Progress 07/28/10 to 07/27/15
Outputs
Progress Report 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. 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. Objective 1. Major objectives of this project were aimed at developing new products from citrus bioactive constituents and specialty food fibers derived from citrus processing waste streams. The first objective involved the study of the metabolism and pharmacokinetics of selected citrus peel flavonoids in an effort to understand the chemical factors influencing bioavailability and efficacy of these compounds. To this end, metabolites of citrus peel compounds, including the main flavanone glycosides eriocitrin and hesperidin, and the diverse polymethoxylated flavones, termed the polymothoxylated flavones, were purified and identified from samples obtained from pharmacokinetic investigations in rats and in humans. Early studies focused on the detection and identification of the main metabolites of nobiletin and tangeretin in rats. In addition to the identification of these compounds, the pharmacokinetic time courses of these compounds and of their metabolites were characterized. These studies showed the occurrence of major differences in the metabolism and pharmacokinetics of the polymothoxylated flavones and the flavanone glycosides. Yet, upon further chemical breakdown of these compounds, strong similarities in the chemical species present in the animal tissues were discovered. In a different 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 high performance liquid chromatography 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. Negligible amounts of ring fission products occurred, thus eliminating this class of compounds as major contributors to beneficial biological actions in humans. The rates of appearance of the main metabolites in human plasma samples were measured and studied as a function of different orange juice extraction techniques (fresh squeezed versus commercially processed juice) . The two juices had notably different profiles of soluble and precipitate compounds, yet the results showed no difference in bioavailability of the orange juice compounds in the two styles of juices. The results of these studies provided evidence of additional metabolites originating from the minor-occurring polymethoxylated flavones and hydroxycinnamates in orange juice (also potentially healthful compounds). This is the first description of polymethoxylated flavone metabolites from orange juice measured in human studies. This is notably different from observations made with similar citrus peel flavonoids fed to rats. 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. In a separate study, the metabolites of eriocitrin, the major flavonoid in lemons, were analyzed in rat urine and blood samples in a pharmacokinetics feeding trial. Biological activities of these compounds were studied in several in vivo inflammation trials in mice fed a special high-fat diet and dramatic protective anti-inflammatory effects occurred. The induction of two powerful proinflammatory signaling proteins, IL-6 and MCP-4 was blocked. Recent work involved large scale purifications of three different polymothoxylated flavones for additional rat feeding trials with the aim to characterize the wide range of metabolites, which have thus far been detected in 4 main classes. Fifteen compounds were isolated and will be evaluated for their chemical structures and subsequently used in in vitro studies. Since these are metabolites already occurring in animals, the small-animal trials can be eliminated. Progress was also made in understanding the chemical mechanisms of grapefruit-drug interactions caused by grapefruit juice phytochemicals. A new fungal metabolite of an important grapefruit furanocoumarin was detected and structurally characterized, and was found to lack CYP3A4 inhibitory activity. CYP3A4 inhibitory activity of most grapefruit furanocoumarins is responsible for the grapefruit/drug interactions that occur in humans consuming both grapefruit and certain prescription medications. It was hypothesized that the ability to modify these grapefruit compounds by fungi may lead to new technologies to eliminate these deleterious drug interactions in humans. The sequestration of grapefruit furanocoumarins by foods was investigated by characterizing the binding between these compounds and foods with contrasting protein, fat, and carbohydrate compositions. Individual grapefruit furanocoumarins exhibited contrasting affinities to foods, where the lipophilic bergamottin and several structurally related dimers bound to foods more tightly than the more polar 6',7'-dihydroxybergamottin. From the investigation of different classes of