Location: Dairy and Functional Foods Research
2019 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 three objectives which address NP306 Component 1: Foods, Problem Statement 1.B: New Bioactive Ingredients and Health-promoting Foods. For Objective 1, studies were conducted on the use of bacteriocin-producing lactic acid bacteria (LAB) as bioprotective cultures for inhibiting the growth of Listeria in fermented skim or whole milk stored at refrigeration temperature. Milk fermented with Streptococcus thermophilus strain B59671 dropped in pH but Listeria continued to survive. Supernatants collected from fermented milk showed thermophilin 110 activity in skim milk when S. thermophilus 13 was used as the target bacterium, however it was not detected in whole milk. Thermophilin 110 was confirmed to inhibit the growth of Listeria grown in microbiological medium at 37C, thus further studies are needed to increase the efficacy of thermophilin 110 against Listeria within dairy foods. Milk fermented with a combination of S. thermophilus B59671 and Lactobacillus plantarum TSH076, which naturally produces pediocin, showed a 3-4 log reduction of Listeria within fermented milk. In addition, a preliminary study in collaboration with an ERRC food safety laboratory showed that whey recovered from yogurt produced using these two strains was able to inhibit the growth of Campylobacter jejuni. Studies using pediocin, enterocin and thermophilin-producing LAB resulted in the isolation of enterocin-resistant Listeria and pediocin-resistant enterococci; however, thermophilin resistant bacteria have not been isolated in our studies. The enterocin and pediocin-resistant isolates were added to our culture-collection. Collaborative studies with Carnegie Mellon University identified a gene cluster potentially encoding bacteriocins within a S. thermophilus strain. A mutant strain was developed by deleting the entire gene cluster and resulted in the loss of antimicrobial activity. Studies are ongoing to identify the gene(s) required for bacteriocin activity. Collaborative studies were initiated with ARS scientists at the U.S. National Poultry Research Center to test the effectiveness of thermophilins against enterococcal chicken pathogens.
With regard to Objective 2, studies continued on the characterization of Lactobacillus strains shown to grow using fructo-oligosaccharides or inulin as sole carbon sources. Studies showed that growth temperature affected prebiotic utilization, and several strains required anaerobic growth conditions for catabolism of prebiotics. All 18 strains tested were shown to produce acetic and propionic acids, however only 4 Lactobacillus strains were identified which produced butyric acid in the presence of either prebiotic. Lactobacillus rhamnosus GG was also shown to produce butyric acid, but only in the presence of inulin. Studies are ongoing to determine if the presence of fructo-oligosaccharides or inulin effect the growth of lactobacilli strains in skim milk.
Under Objective 3 and a CRADA, an invention disclosure was approved and patent application written for fermented tea (Kombucha) key process parameters. Kombucha is a healthy beverage that functions as a synbiotic due to the prebiotic nature of tea polyphenols that shift the microbial composition during fermentation based on the green and black tea used. In CRADA research, we observed that the cranberry pink color was retained on preparative HPLC columns during elution with water. This confirmed earlier research that the interaction between cranberry xyloglucan oligosaccharides and proanthocyanidin pigments was non-covalent and most likely due to hydrogen bonds. Both cranberry xyloglucan oligosaccharides and proanthocyanidins are known for their anti-adhesive properties from previous research. Cranberry oligosaccharides collected by preparative HPLC contained silver that leached from the preparative HPLC column. It was not possible to completely remove the silver from these preparative HPLC fractions. Therefore, prebiotic analysis was not performed on the cranberry fractions due to the antimicrobial activity of silver. In CRADA research, all lactic acid bacteria grew equally well when carrot fiber pectic oligosaccharides were the sole carbon source. This indicated that the free glucose remaining in these fractions was utilized for growth rather than the carrot oligosaccharides. In MTRA research, characterized blueberry fiber which is largely insoluble dietary fiber that contains some pectic oligosaccharides. In collaboration with Wake Forest University, we characterized two resistant starch samples from acorn and Sago Palm that have prebiotic properties. The Sago Palm molecular weight was 1000x lower than the acorn resistant starch which fits with the gut microbiome studies conducted at the Wake Forest Medical School using these samples.
To continue our effort to increase the use of whey proteins in foods, we made progress in researching them as food grade, clean-label stabilizers. Whey proteins are known to be sensitive to heat, particularly at low pH as well as when subjected to thermal processing and storage leading to undesirable traits such as turbidity, precipitation and gelation. Pectin from citrus peels and sugar beet pulp, which are known to have prebiotic dietary fiber properties, has been investigated with whey protein enriched beverage applications through simple mixing and low-grade heating. The results demonstrated, at the molecular level, that both low-methoxyl citrus pectin and sugar beet pectin can act as effective stabilizers to improve the storage and thermal stability of whey proteins when used in beverages (up to 3%), even at low pH (3.2). The emulsification stability of the protein beverages was also greatly extended by pectin-whey protein combinations at both acidic and neutral pH conditions when stored at elevated temperature (60 °C) for up to one week.
Accomplishments
1. Gene sequencing of antimicrobial-producing dairy bacteria. Antibiotic resistant microbes are a significant threat to our food supply and new compounds are needed to control pathogens. Generally recognized as safe bacteria that are used for dairy products produce protein fragments with antimicrobial activity. ARS researchers at Wyndmoor, Pennsylvania completely sequenced the DNA from these bacteria to identify the genes needed to produce antimicrobial protein fragments with activity against foodborne and human pathogens. An agreement was established with Carnegie Mellon University to collaborate on studies to identify antimicrobial genes and regulatory mechanisms which govern their expression. These studies will develop new methods to increase antimicrobial production to improve the safety and quality of our food supply.
Review Publications
Qi, P.X., Chau, H.K., Hotchkiss, A.T. 2019. Molecular characterization of interacting complexes and conjugates induced by the dry-state heating of beta-lactoglobulin and sugar beet pectin (SBP). Food Hydrocolloids. 91:10-18. https://doi.org/10.1016/j.foodhyd.2019.01.010.
Renye Jr, J.A., Needleman, D.S., Steinberg, D.H. 2019. Complete genome sequences of bacteriocin-producing streptococcus thermophilus strains ST016 and ST109. Microbiology Resource Announcements. 8:1-2. https://doi.org/10.1128/MRA.01336-18.
Fishman, M., Chau, H.K., Hotchkiss, A.T., Garcia, R.A., Cooke, P.H., White, A.K. 2019. Effect of long term cold storage and microwave extraction time on the physical and chemical properties of citrus pectins. Food Hydrocolloids. 92:104-116. https://doi.org/10.1016/j.foodhyd.2018.12.047.
