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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Dairy and Functional Foods Research » Research » Research Project #419745


Location: Dairy and Functional Foods Research

2011 Annual Report

1a. Objectives (from AD-416)
Identify food-grade lactic fermentation and probiotic microorganisms that produce unique bioactive peptides and proteins with potential applications as functional food ingredients. 1a. Identify natural antimicrobial products of food grade microbes to protect foods against spoilage and hazardous microorganisms. 1b. Identify antimicrobial products of food grade bacteria useful in functional food applications to reduce the risk of dental caries (caused by streptococci) and bacterial skin infections (caused by propionibacteria). 1c. Identify and develop food-grade microbes for the production of bioactive components of natural proteins of animal and plant origin to reduce the risk of chronic health problems (obesity/cancer) and to enhance immune responses to infections. 1d. Modify and adapt available gene technologies and production parameters to maximize output of bioactive peptides and proteins. Develop microbial technologies to produce or co-produce unique bioactive peptides and proteins to improve food functionality and for use in functional foods. 2a. Develop microbial technologies for the production and co-production of compatible bioprotective (pathogen control), antitumor (cancer cell control) and health promoting (oral hygiene, stomach ulcers, skin infections) gene products in food fermentation and probiotic microbes. 2b. Develop microbial technologies for the transfer of capacity for functional food ingredient production to other bacteria used as essential starter cultures by dairy food industries. 2c. Develop, modify and adapt strategies to assure culture survival and retain efficacy of bioactive ingredients in functional foods and non-food products (oral hygiene, and skin health).

1b. Approach (from AD-416)
Lactic fermentation and probiotic cultures will be screened for the production of bioactive peptide and protein products that improve the nutritional quality and functionality of foods and also protect foods by controlling the growth of contaminating bacteria. Natural peptide products also will be tested for effects on abnormal cell proliferation and induction of immune response in selected animal cell cultures to explore possible applications in the improvement of human health. Microbial biotechnology, enzyme, genetic and proteotnic technologies will be designed or adapted for developing lactic fermentation and probiotic cultures (streptococci, lactococci and lactobacilli) as microbial cell factories for the larger-scale production of potentially useful bioactive peptide and protein gene products. The effects of prebiotic formulations on the growth and productivity of lactic fermentation and probiotic bacteria in milk environments will be evaluated in prototype food systems. In addition, culture performance in whey effluents of cheese manufacturing will be assessed and improved.

3. Progress Report
Using state-of-the-art gene detection and sequencing technologies, starter cultures used in industrial production of cheeses and other fermented products were screened for the presence of gene clusters that appear to be essential for regulating the synthesis of antimicrobial peptides. For testing the presence of each component in the gene cluster, special gene sequences were designed to produce multiple copies of each component to improve detectability. The cultures testing positively were then evaluated by various bioassay techniques against all the other strains to establish the activity spectrum of each lead. DNA sequencing techniques were also applied to defining the molecular properties of a benign cheese-associated bacterium that produced a unique antimicrobial peptide with antilisterial activity as demonstrated by biological assays. Techniques used in biomolecular analysis provided information on the location of genes responsible for the production and transport of the natural bactericidal peptide. The DNA molecule carrying the gene components was processed further by genetic and biotechnology techniques and its key elements were transported into other dairy starter bacteria to produce the peptide. Gene detection and sequencing technologies were used to identify dairy cultures for the presence of glutamate decarboxylase that converts glutamic acid into gamma-aminobutyric acid (GABA), a bioactive peptide with potential uses in treating depression, insomnia and alcoholism, and as a stimulant of immune response and insulin secretion. In several cultures only shortened and inactive versions of the gene could be detected. In cultures carrying the complete gene and displaying measurable decarboxylase activity, the use of molecular probes permitted the correct positioning of the gene on the genome of several dairy starter cultures and the identification of neighboring sequences that allow the excision and transfer of this valuable genetic element into non-enzyme producing cultures. Molecular biology and biochemical genetic techniques were used to develop an in-house gene transport vector for carrying new genes into targeted food grade bacteria, including genes for the antilisterial peptide durancin GL and the enzyme glutamic acid decarboxylase that generates GABA. Further adaptation of the transport vector by fitting it with special features was aimed at incorporating the new genes into the genome of the new host bacterium to ensure its retention and stability under changing environmental conditions.

4. Accomplishments
1. Produced antimicrobial proteins in food-related bacteria. Some bacteria used in the production of dairy foods, are equipped to produce small antimicrobial proteins called peptides, which can benefit consumers by preventing the growth of harmful bacteria that may lead to outbreaks of foodborne illness. ARS researchers at Wyndmoor, PA, identified genes required for the production of antimicrobial peptides in several food-associated bacteria. However some strains of these bacteria are missing the genes and unable to produce the peptides, thus methods were developed to transfer genes required for the production of antimicrobial peptides into these strains. A transport molecule was constructed to deliver a newly characterized gene, required for the production of an antimicrobial peptide, into S. thermophilus, which is commonly used in the production of yogurt and cheese. The resulting bacterium was shown to produce an antimicrobial peptide and inhibited the growth of Listeria, a bacterium known to cause severe foodborne illness. This example demonstrates the potential for using this technology to improve the functional value of food-grade bacteria.

2. Natural bioactive compounds produced by food-grade bacteria. Gamma-aminobutyric acid (GABA) is an amino acid which has been shown to reduce stress and high blood pressure, and protect against diabetes in humans. ARS researchers at Wyndmoor, PA, identified several strains of bacteria commonly used in the production of yogurt and cheese which possess the gene required for production of GABA. Using a newly improved DNA transport molecule, the gene was transferred into a food-grade bacterium normally unable to produce this amino acid. The newly developed bacterium was shown to produce GABA. This method of gene transfer is expected to allow the bacterium to maintain its food-grade status thus allowing for potential commercial applications. Fermented foods made with GABA-producing bacteria are expected to benefit consumers due to their increased health-promoting properties.

3. Developed improved gene transport molecules for fermentation cultures. The transport of new genes into food grade cultures is possible by a variety of techniques but gene stability can be substantially improved by incorporation into the chromosome. ARS researchers at Wyndmoor, PA, developed new strategies to accomplish gene stabilization and expression by equipping well-studied in-house transport molecules with new features that helped the incorporation and maintenance of the new genes in the genome of yogurt and cheese starter cultures. The use of the new strategies resulted in the chromosomal integration of the antilisterial peptide durancin GL gene and the gene for glutamate decarboxylase which is essential in gamma-amino butyric acid or GABA formation. The presence of the genes in the chromosome stabilizes and enhances starter culture performance by reducing chance gene loss due to the stressful conditions of industrial fermentations. This benefits providers of starter cultures who can offer reliable cultures with novel health-promoting and foodborne-illness fighting properties to manufacturers of food products prepared by fermentation processes.

Review Publications
Brito, M.A., Somkuti, G.A., Renye Jr, J.A. 2011. Production of antilisterial bacteriocins by staphylococci isolated from bovine milk. Journal of Dairy Science. 94:1194-1200.

Renye Jr, J.A., Somkuti, G.A., Garabal, J.I., Du, L. 2011. Heterologous production of pediocin for the control of Listeria monocytogenes in dairy foods. Food Control. 22:1887-1892.