Location: Dairy and Functional Foods Research
2017 Annual Report
Objectives
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).
Approach
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.
Progress Report
Progress was made on all objectives, all of which fall under National Program
306 – Quality and Utilization of Agricultural Products, Component I, Food. Progress on this project focuses on Problem Statement B - New Bioactive Ingredients and Functional Foods and C - New and Improved Food Processing Technologies.
Completed preliminary studies to show that a partially purified antimicrobial compound, naturally produced by a yogurt bacterium, could inhibit the growth of a bacterial species commonly associated with the development of dental cavities (objective 1). It was also shown that the presence of the yogurt culture could prevent the dental pathogen from forming a biofilm under laboratory conditions. In addition, antimicrobial compounds produced by yogurt bacteria were shown to prevent the growth of a harmful bacterium which causes strep throat. These studies suggest the potential for using yogurt bacteria as probiotics to prevent infections in humans. Genetic studies identified the gene which encodes the antimicrobial compound; and the complete genome of four yogurt bacteria were sequenced to allow for future molecular studies to identify mechanisms which regulate the production of the antimicrobial compound (objective 1). Studies were also initiated to test if milk could be used as a fermentation medium for production of the antimicrobial compound (objective 1). Results showed that the antimicrobial could be produced in raw whole milk or pasteurized skim milk; however production was not observed in pasteurized whole milk (homogenized) and raw milk which was exposed to a process called microfluidization (high pressure homogenization). It is possible that milk processing, such as conventional homogenization and microfluidization, could release a natural component of milk which inactivates the antimicrobial compound. Studies are ongoing to identify which components of milk could interact with the antimicrobial (objective 1). Preliminary spectroscopic studies in aqueous solution were performed on a crude preparation of bacteriocin to test if these antimicrobial compounds are rendered inactive within some dairy foods due to their interactions with milk components (objective 1). Although the results didn’t indicate strong binding interaction between the major milk protein, Beta-lactoglobulin and the bacteriocin, further clean up of the prep is needed. In addition, experiments using phospholipid as the model membrane interacting medium will be conducted. Collaborative studies are ongoing with the Buffalo Research Institute in China to identify novel bacteria, indigenous to buffalo milk, which naturally produce antimicrobials. Preliminary studies were performed to demonstrate if citrus pectins and smaller pectin fragments could support the growth of dairy (yogurt) bacteria. Results showed that some pectins could support minimal growth, and allow for the production of natural antimicrobials (objective 2).
Whey proteins are used in a diverse and expanding range of food products, including sports beverages, nutritional supplements, infant formula, pharmaceutical formulations, and non-food such as personal care products. In previous work, whey proteins were reacted with sugar beet pectin to improve their solubility, functional properties, and stability. Progress was made recently to provide an understanding of the molecular mechanism between the protein and pectin leading to the improved properties observed in the final products. The two main proteins of whey, Beta-lactoglobulin and Alpha-lactalbumin, were each reacted with sugar beet pectin at controlled temperature, humidity and time. Studies of the reacted products provided insights on how individual protein component of whey contributed to the overall observed improvement of the properties. It is anticipated that the results and knowledge gained from this work will help the dairy processing industry, dairy ingredient manufacturer, nutritional food supplement industry, high-protein health food products and sports beverage industry to design products with tailored and targeted applications. This knowledge will also be used to understand the interaction between milk components and antimicrobials (objective 1) as well as the effect of pectin on antimicrobial production (objective 2). Corn arabinoxylan was prebiotic in human mixed fecal fermentations (growth of both Bifidobacteria and Lactobacilli) and enzymatic hydrolysis to reduce the arabinoxylan molecular weight didn’t alter the arabinoxylan prebiotic properties (Objective 3). Using our HPLC method, cranberry xyloglucan oligosaccharides were present in 13 commercial cranberry products, but none were detected in apple paste. CRADA partner has adopted our HPLC method for quality control of cranberry juice products. New Kombucha (fermented tea) products were commercially released based on our CRADA research. These new Kombucha products have prebiotics and probiotics added following fermentation. Dietary fiber composition and functional properties were determined for tomato and carrot pulp for a CRADA partner. The carrot pulp consisted of a largely insoluble, pectin-rich dietary fiber. The modified citrus pectin (bioactive dietary fiber produced by an ARS researcher in Ft. Pierce, Florida) carbohydrate composition was determined (Objective 3). The rhamnogalacturonan and galacto-oligosaccharide structure of this modified citrus pectin binds to galectin-3 at a lower minimum inhibitory concentration compared to the leading commercial modified citrus pectin currently on the market.
Accomplishments
1. Dairy antimicrobials for personal care products. Due to the growing concern over the number of harmful bacteria which have become resistant to commonly used antibiotics, there is a need to identify alternative antimicrobial compounds. ARS researchers at Wyndmoor, Pennsylvania have demonstrated that an antimicrobial compound, naturally produced by a bacterium used to produce yogurt and cheese, can inhibit the growth of several harmful bacteria known to cause dental cavities, acne and sore throat; as well as invasive life threatening infections. A U.S. patent was issued (Patent # 9,598,471) for the compound’s unique antimicrobial activities, and the potential methods for utilizing this compound to treat infections. The generally recognized as safe (GRAS) status of this yogurt bacterium suggests that either the bacterium or the antimicrobial compound could be used in personal care products to improve oral health and skin care. This invention demonstrates the many benefits of dairy cultures, whether consumed in yogurt or cheese, and also now for use in personal care products.
Review Publications
Di, R., Vakkalanka, M.S., Onumpai, C., Chau, H.K., White, A.K., Rastall, R.A., Yam, K., Hotchkiss, A.T. 2017. Pectic oligosaccharide structure-function relationships: prebiotics, inhibitors of Escherichia coli O157:H7 adhesion and reduction of Shiga toxin cytotoxicity in HT29 cells. Food Chemistry. 227:245-254.
Qi, P.X., Xiao, Y., Wickham, E.D. 2017. Changes in physical, chemical and functional properties of whey protein isolate (WPI) and sugar beet pectin (SBP) conjugates formed by controlled dry-heating. Food Hydrocolloids Journal. doi: 10.1016/j.foodhyd.2017.01.032.
Cameron, R.G., Chau, H.K., Hotchkiss, A.T., Manthey, J.A. 2017. Recovery of pectic hydrocolloids and phenolics from Huanglongbing related dropped citrus fruit. Journal of the Science of Food and Agriculture. 97:4467-4475. https://doi.org/10.1002/jsfa.8310.
Li, L., Renye Jr, J.A., Zeng, Q., Huang, L., Ren, D., Tang, Y., Yang, P. 2016. Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing. Journal of Dairy Science. 99:7016-7024. doi: 10.3168/jds.2016-11041.
Qi, P.X., Xiao, Y., Wickham, E.D. 2016. Stabilization of whey protein isolate (WPI) through interactions with sugar beet pectin (SBP) induced by controlled dry-heating. Food Hydrocolloids. 67:1-13. doi: 10.1016/j.foodhyd.2016.1012.1032.
Cameron, R.G., Chau, H.K., Hotchkiss, A.T., Manthey, J.A. 2017. Release and recovery of pectic hydrocolloids and phenolics from culled citrus fruits. Food Hydrocolloids. 72:52-61. doi:org/10.1016/j.foodhyd.2017.05025.
Van Hekken, D.L., Tunick, M.H., Renye Jr, J.A., Tomasula, P.M. 2017. Characterization of starter-free Queso Fresco made with sodium-potassium salt blends over 12 weeks of 4 degrees C storage. Journal of Dairy Science. doi: 10.3168/jds.2016-12081.
