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  • Association of foodborne pathogens with Capsicum annuum fruit

    and evaluation of the fruit for antimicrobial compounds

    Karleigh Rose Huff

    Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State

    University in partial fulfillment of the requirements for the degree of

    Doctor of Philosophy

    in

    Food Science and Technology

    Renee R. Boyer, Chair

    Cynthia J. Denbow

    Sean F. O‟Keefe

    Robert C. Williams

    September 7 th

    , 2011

    Blacksburg, VA

    Keywords: Capsicum annuum, growth and survival, liquid chromatography, foodborne

    pathogenic bacteria, bacterial growth curve

  • Association of foodborne pathogens with Capsicum annuum fruit

    and evaluation of the fruit for antimicrobial compounds

    Karleigh Rose Huff

    ABSTRACT

    Hot peppers are gaining popularity in the United States as both a vegetable and a

    spice. In 2008, jalapeño peppers were involved in a multistate outbreak of Salmonella

    Saintpaul. This is the first outbreak implicating jalapeños as a vehicle for foodborne

    illness. Hot peppers contain many compounds thought to possess antimicrobial

    characteristics. This research was conducted to provide more information on the

    interactions of pathogenic bacteria and jalapeño peppers, as well as to identify properties

    of Capsicum annuum that affect bacterial survival, growth, and inhibition.

    Behavior of pathogens associated with jalapeños was investigated by inoculating

    jalapeño fruits with a cocktail of Listeria monocytogenes, Salmonella enterica, or

    Escherichia coli O157:H7 on the intact external surface, injured external surface, or

    intact internal cavity and storing the jalapeños at 7°C or 12°C. Intact external jalapeño

    surfaces did not support the growth of the bacteria tested under storage conditions of 7°C.

    However, L. monocytogenes populations remained detectable throughout the 2 week

    study. At 7°C, pathogenic bacteria were able to survive but not grow on injured and

    internally inoculated jalapeños, but populations increased at 12°C (p=0.05). The most

    supportive growth environment for the pathogenic bacteria was the internal cavity of

    jalapeños held at 12°C. This study demonstrated the importance of intact uninjured

    produce and proper storage temperatures for food microbial safety.

    Inhibitory properties of jalapeños were studied by making extracts from fresh

    jalapeño peppers to test for antimicrobial activity. A disk diffusion assay determined that

  • iii

    the extracts were capable of inhibiting the growth of the pathogenic bacteria tested.

    Listeria monocytogenes was especially sensitive to the extracts. Jalapeño extracts were

    fractionated using HPLC and used for inhibition assays using disk diffusion and growth

    curve generation. Two fractions stimulated bacterial growth (p=0.05), while two other

    fractions inhibited bacterial growth. The inhibitory fractions were separated further using

    HPLC and tested for antimicrobial activity. Fraction E1 suppressed the growth of L.

    monocytogenes. HPLC-MS analysis revealed that Fraction E1 contained compounds

    known as capsianosides. To prove that inhibition is caused by capsianoside(s) and

    determine minimum inhibitory concentrations, a method to isolate the pure compound

    should be developed.

  • iv

    Dedication

    This is dedicated to Ian Bacon for all the sharing of ideas and honest feedback he

    provided. I could not have done it without him.

  • v

    Acknowledgements

    I have had an excellent experience in the Department of Food Science and

    Technology at Virginia Tech. There are a number of people that I would like to thank.

    First, Dr. Renee Boyer, who has been my advisor and mentor. Dr. Boyer has given me

    many opportunities to grow as a professional and get involved in activities outside of

    research. She has offered advice and knowledge throughout my doctoral studies, and I am

    very grateful to her as an advisor.

    I would like to thank my other committee members: Dr. Robert Williams, Dr.

    Sean O‟Keefe, and Dr. Cindy Denbow. Thank you for your willingness to help me with

    my research and with my career. Your advice has been invaluable.

    Thank you to the faculty and staff at the Department of Food Science and

    Technology for the opportunities to teach, learn, and share. Thank you to Dr. Kevin

    Holland and Dr. Paul Sarnoski for their willingness to help me with all things HPLC. I

    would still be staring at that piece of equipment in awe and confusion without you.

    Thank you to the graduate students in the department for creating a fun and

    friendly work environment. Special thanks to Mona Kumar and Sabrina Liles for their

    lasting friendships. I‟d also like to thank Courtney Klotz for all of her contributions and

    hard work as part of her undergraduate research with me.

    Lastly, thank you to my family (Mom, Dad, Kalow, Klayton, Mandi, Molly, and

    all the Huff‟s, Donaldson‟s, Bacon‟s, and Danielson‟s) for their support. I know my

    parents wish I could have found a school a little closer to home for my doctoral program,

    but they showed their support every step of the way. Thank you to Ian for being my most

    honest critic and for helping me be a better scientist and person.

  • vi

    Table of Contents

    Abstract ............................................................................................................................... ii

    Dedication .......................................................................................................................... iv

    Acknowledgements ..............................................................................................................v

    Table of contents ................................................................................................................ vi

    List of figures ..................................................................................................................... ix

    List of tables ....................................................................................................................... xi

    Chapter 1: Introduction ........................................................................................................1

    References ....................................................................................................5

    Chapter 2: Review of the literature ......................................................................................7

    The current state of food safety ...............................................................................7

    Increased incidence of produce related outbreaks .......................................7

    Interactions of bacterial pathogens with fresh produce .............................10

    Risks from farm to fork..............................................................................13

    Listeria monocytogenes: Basic characteristics .......................................................14

    Association of L. monocytogenes with fresh produce ...............................16

    Pathogenic Escherichia coli: Basic characteristics ................................................17

    Enterohemorrhagic E. coli (EHEC) ...........................................................19

    Association of E. coli O157:H7 with fresh produce ..................................20

    Salmonella: Basic characteristics ...........................................................................21

    Association of Salmonella enterica with fresh produce ............................23

    Plants as a host for pathogenic foodborne bacteria ................................................23

    Summer 2008 outbreak of Salmonella associated with jalapeños .............24

    Capsicum: Fact and folklore ..................................................................................26

    Pepper production and harvesting measures ..........................................................27

    Properties associated with Capsicum species ........................................................29

    Capsicum annuum as a source of natural antimicrobials ...........................33

    Foodborne pathogenic bacterial interactions with peppers ....................................35

    References ..............................................................................................................40

    Chapter 3: Effect of storage temperature on survival and growth of foodborne

    pathogens on whole, damaged, and internally inoculated jalapeños (Capsicum annuum

    var. annuum) ......................................................................................................................51

    Abstract ..................................................................................................................52

  • vii

    Materials and methods ...........................................................................................54

    Results...................

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