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United States Department of Agriculture

Agricultural Research Service


Location: Dairy and Functional Foods Research

2012 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 proteomic 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:
As per Objectives 1A and 1C, newly acquired strains of lactic fermentation and probiotic (LFPB) cultures were systematically evaluated. Design of experimental strategies was intiated to evaluate immunoregulatory effect of LFPB srains. Gene detection and DNA sequencing technologies were used to find gene clusters required for the synthesis of antimicrobial peptides produced by lactic fermentation bacteria used in cheese and yogurt production. Special gene sequences were designed to test the presence of each component in the gene cluster, and DNA sequencing techniques were applied to establish the architectural integrity of genes essential for synthesis. The cultures testing positively were then evaluated by various bioassay techniques against all the other strains to establish the activity spectrum of each lead. As per Objective 1C, DNA sequencing techniques were also applied to complete research on the molecular architecture of genes involved in enzymes responsible for the production of gamma-aminobutyric acid (GABA) in lactic cultures that may impart additional health benefits to consumers. As per Objectives 1A, 1C and 1D, biotechnology and biochemical genetic techniques were used to develop a DNA transport vector to replace the blpC component in the genome of lactic cultures that has an essential function in regulating the expression of genes involved in bacteriocin production. Further, proteins synthesized to mimic blpC successfully restored bacteriocin production when supplied to growing bacteriocin-nonproducing cells. This provided unequivocal proof for the pivotal role the blpC protein plays in turning on bacteriocin production in lactic bacteria. Survey of the culture collection also revealed two additional lactic cultures that responded to the synthetic peptide by producing bacteriocins with potentially novel properties. As per Objective 1A, collaborative work was initiated on developing conditions to evaluate the immunoregulatory properties of lactic cultures when cultured together with eukaryotic cell lines, with initial emphasis on the production of anti-inflammatory cytokines by rat epithelial cells.

4. Accomplishments
1. Developed special DNA transport molecule. Production of antimicrobial proteins (bacteriocins) in lactic fermentation bacteria is regulated by a complex group of genes (blpABC) but the role of individual genes is poorly defined. ARS researchers at Wyndmoor, Pennsylvania designed a DNA transport molecule (vector) to replace the blpC gene in the chromosome of lactic cultures, leading to the loss of bacteriocin production. Bacteriocin production was successfully restored when a synthetic 30-amino acid protein that is equivalent to the blpC gene product was supplied to the blocked culture. This provided strong evidence for the essential role of the blpC gene product in regulating bacteriocin production and expression in dairy fermentation cultures. The vector molecule is also being used to remove additional components of the blp gene complex in order to identify those that are required for the production of bacteriocin and immunity proteins that protect the producing cultures from the lethal effects of the bacteriocin.

2. Determined role for individual genes in bacteriocin production. DNA transport molecules may be useful in removing and transferring individual genes from the gene complex (blpABC) regulating bacteriocin production. ARS researchers at Wyndmoor, Pennsylvania found that the DNA transport molecule pMEU5a was useful in determining the role of selected blpABC components in bacteriocin synthesis. A native DNA sequence (ST2201) from the chromosome of the yogurt fermentation bacterium Streptococcus thermophilus was fused to individual blp subunits to promote their expression in new host cultures. The ST2201-blp fusions were successfully transferred to the pMEU5a vector and four blp genes were transferred into a bacteriocin-negative strain. The engineered strain could produce bacteriocin only if the blpB gene which facilitates the secretion of the bacteriocin was intact. This confirmed the need to check by DNA sequencing techniques the structural integrity of the blpB gene in cultures intended to be recipients of bacteriocin genes.

3. Identified new bacteriocin-producing lactic bacteria. Antimicrobial proteins (bacteriocins) are food grade natural products with potential applications protecting foods against bacterial contamination. ARS researchers at Wyndmoor, Pennsylvania identified several strains of lactic cultures with capacity to produce antimicrobial bacteriocins. Under regular growth conditions, the cultures do not produce bacteriocins at a detectable level but respond to the addition of the blpC gene product, a 30-amino acid protein that controls the functionality of the blpABC gene complex. The findings indicated that in several strains, the blpC gene is not producing adequate amounts of the 30-amino acid protein to turn on bacteriocin synthesis. The addition of the synthetic protein to screening strategies offers a new way to detect bacteriocin-producing strains of lactic fermentation bacteria that may yield novel bacteriocins with unique antimicrobial range and properties with useful applications in food protection against bacterial contamination.

Review Publications
Du, L., Somkuti, G.A., Renye Jr, J.A., Huo, G. 2012. Properties of durancin GL, a new antilisterial bacteriocin produced by Enterococcus durans 41D. Journal of Food Safety. 32:74-83.

Renye Jr, J.A., Somkuti, G.A. 2012. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus. Biotechnology Letters. 34:549-555.

Brito, M.A., Somkuti, G.A., Renye Jr, J.A. 2011. Isolation of bacteriocin-producing staphylococci from Brazilian cheese. Journal of Food Safety. 31:365-370.

Du, L., Somkuti, G.A., Renye Jr, J.A. 2012. Molecular analysis of the bacteriocin-encoding plasmid pDGL1 from Enterococcus durans and genetic characterization of the durancin locus. Microbiology. 158:1523-1532.

Somkuti, G.A., Renye Jr, J.A., Steinberg, D.H. 2012. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110. Journal of Industrial Microbiology and Biotechnology. 39:957-963.

Last Modified: 2/23/2016
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