukes, Dr.?Nazimuddin?Mohhamed, Quantitative Determination Of Aflatoxins In Corn Starch, Peanut Butter And Almond From Local Grocery Stores Using HPLC. Proceedings at the INBRE conference held at the South Carolina State University on July 25, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Sasha Prioleau, Dr. James B. Stukes, Dr. Nazimuddin Mohammed, Rapid Analysis Of Aflatoxins Found In Pasta Products And Vicam Corn. Proceedings at the INBRE conference held at the South Carolina State University on July 25, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: DEASIA GATHERS, Dr. N. Mohammed and Dr. J.B. Stukes, The Effects Of Ozone Treatment On The Reduction Aflatoxins In Corn. Proceedings at the 77th PAWC conference held at Tuskegee University on December 6, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: J. B. Stukes*, N. Mohammed, S. Powell, and M. Roper. The Effects of Ozone Treatment on the Reduction Aflatoxins in Corn and Peanuts.March 29-April 3, 2019 ARD symposium, Jacksonville, Fl.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: J.L. Cortese*, J.B. Stukes, N. Mohammed, and M. Roper. A Comparison of Aflatoxins Levels in Corn Oil Obtained from Local Grocery Stores.March 29-April 3, 2019 ARD symposium, Jacksonville, Fl.

Progress 08/01/18 to 09/30/18

Outputs
Target Audience: SCSU Faculty SCSU Students 1890 Extension agent Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has allowed for the preliminary experiments for testing the presence of aflatoxin associated with wheat and soybean products purchased from local grocery stores. Moreover, the results were presented at The Louis Stokes South Carolina Alliance for Minority Participation Program (LS-SCAMP) conference. How have the results been disseminated to communities of interest?Poster presentations entitled "Determination of Aflatoxins in Corn Oil" at the LS-SCAMP conference held at South Carolina State University on November 9, 2018 as well as the Annual Biomedical ResearchConferencefor Minority Students (ABRCMS) conference held on November 14, 2018 in Indianapolis, Indiana. What do you plan to do during the next reporting period to accomplish the goals? Equipment such as High Performance Liquid Chromatography (HPLC) and evaporator will be installed. Training will be arranged to learn the HPLC operating procedures for faculty and students. Preparation of internal standard curve using standard aflatoxin. Collection of wheat, soybean and cottonseed samples from local farmers.

Impacts
What was accomplished under these goals? Two undergraduate students were hired to work on the project. Equipment such as High Performance Liquid Chromatography (HPLC) and evaporator were ordered. Chemicals and supplies were ordered. Since this is a new project, the HPLC equipment and supplies have been ordered but have not been received at this time. Preliminary experiments were processed on wheat and soybean products purchased from local grocery stores utilized the Vicam reader to determine base line aflatoxin readings. Aflatoxin levels in wheat whole flour and tofu were 1.593±0.252 and 1.127±0.108 ppm respectively, which are safe for human consumption.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: 1. J. B. Stukes*, N. Mohammed, S. Powell, and M. Roper. The Effects of Ozone Treatment on the Reduction of Aflatoxins in Corn and Peanuts. 1890 Research, Department of Biological and Physical Sciences, South Carolina State University, Orangeburg, SC 29117. Presentation at the 2019 ARD Research Symposium, Jacksonville, FL. March 30 - April 3, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: 2. J. L. Cortese*, J. B. Stukes, N. Mohammed, and M. Roper. A Comparison of Aflatoxins Levels in Corn Oil Obtained from Local Grocery Stores. 1890 Research, Department of Biological and Physical Sciences, South Carolina State University, Orangeburg, SC 29117.Presentation at the 2019 ARD Research Symposium, Jacksonville, FL. March 30 - April 3, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: 3. Jazmyne Cortese, James B. Stukes, Nazimuddin Mohammed and Myrandi Roper, Determination Of Aflatoxins In Corn Oil. Proceedings at the South Carolina State University LS-SCAMP conference held at South Carolina State University on November 9, 2018.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: 4. Jazmyne Cortese, James B. Stukes, Nazimuddin Mohammed and Myrandi Roper. Determination Of Aflatoxins In Corn Oil. The Annual Biomedical Research Conference for Minority Students (ABRCMS) conference held on November 14, 2018 in Indianapolis, Indiana.