1a. Objectives (from AD-416):
1. Identify and characterize biologically active compounds in citrus peel and associated processing byproducts for potential as value-added products to promote human health. a) Advance the discovery, isolation, and structural characterization of compounds from peel, molasses, and ethanol conversion residues and from lipid-soluble constituents of peel oil residues for biotesting purposes. b) Discover new beneficial pharmacological actions of citrus byproduct compounds, validate these biological actions in animals, and characterize the associated modes of action, pharmacokinetics and bioavailability. 2. Identify citrus processing waste compounds that can be used as value-added products to control major citrus and other plant diseases. a) Discover anti-microbial compounds from citrus processing waste. b) Characterize the influences of citrus byproduct compounds on pathogen virulence and microbial ecology. 3. Develop economical recovery methods for biologically active classes of compounds in citrus processing waste. 4. Develop value-added food materials from polysaccharide constituents of citrus byproduct waste streams.
1b. Approach (from AD-416):
Develop new value-added uses of citrus processing byproducts by exploiting the bioactive constituents (secondary metabolites) and specialty food fibers of these citrus processing materials. Fractions enriched in specific phenolic compounds will be isolated and evaluated as potential value-add materials for food and health applications. New, untested compounds, novel compositions with other citrus compounds, and new biological applications will be pursued. To achieve this, research will be directed towards 1) discovering new beneficial biological actions of citrus byproduct compounds, 2) validating these biological actions in vivo, 3) characterizing modes of action, pharmacokinetics, and bioavailability of bioactive citrus byproduct derived materials, and 4) producing specialty fibers to fill a wide range of high value food applications. Approaches to discover new biological actions will extend to studies of the influences of citrus processing waste compounds on microbial pathogen virulence and ecology, with an aim towards controlling microbial pathogens in citrus production. Broad profiles of compounds will be tested against plant pathogens, with an emphasis on important citrus microbial pests. Another direction to this work is the development of new syntheses and analytical methods for the successful production of pectin materials possessing wide ranges in functionalities to fill high-value food and pharmaceutical applications. Emphasis will be placed on the production of these specialty fibers through selected actions of known hydrolytic enzymes and through site specific chemical modifications. Critical to any future commercialization of value-added citrus byproduct materials is the development of economical recoveries of the modified polysaccharide fibers and secondary metabolites. Effective fractionations of byproduct streams into specific classes of secondary metabolite compounds and structural polysaccharides will be developed.
3. Progress Report:
This project is aimed at developing new value-added products from citrus bioactive constituents and specialty food fibers from citrus processing residues. As part of this project, the metabolism and pharmacokinetics of citrus peel flavonoids were characterized in rats and humans. Such analyses are critical to understanding the efficacy of these compounds in animal trials. Citrus flavonoids are rapidly and extensively metabolized into a variety of new forms, and knowledge of the rates of metabolism and metabolite bioavailabilities is critical to the design and selection of protocols useful in establishing beneficial actions in mammals. These studies are associated with Objective 1: "Identify and characterize biologically active compounds in citrus peel and associated processing byproducts for potential as value-added products to promote human health.” In a human clinical trial conducted by a collaborator at Sao Paulo State University, 24 volunteers ingested two different doses of citrus flavonoids and extracts of plasma and urine samples were prepared for analyses by HPLC-mass spectrometry. The metabolites in these tissue extracts were subsequently quantitatively and qualitatively analyzed. Flavonoid metabolites in humans exclusively occurred as sulfate and/or glucuronic acid conjugates. Isolation of metabolites of citrus peel polymethoxylated flavones allowed the quantification of this class of flavonoids in the human samples. These preliminary pharmacokinetic data will be immediately applicable to efforts to identify and validate beneficial biological effects of these citrus compounds in emerging new commercial products. Important progress was also made in the analysis of metabolites of major flavonoids from lemons fed to rats. Biological activities of these compounds in rats were substantiated. Associated with Objective 2, “Identify citrus processing waste compounds that can be used as value-added products to control major citrus and other plant diseases”, studies on the biological and chemical ecology of the canker lesion were continued. Three of the four main compounds formed in response to the infection of citrus tissues by the pathogenic bacterium Xanthomonas citri were identified and quantified and three additional structurally-related compounds or “phytoalexins” were also identified as well as other disease-induced compounds newly observed in citrus. Associated with Objective 4, “Develop value-added food materials from polysaccharide constituents of citrus byproduct waste streams” progress was also made pertaining to value-added chemical research into the polysaccharide structures of citrus byproducts. Pectin methylesterase present in a commercial papaya enzyme extract was used to demethylate a model pectin molecule. The resulting modifications to the pectin nanostructure have been characterized. The results indicate that reaction conditions (i.e., pH and enzyme/substrate ratios) affect the introduced nanostructural motifs.
Kim, Y., Williams, M.A., Galant, A.L., Luzio, G.A., Savary, B., Vasu, P., Cameron, R.G. 2013. Nanostructural modification of a model homogalacturonan with a novel pectin methylesterase: Effects of pH on nanostructure, enzyme mode of action and substrate functionality. Food Hydrocolloids Journal. 33:132-141.