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Develop a Purified Bitter Leaf (Vernonia Amygdalina) Extract with Antimicrobial Properties and a PCR-Based Rapid Method for Detecting of Foodborne Pathogens


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. <P>

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.

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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.

APPROACH: 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.

Besong, Samuel
Delaware State University
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