macromolecules in foods, water- soluble proteins were found to be the major constituents responsible for furanocoumarin sequestration. Studies using bovine serum albumin as a model protein demonstrated the dissociation of grapefruit furanocoumarins from the insoluble juice cloud particles and the subsequent formation of water-soluble bovine serum albumin-furanocoumarin complexes. Fluorescence binding assays further demonstrated the binding of bergamottin and 6',7'- dihydroxybergamottin to bovine serum albumin. These results demonstrate that proteins can be sequestration agents of these important dietary furanocoumarins. Objective 2. Progress was also made in two separate projects to detect biomarker compounds for citrus canker and Huanglongbing diseases. Key differences were detected in the profiles of compounds of healthy versus Huanglongbing-affected leaves and juice. Of particular importance was the discovery of the elevated levels of the bitter-tasting limonoids in Huanglongbing-symptomatic fruit. This marker of Huanglongbing-off-flavor has been widely adopted by the industry. The limonoids are first biosynthesized in the phloem, then transported to other plant tissues, and it is in the phloem that the Huanglongbing bacterial pathogen occurs. It was hypothesized that certain compounds in the phloem are essential to the growth of the Huanglongbing-causing bacterium. Extensive high performance liquid chromatograpy studies were done to identify and catalog these compounds including the energy requiring nucleotides, nucleosides, amino acids, and the secondary natural products, flavonoids, and hydroxycinnamates. High levels of the bitter-tasting limonin and nomilin were detected in the phloem tissues. Other work evaluated natural compounds for their antimicrobial activity against the citrus canker-causing bacterium Xanthomonas citri and problematic decay pathogens. Our investigations showed that the chemical make-up of canker lesions changes as the canker ages, and that there are increased levels of fluorescent coumarins in the lesions. 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. Three additional compounds structurally related to these �phytoalexins� were identified. Other disease-induced compounds, including compounds newly observed in citrus, were detected and partially isolated. Objective 3. Progress was also made in enhancing the recovery of citrus peel bioactive compounds from steam-explosion technology. Steam explosion of either whole fruit or peel greatly improves pectin extraction, as well as the extraction of many other fruit compounds. New finding on the contents of water washes of steam-exploded fruit and peel, point to high recoveries of the fruit�s water soluble hydroxycinnamates, limonoid glucosides, and almost a third of the total polymothoxylated flavones. These clarified water washes could provide major portions of the world demand for many of these compounds based on mass balance studies of these wash fractions and total crop production. An additional application of this method is the product recovery from the fruit affected by the Huanglongbing disease. Due to this disease, large percentages of the total crop are lost to fruit drop, undersized fruit, or poor quality fruit. The recovery of the marketable compounds from the steam-explosion process represents a new opportunity for valuable co- product recovery from this �lost� portion of the citrus crop. Objective 4. The nanostructure of the pectin molecule is important for development of pectin products for food, medical or industrial use. Research into the polysaccharide structures of citrus byproducts used pectin methylesterase from commercial papaya enzyme extract to demethylate model pectin molecules. The resulting modifications to the pectin nanostructure were characterized, and indicate that reaction conditions (i.e., pH and enzyme/substrate ratios) affect the introduced nanostructural motifs. Accomplishments 01 Inhibition of inflammation occurring with high-fat diet in mice. Citrus compounds, termed flavonoids, have been shown to inhibit inflammation in certain animal assays and the study was conducted to test if these compounds could inhibit the oxidative stress and systemic inflammation induced by high-fat diet in mice. ARS researchers at Ft. Pierce, Florida and at Sao Paulo State University showed that the examined citrus flavonoids had protective effects against the inflammation and oxidative stress caused by the high-fat diet in mice and, therefore, prevented metabolic alterations associated with the development of cardiovascular diseases. This study supports the use of citrus as a health-promoting food, and increases the demand and value for these citrus byproduct flavonoids.
