The microbial composition of foods and beverages is strongly linked to human health and well-being. This is evident during food-borne outbreaks caused by the entry of human pathogens into food supply chains. Conversely, growth of certain types of bacteria in food matrices is essential for the production of fermented products such yogurt, cheese, olives, and sausage or for delivery as probiotics intended to improve health. <P>
These two opposing (detrimental and beneficial) facets of microorganisms in foods are important to food industries in California which want to ensure their products are safe, appealing, and provide optimal health benefits. However, we currently have an incomplete understanding of how to fully control the microorganisms associated with food environments. <P>
The current proposal aims to improve our understanding of these microorganisms by focusing on the identification and adaptations of lactic acid bacteria (LAB) to food systems. LAB are Gram positive, fermentative, non-pathogenic bacteria typically associated with agriculturally-relevant plants, animal digestive tracts, and animal- products (i.e. milk) (Stiles and Holzapfel, 1997). These organisms are best understood for their roles in numerous industrial and artisanal dairy, meat, and plant fermentations resulting in food products which are integral to American diets (Hutkins, 2006). LAB confer much of the taste, aroma, and texture of these fermented foods. Additionally, LAB are able control the amounts of human pathogens in food matrices as a result of their production of organic acids (e.g. lactic acid) and specific antimicrobial compounds (e.g. bacteriocins) (Hatakka and Saxelin, 2008). <P> In addition to their roles in food production, certain strains of LAB are recognized for their capacities to confer health benefits in mammalian gastrointestinal tracts. These LAB are termed probiotics and have been associated with the prevention and treatment of disease including infectious diarrhea, inflammation, allergy and respiratory infections (Floch et al., 2008). Therefore the amounts and strains of LAB in foods should be carefully regulated to ensure optimal levels of their metabolism and survival required for food processing and health benefits in the gut. Knowledge of the adaptations and identities of LAB in food systems would afford advantages to numerous CA agricultural commodities and food companies. <P>In this context the following objectives were formulated: <OL> <LI> Quantify and identify species of lactic acid bacteria associated with agriculturally important plants. <LI> Determine the ecological fitness of LAB for growth and survival on plants and plant materials. <LI> Perform molecular genetic analysis on specific LAB strains to identify plant-specific phenotypes. <LI> Identify mechanisms of LAB strain-specific inhibition of human pathogens in food-relevant systems. <LI> Determine the contributions of the foods/beverages to probiotic Lactobacillus survival and functional effects in the mammalian gut.
NON-TECHNICAL SUMMARY: The aim of this Hatch project is to make a contribution to the development of food products involving lactic acid bacteria which are both safe and healthy. At its base, the project is about the exploitation of naturally occurring lactic acid bacteria to modify the taste and texture properties of fermented foods, limit the growth of human pathogens, and confer health benefits upon ingestion and entry into the human digestive tract. <P>
The proposed study parallels and complements ongoing research in the Marco lab that focuses the adaptations of lactic acid bacteria in their natural ecosystems. These studies are important for ensuring the appropriate amounts and activities of these industrially and medically relevant bacteria in foods and the human gut. The project's overall objective is to explore that potential in a systematic manner specifically as it relates to the interactions of LAB with food matrices (dairy- and plant-based products) in fermented and minimally processed foods which result in improved quality assurance and safety in food production and optimal function in the gut.
APPROACH: 1) The amounts and identities of LAB which are naturally associated with agriculturally relevant plants (including leaves, fruits, and plant tissues) and fermented plant products (including olives) will be determined by culture-dependent and -independent approaches. Culturable LAB will be quantified and isolated by plating serial dilutions of plant leaf, fruit washes, and tissue macerates onto semi-selective media. Culture-independent analysis of LAB in the phyllosphere targeting the 16S rRNA gene will afford quantitative measurements of amounts of LAB which are present in low quantities or are otherwise prove difficult to cultivate in the laboratory. <P>2) We will quantify the ability of individual LAB strains to grow and survive on plant-surfaces and plant-associated environments. To determine if certain LAB strains are specifically adapted for plant environments, these assessments will compare strains which were directly isolated from plants and plant fermentations to those which originated from dairy-environments and mammalian gastrointestinal tracts. Emphasis will be on using strains which are genetically tractable and for which genome sequences are known. <P>3) Molecular genetic analysis will be performed on selected strains of LAB to identify their specific functional traits relevant for performance in plant ecosystems and potentially the mammalian gut. Real-time RT-PCR and transcriptional profiling will be used to quantify LAB gene expression during growth in association with plants. The plant-inducible genes will also be targeted for knockout mutagenesis to determine their roles in LAB fitness in plant environments and/or production of fermented plant-based food products. <P>4) This objective will identify potential relationships between LAB and the persistence of human pathogens on food. Plant-associated LAB will be screened for their abilities to inhibit the growth of E. coli O157:H7 in laboratory culture medium. Isolates with growth inhibitory capacities will then evaluated to determine the specific cell components or metabolites conferring antimicrobial effects (e.g. production of acid, specific proteins, or metabolites). The efficacy of these organisms for controlling the growth of the human pathogens on agriculturally important products such as lettuce. <P>5) The effects of food matrices and host diet on probiotic performance in the gut will be evaluated in mouse model systems. The probiotic Lactobacillus strains will be provided to mice in milk, fermented milk, plant materials, or saline to conventionally-raised mice fed either standard chow or formulated diets designed to mimic typical foods consumed in the West. The amounts of the inoculated strain able to survive gastrointestinal tract transit will be measured in fecal samples collected from the mice. The amounts and activity levels of the probiotic strains will also be examined in vivo to determine the effects of their nutritional environment on gene expression and survival from the stomach to the colon.