Location: Dairy and Functional Foods Research2013 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:
DNA sequencing techniques were used to identify and characterize the cluster of genes required for production of antimicrobial peptides within lactic fermentation and probiotic bacteria (LFPB). The mapped gene clusters were shown to differ significantly from sequences reported in the literature. Semi-quantitative PCR techniques were further used to characterize the expression of several genes within the gene clusters by measuring the amount of RNA present. Expression levels of targeted genes were shown to correlate with the level of antimicrobial activity observed in bioassays. DNA transfer molecules were developed to express individual components of the antimicrobial gene cluster in other LFPB. Gene fusions were created to fit potential antimicrobial peptides with a new leader sequence to allow for secretion of these peptides from various LFPB hosts. Vectors have been introduced into LFPB hosts and studies are ongoing to demonstrate recombinant gene expression. DNA vectors have also been designed to inactivate components of the antimicrobial gene cluster in LFPB. An integrative vector was developed to permanently remove genes from within the antimicrobial gene cluster. The vector was used to delete a gene (blpK) thought to encode an antimicrobial peptide; however removal of the gene did not impair antimicrobial activity. To inactivate genes which could not be removed from the cluster another inactivation vector was constructed to express an anti-sense RNA and prevent expression of targeted peptides. Screening of LFPB for immunoregulatory activity was performed by co-culturing bacteria with rat epithelial cells and testing for the production of molecules known to regulate pro- or anti-inflammatory responses. The initial test used to monitor expression of these molecules proved inconclusive, thus an alternative approach using semi-quantitative RNA analysis is currently being developed to screen for LFPB induced changes in the immune response of epithelial cells. Environmental conditions were tested to see if they influenced the expression of antimicrobial peptides in LFPB. Antimicrobial activity was similar when LFPB were grown in presence of lactose or glucose; however the activity was shown to differ for some LFPB strains if the base medium was changed. In addition, strains requiring the use of an induction peptide to activate antimicrobial activity were shown to be less responsive when grown in reconstituted milk. Studies are ongoing to determine if the lack of activity was due to altered gene expression or sequestration of the antimicrobial peptides. Work was initiated to investigate the potential for co-culturing multiple LFPB strains which naturally produce antimicrobial peptides, and develop methods for cloning alternative antimicrobial molecules (eg. endolysins).
1. Characterized the components of a production system required for the expression of natural antimicrobials in yogurt bacteria. A group of genes required for the production of a natural antimicrobial in yogurt cultures has been identified, but it remains unclear why very few yogurt cultures naturally produce the potential antimicrobial. ARS researchers at Wyndmoor, Pennsylvania characterized this group of genes in four yogurt cultures and studied their expression. Although the components known to regulate production were identical in all cultures, their expression was significantly higher in the two strains which naturally produced the antimicrobial. These two strains have potential applications in protecting foods from bacterial contaminants.