Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Sep 1, 2010
Project End Date
Aug 31, 2015
Grant Year
Project Director
Besong, SA, A.
Recipient Organization
DOVER,DE 19901
Performing Department
Human Ecology
Non Technical Summary
American consumers do enjoy the safest and healthful food supply in the world, yet foodborne illness, a preventable and underreported disease, continues to pose serious health. Despite the multibillion dollar investment by the United States government to improve the safety of the nation's food supply, there are still reported cases of people getting sick from consuming contaminated foods. The recent salmonella outbreak that is linked to the consumption of raw alfalfa sprouts and caused the infection of 22 people in 10 states (CDC report, May 21, 2010) indicates that foodborne illnesses continue to be a major health and economic problem for individuals, businesses and the nation. Global concern of foodborne illness, particularly from ingestion of foodborne bacteria, necessitates a rapid detection within food and environmental matrices. Rapid identification of pathogens is crucial for effective control of potential outbreak from foodborne pathogens. Therefore, additional efforts should be invested in the discovery of antimicrobial compounds/bioactive components from plants that can be added to food products as additives to prevent the growth of pathogens. In addition, food contamination can be prevented by developing a rapid method for detecting foodborne pathogens postharvest. On the other hand, online sales (ecommerce) of ready-to-eat foods have increased significantly in recent years and there is potential for continued growth. The internet has provided a unique opportunity for small food manufacturers to sell their food products directly to consumers at a lower price. This new system of selling is booming, yet food safety information related to the quality of products in this emerging market is either scarce or not available. Food producers who only sell their products directly to consumers may avoid a number of industrial food safety management practices. Since food products from eCommerce/online vendors could reach consumers directly, without going through the traditional wholesale and retail distribution routes, they may also bypass existing federal and state food safety monitoring programs. Therefore, it is important to search for antimicrobial components in plants like bitter leaf (Vernonia amygdalina) that can be used as food additives to prevent food contamination by pathogens by using the land-grant tri-partite approach (research, education and outreach). A critical component of this project is to provide a unique opportunity for students to increase their knowledge and skills in the field of Food Microbiology/Food Safety. Demographic changes in the US require academic institutions to recruit and train more minority scientists for jobs in Food and Agriculture industries and help to meet the needs for diversity in this global economy.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
The goals of this project are to: (1) assess the potential of antimicrobial compounds from bitter leaf and as a food additive/spray that can be spayed on fresh fruits and vegetables to prevent the growth of food borne pathogens and (2) develop a rapid method for detecting foodborne pathogen and train a new cadre of food microbiologists. Specific objectives are: 1) Conduct a risk analysis on the potential for contamination of ready-to-eat food products with pathogen from internet vendors, 2) develop a purified extract of bitter leaf containing antimicrobial properties that can be sprayed on fresh fruits and vegetables, 3) assess antimicrobial properties of bitter leaf extract and 4) develop a real-time PCR rapid detection method and train a new cadre of food microbiologists.
Project Methods
To prepare bitter leaf extract, Fresh bitter leaves will be cut into small pieces and then mixed with sufficient amount of anhydrous sodium sulfate in 1L Pyrex storage bottles. After adding chloroform, bottles will be sealed with caps and then be vigorously shaken for 2 hr in a reciprocating shaker. The chloroform extracts will be passed through glass wool in funnels and collected into a new set of storage bottles. The obtained extracts will be evaporated using a Labconco RapidVap vacuum evaporation system to get rid of chloroform. The evaporation temperature will be controlled below 40 degree celsius. To separation the extract, a portion of the obtained solid extract will be dissolved in chloroform and preliminarily separated using silica gel columns. The separation solvent system will be optimized based thin layer chromatography (TLC). Each collected portion will be evaporated and the obtained residues will be used for animal hypoglycemic assay. The portion with anti-microbial properties will be further separated using silica gel columns and components will be collected for animal study. To identify bioactive components in the extract, components with anti-microbial properties will be derivatized and analyzed in gas chromatography mass spectrometer or directly analyzed in liquid chromatography mass spectrometer. To confirm the chemical structure, the component(s) will be further purified and then analyzed in nuclear magnetic resonance (NMR). To develop a rapid method for detecting foodborne pathogens, DNA-based tests and assays that are commercially available will be modified to speed up the development of a user friendly technique as described by Dziezak (1987), Fung, (1991), Fung et al. (1988), Ibrahim (1986), and Stager and Davis (1992). Some of the commercially available nucleic acid-based assays, rapid methods, and antibody-based assays (Cox et al. 1987, Feng et al. 1982) will provide essential tools that will be needed to develop a unique pathogen detection method.

Progress 09/01/10 to 09/30/10

Target Audience:Target audience are undergraduate and graduate students, post-doctoral fellow and visiting scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: Three graduate students in Food Microbiology were supported for their research projects from this grant. Through this project, graduate research students were trained in experimental techniques and exposed to critical experiences in field study. Also, they were trained in areas such as professional writing and presentation skills. In addition, the PI mentored a research student in order to maximize one-on-one educational influence. Professional development: Graduate students and PIs developed their professional abilities by attending conferences. Three graduate students attended the Beltsville Area Graduate Student Agricultural Research Symposium, Beltsville, MD in 2011. One graduate student, Ms. Talaysha Lingham, received a second place award in poster competition at the DSU Graduate Research Symposium in 2012. The PI participated in several professional meetings (ARD, KosFoST, and IAFP) and made research presentations, and developed research collaboration with peers from other academic institutions and federal agencies. How have the results been disseminated to communities of interest?Outcomes of this project were disseminated through graduate student thesis, professional meetings, publications and the PI's web sites. Technical results were presented at domestic and international scientific meetings and conferences. Manuscripts resulting from this project were submitted and published in peer-reviewed international journals. Publications generated from this project were also advertised at the PI's website at DSU to provide maximum dissemination of project outcomes. What do you plan to do during the next reporting period to accomplish the goals?We plan to develop a DNA based amplification method by using the isothermal amplification technique. In detail, we will develop a RPA assay to detect pathogens and spoilage bacteria and will apply this methodology to raw and processed foods. We also plan to develop cutting-edge techniques using the Next Generation Sequencing (NGS) system to study microbiome associated with food spoilage and to train graduate student in the Food Microbiology lab at Delaware State University.

