Location: Dairy and Functional Foods Research2016 Annual Report
1a. Objectives (from AD-416):
1: Develop strains of dairy lactic acid bacteria (LAB) that excrete bioactive peptides and proteins which inhibit the growth of food-borne pathogens (Listeria), and/or the bacteria associated with non-food related diseases of the oral-pharyngeal cavity (streptococci), skin (propionibacteria) and gastrointestinal tract (clostridia). 1a. Characterize the broad spectrum antimicrobial activity of bacteriocins produced by dairy lactic acid bacteria, and investigate methods for optimizing their production for use in food and non-food applications. 1b. Investigate the molecular structures of bacteriocins produced by dairy lactic acid bacteria and elucidate mode of action pertaining to their antimicrobial activities. 2: Identify prebiotic and probiotic combinations which influence human health through interaction with bacteria from the gut microbiota and/or intestinal epithelial cells. 3: Identify dietary fiber and prebiotics from pectins and hemicelluloses in sugar beet, citrus, cranberry and energy crop biomass with additional bioactivity including anti-adhesion of pathogenic bacteria to epithelial cells and immunomodulation (anti-inflammation, cytokine expression).
1b. Approach (from AD-416):
A multi-disciplinary approach will be used to study bioactive food ingredients that influence the gut microbiome, and inhibit the growth of bacterial pathogens. We will develop prebiotic, probiotic and anti-microbial compounds produced by dairy lactic acid bacteria (LAB) as well as plant cell wall oligosaccharides. The potential for LAB bacteriocins to prevent contamination of foods, and infections within the gut and oral cavity as well as on the skin will be investigated. Novel prebiotics will be developed as another bioactive intervention used to control food-borne pathogens and to promote health. Protein structure-function relationships will be determined both for bacteriocins and the interaction between dietary fiber carbohydrates and dairy proteins. The probiotic properties of LAB, the effects of prebiotics on these beneficial bacteria, and the combination of the two as synbiotics will be investigated. The interface between how combinations of prebiotics and probiotics influence gut bacteria and epithelial cells will be investigated in model studies. Additional health-promoting bioactivities (anti-adhesion of pathogens and immunomodulation) of dietary fiber and plant cell wall oligosaccharides will also be examined.
3. Progress Report:
Dairy lactic acid bacteria were tested for their ability to naturally produce bioactive molecules which inhibit the growth of bacteria that cause food spoilage and human disease (Objective 1). One yogurt bacterium was shown to produce a bioactive molecule that inhibits the growth of a bacterium known to cause tooth decay in humans. The antimicrobial molecule was partially purified and shown to prevent the formation of a bacterial film associated with oral disease. The yogurt bacterium is being tested as a potential oral probiotic (Objective 1a). The same yogurt bacterium was shown to inhibit the growth of Listeria, which is known to cause foodborne illness. Initial studies have shown that growth temperature and nutrients can affect the production of bioactive molecules by this yogurt bacterium; and the addition of food-grade buffering agents were shown to increase production of these molecules by 2 to 8-fold in milk and whey protein solutions. Current research is aimed at identifying conditions which will allow for optimal production of these bioactive molecules for food safety applications (Objective 1b). Bioactive molecules with antimicrobial properties were chemically synthesized and initial analysis was performed to determine the solubility and protein conformation within organic solvents. Once an appropriate solvent was determined, the peptides were tested for antimicrobial activity. Two of these molecules were shown to have antimicrobial activity; but their natural production varied in different yogurt bacteria. Molecular studies are ongoing to determine mechanisms which regulate their production. Analysis of synthetic molecules is expected to improve methods for purifying bioactive molecules (Objective 1c). Collaborative studies are ongoing to determine if yogurt bacteria from our in-house collection can elicit an immune response by cells which line the gastrointestinal tract. Preliminary results suggest that