Impacts
(N/A)
Publications
- Silveira, J.Q., Cesar, T.B., Manthey, J.A., Baldwin, E.A., Bai, J., Raithore, S. 2014. Pharmacokinetics of flavanone glycosides after ingestion of single doses of fresh-squeezed orange juice versus commercially processed orange juice in healthy humans. Journal of Agricultural and Food Chemistry. 62:12576-12584.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. Objective 1: Progress was made in the chemical isolation and structural elucidation of metabolites of several of the major flavanone glycosides in the peels of oranges and lemons. These components can impart health benefits in humans but only if adequately absorbed. Bioavailability is often evaluated by measuring the metabolites of these compounds in humans after ingestion. However many of these metabolites occur in human blood serum samples at only trace concentrations and analytical detection techniques were needed for these compounds. These techniques were developed and the metabolites were discovered and measured in rat and human samples. For the metabolites at the lowest concentrations, preliminary structural analyses were achieved. The rates of appearance of the main metabolites in human plasma samples were measured and studied as a function of different orange juice extraction techniques (fresh squeezed versus commercially processed juice). The results of these studies provided evidence of additional metabolites originating from the minor-occurring polymethoxylated flavones and hydroxycinnamates in orange juice (also potentially healthful compounds). This is the first description of polymethoxylated flavone metabolites from orange juice measured in human studies. The major flavanone metabolites in rat whole blood extracts were also studied and seven compounds were isolated to purity. One metabolite has been identified and six metabolites are in line for similar structure elucidations. Four of the compounds were submitted to a contract research laboratory for an in vitro test for the inhibition of inflammation in human white blood cells. Minor metabolites in rat whole blood extracts have also been detected and studied in several pharmacokinetic studies in rats. These pharmacokinetic studies showed that none of the originally dosed flavanones appeared in the blood serum, rather, only modified metabolites occurred. This contrasted with previous findings made with the polymethoxylated flavones. Later time points in the pharmacokinetics studies showed the appearance of a second generation of metabolites derived from the original molecules. Progress was also made in the study of the uptake and pharmacokinetics of compounds isolated from orange oil. Gram quantities of four compounds were isolated and submitted for a pharmacokinetic study in rats. Some of these compounds represent new targets for value-added health benefiting compounds from orange peel. Objective 2: Citrus greening or Hunaglongbing (HLB) disease is devastating the citrus industry. The phytochemicals in orange tissues have been shown to be affected by the HLB disease and these compounds affect fruit and peel quality. Some of the flavor affecting compounds are transported via the stem phloem. It is in the phloem that the HLB bacterial pathogen occurs. Compounds in the phloem might be important to the growth of the bacteria. Methods to detect the compounds in citrus phloem extracts were developed and phloem extracts were analyzed. These extracts were also shown to be rich in certain chemicals, which may play roles in fruit quality. The quantitation of these chemicals in the phloem extracts was initiated. Objective 4: Pectin is a major and high value component of citrus peel. The nanostructure of the pectin molecule is important for development of pectin products for food, medical or industrial use. Progress was made on the study of pectin nanostructure. Reaction variables, i.e. presence or absence of monovalent cations and enzyme/substrate ratios, were tested for their effects on pectin nanostructure during enzymatic modification. The effect of the method of preparing a pectin with certain desired characteristics on the reaction conditions involving enzymes was explored. Accomplishments 01 Juice extraction techniques do not influence the pharmacokinetics and bioavailability of orange juice flavanones in humans. Different juice extraction techniques produce significantly different concentrations and solubility profiles of healthful flavonoids. Most of the flavonoids in commercially processed juice occur precipitated in the juice cloud, while most of the flavonoids in freshly squeezed juice occur in solution. Consumption of both styles of orange juice produced similar rates of appearance for the flavonoid metabolites, indicating that the bioavailability of citrus flavonoids is not influenced by the juice extraction techniques. However, fresh-squeezed juice was shown to contain higher levels of a class of flavonoids, termed the polymethoxylated flavones, which have exhibited potentially beneficial health effects in in vitro assays.
Impacts
(N/A)
Publications
- Hijaz, F.M., Manthey, J.A., Folimonova, S.Y., Davis, C.L., Jones, S.E., Reyes-De-Corcuera, J.R. 2013. An HPLC-MS Characterization of the Changes in Sweet Orange Leaf Metabolite Profile following Infection by the Bacterial Pathogen Canditatus Liberibacter asiaticus . PLoS One. DOI: 10. 1371/journal.pone.0079485.
- Galant, A.L., Widmer, W.W., Luzio, G.A., Cameron, R.G. 2014. Characterization of molecular structural changes in pectin during juice cloud destabilization in frozen concentrated orange juice. Food Hydrocolloids Journal. 41:10-18.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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.
