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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Dairy and Functional Foods Research » Research » Research Project #428809

Research Project: Bioactive Food Ingredients for Safe and Health-Promoting Functional Foods

Location: Dairy and Functional Foods Research

2020 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:
This is the final report for the project 8072-41000-100-00D which ended June 3, 2020. The new NP306 project entitled “New Bioactive Dairy Products for Health-Promoting Functional Foods” was approved and is being established. For Objective 1, research focused on the production of bacteriocins, which are antimicrobial peptides naturally produced by a variety of bacterial species to provide a competitive advantage for growth in a specific niche. Production of these antimicrobial peptides by food-grade lactic acid bacteria has led to their investigation as alternatives to chemical preservatives in foods or alternatives to antibiotics for human or animal health. This project continued the characterization of novel bacteriocins produced by the dairy culture Streptococcus thermophilus, and a collaborative effort with Carnegie Mellon University has identified a cluster of genes required for bacteriocin expression from a strain in our collection. Work is continuing to identify the peptide and characterize its activity spectrum. Work has continued to identify potential applications for thermophilin 110, the bacteriocin produced by S. thermophilus B59671. Results showed it inhibited growth and biofilm formation of the oral pathogen S. mutans; and preliminary results did not show enhanced activity in the presence of other chemicals used in toothpaste (fluoride, triclosan). Thermophilin 110 was also shown to inhibit the growth of enterococcal isolates, including strains of Enterococcus cecorum, a poultry pathogen. Fermentates recovered from milk fermented with S. thermophilus B59671 and Lactobacillus plantarum TSH076 showed activity against both Gram-negative and -positive foodborne pathogens. These studies are ongoing with collaborators at U.S. National Poultry Research Center (Athens, GA) and colleagues at ERRC (NP108). Resistant mutant have not been identified for target bacteria susceptible to thermophillin 110; and thermophilin 110 has been shown to inhibit pediocin-resistant enterococci, suggesting it can be used in combination with other bacteriocins for food safety applications. Objective 2 research focused on the identification of Lactobacillus strains from within our in-house collection capable of growth when fructo-oligosaccharides, pectin or cranberry oligosaccharides were provided as the sole carbon source. Several strains were identified and shown to produce lactic acid, as well as short chain fatty acids, specifically acetic, butyric and propionic acids. These strains will be used in future studies to assess the prebiotic activity of additional plant oligosaccharides. Additionally, collaborative studies with a visiting scientist from CIAD (Mexico) showed identified strains of Bifidobacteria which grew using arabinoxylan as a sole carbon sources; and showed the potential for using arabinoxylan gels for encapsulating potential probiotics or bacteriocin-producing lactic acid bacteria. Under Objective 3, new sources of dietary fiber and prebiotics were identified and their structures were compared with anti-adhesive and anti-inflammatory oligosaccharides. The first in vivo prebiotic activity for citrus and sugar beet pectic oligosaccharides was identified using an in ovo chicken model. The structure-function relationships of citrus pectic oligosaccharide samples for prebiotic, pathogen anti-adhesive and colon cancer cell apoptosis and anti-proliferation activity was defined. Cranberry oligosaccharides with pathogen anti-adhesive activity also produced butyrate during human fecal fermentation. Cranberry oligosaccharides were isolated by preparative HPLC and the pectic oligosaccharide structure was characterized. These cranberry oligosaccharides were present in cranberry juice samples and a CRADA partner adopted the technology to determine oligosaccharide levels in commercial products. Citrus fiber was able to emulsify vegetable oil with maltodextrin as a carrier prior to spray-drying in our pilot plant that was highly stable for a year of storage. Fermented tea (Kombucha) was analyzed to determine if it contains probiotic bacteria or has health-promoting properties. Kombucha is a healthy beverage synbiotic since it delivers bioactive tea polyphenols in a cellulose matrix plus acetic acid and gluconic acid, not because of known probiotic bacteria based on meta-genomic sequencing analysis. In CRADA research, the key Kombucha process parameters including the type of tea, age of inoculum, presence or absence of solid inoculum and specific interfacial area were evaluated and correlated with chemical (sugars, acids, alcohol, catechins, total polyphenols), microbial (bacteria and yeast counts, and composition) and sensory characteristics. In CRADA research, identified carrot fiber pectic oligosaccharide structures with reduced molecular weight following enzymatic treatment as branched, unsaturated rhamnogalacturonan I oligosaccharides with arabinogalacto-oligosaccharide side chains. In MTRA research, characterized blueberry, strawberry and red beet fiber which were largely insoluble dietary fibers with different polysaccharide composition that include pectic oligosaccharides. In collaboration with Wake Forest University, characterized two resistant starch samples from acorn and Sago Palm and a low-methoxy pectin 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. Whey proteins of milk 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, such as emulsion stability, and heat stability. Progress was made recently to provide an in-depth understanding of the molecular mechanism between the protein and pectin, leading to the improved properties observed in the final products. Whey protein is a complex mixture of many proteins with beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) as the two main components, containing over 50% and 25%, respectively. Beta-LG and alpha-LA were each reacted with sugar beet pectin at the same conditions as with whey (done previously), i.e., at the controlled temperature, humidity, and time. The results showed that purified beta-LG and alpha-LA alone, in the absence of other proteins, is insufficient to improve emulsification properties when used in an interacting system with pectin. These studies further elucidated the molecular mechanism determining the functional properties. They also demonstrated that other proteins in whey, less dominating, must contribute to its overall characteristics and heat stability. This study will guide us in developing new formulations containing whey proteins and pectin and perhaps other dietary fibers for improved quality of food and beverage applications.

4. Accomplishments
1. Sugar beet fiber promotes health. Sugar beet fiber is a low-value product of sugar processing used as animal feed. Also, the sugar industry is facing challenges from consumers who increasingly demand a low-carbohydrate diet and the industry can benefit from more income. ARS scientists at Wyndmoor, Pennsylvania, determined that sugar beet fibers possess unique structures that promote the growth of gut bacteria. The unique structure of the sugar beet fiber pectin is responsible for these health-promoting properties. The world's largest pectin producer has applied for a license on an ARS patent covering pectin structure that promotes the growth of health-beneficial gut bacteria.

Review Publications
Hotchkiss, A.T., Qi, P.X., Liu, L.S., Chau, H.K., Cooke, P., Nunez, A., White, A.K., Fishman, M. 2019. Sugar beet pulp fiber is a source of bioactive food and feed ingredients. International Sugar Journal. 121:126-131. Available:
Muhidinov, Z.K., Bobokalonov, J.T., Ismoilov, I.B., Strahan, G.D., Chau, H.K., Hotchkiss, A.T., Liu, L.S. 2020. Characterization of two types of polysaccharides from Eremurus hissaricus roots growing in Tajikistan. Food Hydrocolloids. 205.
Qi, P.X., Wickham, E.D. 2020. Changes in molecular structure and stability of beta-lactoglobulin induced by heating with sugar beet pectin in the dry-state. Food Hydrocolloids. 105:1-11.
Qi, P.X., Wickham, E.D., Xiao, Y. 2020. Chemical composition as an indicator for evaluating heated whey protein isolate (WPI) and sugar beet pectin (SBP) systems to stabilize O/W emulsions. Food Chemistry. 330: 1-9.
Du, R., Qu, Y., Qi, P.X., Sun, X., Liu, Y., Zhao, M. 2020. Natural flagella-templated Au nanowires as a novel adjuvant against Listeria monocytogenes. The Royal Society of Chemistry. Nanoscale. 12:5627-5635.