some yogurt bacteria may induce a pro-inflammatory response in the gut. Collaborative studies are also ongoing with scientists from China to identify novel food-grade bacteria within raw buffalo milk that naturally produce antimicrobial compounds. In a case study, the physical properties of two batches of whey protein concentrate (WPC) were studied under adverse storage conditions to obtain information on shelf life in hot and humid areas. This paper has been published. The interactions between beta-lactoglobulin (beta-LG), a major whey protein in milk, and low methoxyl pectin (LMP), a commercial citrus pectin were investigated. The beta-LG molecules formed complexes with LMP in the presence and absence of sucrose, which is often used to standardize commercial pectin products. Results demonstrated that sucrose facilitated the interactions between beta-LG and LMP, and protected the protein against heat-induced deformation. Results from this study will aid the design of new food formulations and ingredients containing dairy proteins and pectin. We continued to investigate the prebiotic properties (bifidobacteria and lactobacilli) of different pectic oligosaccharide structures using an in ovo chicken model. Now that the first animal data was obtained for the pectic oligosaccharide prebiotic properties, we will seek partners interested in a chicken feeding trial as the next step. An enzymatically hydrolyzed corn arabinoxylan was bifidogenic compared to the negative control (Objective 3) during fecal fermentation. Cranberry xyloglucan oligosaccharides were not bifidogenic (Objective 3) but did produce significant amounts of butyrate short chain fatty acids during fecal fermentation by one donor. Butyrate production has been reported to be associated with colonic health. Cranberry xyloglucan oligosaccharides were present in 25 commercial juice products, but only those produced when a commercial pectinase was used for juice processing. Citrus fiber was able to emulsify vegetable oil with maltodextrin as a carrier prior to spray-drying in our pilot plant. Kombucha is a fermented tea product that is produced by Zygosaccharomyces spp., a sucrose fermenting yeast, and Gluconacetobacter spp., an acetic acid producing bacterium as the major microbial species, but metagenomic sequencing demonstrated that these cultures contained 111 bacterial and yeast species. Processing parameters (fermentation time and temperature, black or green tea, age and state of microbial inoculum (solid or liquid), carbohydrate and organic acid content) were evaluated to produce Kombucha with optimal taste.
5. Significant Activities that Support Special Target Populations:
Renye Jr, J.A., Somkuti, G.A. 2015. Bacteriocins of food grade lactic acid bacteria in hurdle technology for milk and dairy. Editors: Datta, N., Tomasula, P.M., Emerging Dairy Processing Technologies Opportunities for the Dairy Industry. John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO198SQ.UK. Book Chapter. 1:267-290.
Maxwell, E.G., Colquhoun, I.J., Rolin, C., Chau, H.K., Hotchkiss, A.T., Waldron, K.W., Morris, V.J., Belshaw, N.J. 2015. Rhamnogalacturonan I containing homogalacturonan inhibits colon cancer cell proliferation by decreasing ICAM1 expression. Carbohydrate Polymers. 132:546-553. DOI: 10.1016/j.carbpol.2015.06.082
Maxwell, E.G., Colquhoun, I.J., Claus, R., Chau, H.K., Hotchkiss, A.T., Waldron, K.W., Morris, V.M., Belshaw, N.J. 2015. Modified sugar beet pectin induces apoptosis of colon cancer cells via interaction with the neutral sugar side-chains. Carbohydrate Polymers. 136:923-929. DOI: 10.1016/j.carbpol.2015.09.063.
Paul, M., Phillips, J.G., Renye Jr, J.A. 2016. Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide. Journal of Dairy Science. 99(5):3263-3266. doi: 10.3168/jds.2015-10437.
Tunick, M.H., Thomas-Gahring, A.E., Van Hekken, D.L., Iandola, S.K., Singh, M., Qi, P.X., Ukuku, D.O., Mukhopadhyay, S., Onwulata, C.I., Tomasula, P.M. 2016. Physical and chemical changes in whey protein concentrate stored at elevated temperature and humidity. Journal of Dairy Science. 99:2372-2383. DOI: 10.3168/jds.2015-10256.
Chaluvadi, S., Hotchkiss, A.T., Yam, K. 2015. Gut Microbiota: Impact of probiotics, prebiotics, synbiotics, pharmabiotics and postbiotics on human health. Book Chapter in Probiotics, Prebiotics, and Synbiotics: Bioactive Foods in Promoting Health, Watson, R.R and Preedy, V.R. (Eds.), elsevier, New York. pp. 515-523. 2016. DOI: 10.1016/B978-0-12-802189-7.00036-8.