Impacts
(N/A)
Publications
- 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.
- Myung, K., Manthey, J.A., Narciso, J.A. 2013. Protein Sequestration of Lipophilic Furanocoumarins in Grapefruit Juice. Journal of Agricultural and Food Chemistry. 61:667-673.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. This project is aimed at developing new products from citrus bioactive constituents and specialty food fibers derived from citrus processing waste streams. As part of these investigations, studies were performed to discover naturally occurring citrus plant response compounds capable of limiting the deleterious actions of microbial pathogens. Objective 1: Improvements were also made in analytical techniques for the study of citrus by-product compounds. The use of fluorescence detection was explored for three major classes of bioactive constituents. Fluorescence detection allows for the detection of single peaks that were normally unresolved in ultraviolet wavelength monitored, highly overlapping chromatograms. The use of fluorescence detection enhanced the detection of a number of newly observed compounds in citrus, the identities of which are currently under investigation. Objective 2: Studies on the biological and chemical ecology of the canker lesion were continued as well as screens for natural antimicrobials. Recent data show that when the citrus-causing bacterium is �stressed� by treatments (e.g. exposure to antimicrobials) the colony changes. Whereas the Xanthomonas citri subsp. citri (Xcc) produces a yellow gummy substance (xanthin), the �stressed� colony produces a companion cell that is white to opaque. This organism becomes the dominant cell form rather than the canker bacterium. Depending on the treatment (i.e. screens for antimicrobials) there are various ratios of the two cell types which may play a role in the success of the canker bacterium as an epiphyte. When comparing cell population of young and old cankers, young cankers are predominantly Xcc while older cankers are a mix of these and the opaque cells. Data show that the chemical make-up of the lesion changes as well as the canker ages. Data from our screens show that Xcc may be able to process toxic compounds in such a way that the bacterial cells are eventually able to overcome the toxicity and begin to grow. Objective 4: Progress was 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. Accomplishments 01 A predictive model for the relationship between pectin nanostructure and rheological properties. ARS Researchers at Ft. Pierce, FL have develope a predictive model for the relationship between pectin nanostructure and rheological properties. Pectin functionality is dependent on the distribution of charges along its polymeric backbone. Random versus ordered charge distribution affects pectin�s functionality and variation in the amount or topographical order of electrical charge within the polymer chain also affect rheology. We have modified pectin charge distributions using enzymes or chemical processes and developed technolo to statistically describe the introduced nanostructural modifications. Rheological testing of the engineered pectins have enabled us to develop predictive model relating nanostructural parameters to rheological properties. This accomplishment has the potential to allow food processors greater control over texture, gel formation and interaction o pectin with other food components related to flavor and aroma.
Impacts
(N/A)
Publications
- Vasu, P., Savary, B.J., Cameron, R.G. 2012. Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation. Food Chemistry. 133:366-372.
- P�rez, C.D., Fissore, E.N., Gerschenson, L.N., Cameron, R.G., Rojas, A.M. 2012. Hydrolytic and oxidate stability of L-(+) -ascorbic acid supported in pectin films: Influence of the macromolecular structure and calcium presence. Journal of Agricultural and Food Chemistry. 60:5414-5422.
- Manthey,J.A. 2012. Potential value-added co-products from citrus fruit processing. In: Bergeron,C., Carrier,D.J., Ramaswamy,S., editors. Biorefinery Co-Products. Hoboken, NJ: John Wiley & Sons, Ltd. p. 153-178.