What was accomplished under these goals? To understand bacterial spoilage mechanisms on catfish, before and after fillet processing, unidentified bacterial species during storage periods were isolated using various screening approaches. Bacterial Isolation from Catfish After completing six cycles of bacterial isolation from retail and pond sources, 237 unique colonies were isolated. Based on colony morphology the majority of the colonies maintained a circular shape. Colonies frequently appeared yellow, orange, white or translucent in color with slight elevation; from these results, there was an expectation to identify both Pseudomonas and Shewenella species from the fresh catfish fillets. The fillets obtained from the retail source reached a spoilage state much faster than the fillets obtained from the pond source. The growth of bacteria from catfish obtained from the pond sources resembled the standard bacterial growth curve with distinctive lag, log, stationary and death phases. Catfish samples obtained from retail sources displayed a different bacteria growth curve likely due to significant spoilage that took place prior to reaching and being sold in the market. In order to examine antimicrobial activity of natural fermented sources, four concentrations of vinegar were used to treat pathogenic, spoilage bacterial spp. isolated from fish fillets. In addition, characteristics of vinegar were studied through microbial, chemical methods to obtain increasing shelf life and improve sensory properties on catfish fillets. Antimicrobial Activity of Vinegar on Spoilage and Pathogenic Bacteria The different concentrations of vinegar were confirmed for the presence of acetic acid in samples. The antimicrobial activity of vinegar was determined on foodborne pathogens and bacteria isolated from catfish fillets using zone of inhibition and minimum inhibitory concentration tests. Antimicrobial Activity of vinegar was effective in all concentrations (0.5%, 1%, 2.5% and 5%) for foodborne pathogens except for E. coli DH5α. E. coli 0111, Salmonella Typhimurium, Staphylococcus aureus and Serratia marcescens resulted in the largest 5% acidity zone of inhibition and Salmonella Typhimurium had the smallest clear zone measurement. Pseudomonas aeruginosa showed the lowest MIC of 0.1. For the bacteria isolated from catfish, the MIC ranked from 0.03 to 1%. Staphylococcus aureus showed no clear zones. Staphylococcus aureus was the only gram positive bacterium tested in this study and the only bacterium that is resistant to the antimicrobial activity of vinegar. Results from this study indicate that acetic acid is a weak antimicrobial agent for gram positive bacteria compared to gram negative bacteria. Results from this study also show that vinegar product can be used as a substitute ingredient in fishery products and other foods to increase shelf life. To develop simple quantification methods to assess the quality of fish fillet, the MPN-PCR based on DNA amplification was optimized and applied for quantifying Pseudomonas spp. from randomly purchased fillets at retail sources and fresh caught catfishes at university pond. Enumeration of bacterial spp. from fish fillets The results obtained from MPN and MPN-PCR. From the MPN method, judging each tube by turbidity, the data from haddock showed Pseudomonas and total bacteria to be mildly high population. Data obtained from the pond water was high in total bacteria and very low in Pseudomonas spp. Scottish Atlantic salmon showed total bacteria at high population and for Pseudomonas spp. at low population. Catfish from the local supermarket was moderately high in total bacteria and very low in Pseudomonas spp. Tilapia was very high in total bacteria and very low in Pseudomonas spp. Wild catfish was high in total bacteria and for Pseudomonas spp. at mildly high population. The data obtained from the MPN-PCR showed Atlantic salmon as the highest in Pseudomonas spp. detection. Haddock, wild catfish, and catfish from the local supermarket followed higher bacterial populations of Pseudomonas spp. respectively. Tilapia and pond water from DSU aquaculture department was the lowest in Pseudomonas spp. These project contributed significantly to the development of a state-of-the-art research facility at DSU to identify pathogens involve in catfish spoilage and to study catfish spoilage during processing and storage conditions. This project also provided resources to study the antimicrobial properties of vinegar and potential application to increase shelf life of catfish. The use of MPN-PCR method to assess total Pseudomonas spp. And quantification on catfish fillets is an important product that can be used in the private sector. The state-of-the-art research facility at DSU that was developed as a result of this project has trained over 5 graduate students who have joined the workforce, 2 undergraduate students, on post-doctoral fellow and 5 visiting international students.


  • Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: K.D. Maull, M.E. Hickey, and J.L. Lee. The Study and Identification of Bacterial Spoilage Species Isolated from Catfish during Refrigerated Storage. Journal of Food Processing and Technology. 2012. DOI:10.4172/2157-7110.S11-003.
  • Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: Christopher Donald. Development of a quantitative detection method for Pseudomonas spp. on selective fish using MPN, MPN-PCR, and plate count methods.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Lingham, T., S.A. Besong, G. Ozbay and J.L. Lee. Antimicrobial Activity of Vinegar on Bacterial Species Isolated from Retail and Local Channel Catfish (Ictalurus punctatus). Journal of Food Processing and Technology. 2013. DOI:10.4172/2157-7110.S11-001.