- Myung, K., Manthey, J.A., Narciso, J.A. 2012. Biotransformations of 6',7'- dihydroxybergamottin and 6',7'-epoxybergamottin by the citrus-pathogenic fungus Penicillium digitatum diminish cytochrome P450 3A4 inhibitory activity. Bioorganic and Medicinal Chemistry Letters. 22:2279-2282.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Important progress was made in the isolation and identification of metabolites of citrus peel compounds, polymethoxylated flavons (PMFs), in a pharmacokinetic investigation in rats. Pharmacokinetic time courses of distinct profiles of metabolites of citrus peel PMFs were also measured. The PMFs are currently sold as food supplements, and their efficacy is dependent on their metabolism and oral bioavailability. This rat study will contribute to improved use of the PMFs, hence increasing the value of these citrus byproduct compounds. Progress was also made in enhancing the recovery of PMFs from peel waste, initially treated for ethanol production. The PMFs, are present in orange processing waste steams at approximately 0.02�0.03% of the wet weight. Research showed that after hydrolysis and fermentation of available sugars to ethanol for use as a biofuel, the available PMF content in the residue for isolation purposes increased by 150%. It was found that 1 1iter of the food grade resin (XAD 16) used for citrus debittering could remove the PMFs from approximately 200 Liters of hydrolyzed and clarified orange peel extract. However, with the low amounts (0.02�0.05%) of these components present and the need to clarify the orange processing waste extracts prior to treatment, recovery is unlikely to be economically viable. Progress was also made in understanding the chemical mechanisms of grapefruit-drug interactions caused by grapefruit juice phytochemicals. A new fungal metabolite of an important grapefruit furanocoumarin was detected and structurally characterized, and was found to lack CYP3A4 inhibitory activity. CYP3A4 inhibitory activity for most grapefruit furanocoumarins is responsible for the grapefruit/drug interactions that occur in humans consuming both grapefruit and certain prescription medications. The ability to modify these grapefruit compounds by fungi may lead to new technologies to eliminate these deleterious drug interactions in humans. Progress was also made in two separate projects to detect biomarker compounds for citrus canker and HLB diseases. Key differences were detected in the profiles of compounds of healthy versus Huanglongbing (HLB) affected leaves. This will aid in early detection of HLB and contribute to an understanding of the progression of this disease in citrus plant tissues, and to discover mechanisms by which citrus trees cope with the canker- causing bacterium. Work also continued with evaluating natural compounds for their antimicrobial activity against Xanthomonas citri and problematic decay pathogens. 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 affect the nanostructural motifs which may lead to the development of novel pectin products. This project replaced project 6621-41000-014-00D which was a bridging project which continued work from project 6621-41000-012-00D. Accomplishments 01 Chemical responses in grapefruit to bacterial plant pathogens. The bacterial disease in citrus, termed citrus canker, is devastating grapefruit and orange production in Florida for fresh fruit markets. Amo the many different citrus cultivars there is a wide range in susceptibility to this disease, yet the chemical basis for this is poorl known. Extensive analyses of the susceptible grapefruit have provided ke information about the changes in the chemical environments accompanying infection by the canker bacterium. Important marker compounds for canker infection have been found. These marker compounds may be plant defense compounds which may be useful in combating this disease. These discoveri will provide a baseline by which researchers can compare susceptible cultivars (i.e. grapefruit) against less susceptible cultivars, and gain an understanding of the chemical basis of tolerance to this pathogen. Th will enable researchers to develop technologies to combat and mitigate this disease, and restore profitability to the Florida fresh fruit marke
Impacts
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Publications
- Manthey, J.A., Perkins-Viazie, P. 2009. Influences of harvest date and location on the levels of �-carotene, ascorbic acid, total phenols, in vitro antioxidant capacity, and phenolic profiles of five commercial varieties of mango (Mangifera indica L.). Journal of Agricultural and Food Chemistry. 57:10825-10830.
- Cameron, R.G., Luzio, G.A., Vasu, P., Savary, B.J., Williams, M.A.K. 2011. Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from citrus: I. Nanostructural characterization, enzyme mode of action and effect of pH. Journal of Agricultural and Food Chemistry. 59:2717-2724.
- Manthey, J.A., Cesar, T.B., Jackson, E., Mertens-Talcott, S. 2011. Pharmacokinetic study of nobiletin and tangeretin in rat serum by high- performance liquid chromatography-electrospray ionization-mass spectrometry. Journal of Agricultural and Food Chemistry. 59:145-151.
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