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United States Department of Agriculture

Agricultural Research Service

2006 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Citrus is recognized as a dietary component important to human nutrition. However, information about the role of the wide variety of natural chemicals (phytochemicals) present in citrus in human health and nutrition is limited. Relating specific phytochemicals or mixtures of phytochemicals to the improvement of human health would reinforce the importance of citrus in the human diet, promote increased citrus consumption and generally contribute to the improvement of human wellness. The identification of specific citrus phytochemicals beneficial to human health in citrus juice processing waste streams will provide the citrus processing industry an opportunity to realize increased revenues through the reclamation of these biologically active phytochemicals from these waste streams.

Increased demand for citrus with health promoting phytochemicals will expand existing citrus markets and open new markets for citrus varieties containing specific healthful phytochemicals. Understanding the origins of these phytochemicals in citrus will allow the development of genetic and molecular biological methods to enhance the amounts of these chemical compounds in citrus and will provide access to the biological mechanisms associated with the maintenance of citrus quality. Successful manipulation of these processes will lead to an expanded selection of higher quality citrus with increased content of recognized healthful phytochemicals that are beneficial to consumers.

This research project primarily focuses on a specific class of biologically active phytochemicals (limonoids) that occur in large amounts in citrus juice and citrus processing by-products. Limonoids occur in orange juice in amounts comparable to vitamin C and it is estimated that 15,000 tons of limonoids are available annually in by-products of worldwide citrus juice production. Research evidence has established that limonoids possess significant anti-cancer activity in animal tests and these compounds have been shown to become available to act in humans after ingestion. This research project will specifically assess pure citrus limonoids as agents that can act to intercede in preventative actions in humans that result in improved health. The research will concurrently develop methods to reclaim health-promoting limonoids from citrus processing by-products. Information about the health-promoting properties of specific citrus limonoids will also provide the basis for the biological examination of a variety of citrus species to accumulate new information to guide genetic or molecular biological manipulation protocols directed to generating new or improved high quality limonoid enriched citrus varieties.

The project's research activities fall within Problem Area 2a. New Product Technology of Component 2. New Processes, New Uses, and Value-Added Foods of NP306. Customers for the results of this research include citrus processors, citrus growers, the scientific community and the general public.

2.List by year the currently approved milestones (indicators of research progress)
FY05 - Inventory amounts and purity of currently held limonoids, purify existing isolated limonoids, establish synthetic procedures for production of compounds for structure/activity studies with currently held limonoids, identify citrus sources for extraction of new limonoids/secondary metabolites, begin collection of citrus materials, develop bioassay-guided screening protocols, identify and establish bioassay screening resource. Human cholesterol/inflammation study - Marshal resources, establish study protocol and submit for clearance, accumulate limonin glucoside materials and prepare for study. Animal metabolism/toxicity study – Accumulate pure limonoids for study.

FY05 - Samples from commercial citrus processing co-product streams will collect, limonoid glucoside content of co-product streams will be obtained, promising co-product streams will be selected and appropriate materials collected, initial separation of limonoid glucosides based upon patented technology will be conducted, methodology for isolation of pure limonoid glucosides will be formulated.

FY05 - Characterization and engineering of limonoid glucosyltransferase (LGTs)-Complete collaborative DNA and protein sequence analysis of LGT, clone promising enzymes and express as recombinants for additional characterization. Characterization of Limonin D-ring Lactone Hydrolase (LDLHs) - Complete the cloning and recombinant expression of LDLH. Metabolic Profiling - Develop methods for the metabolic profiling of limonoid aglycones and glucosides in citrus cultivars and in collaboration with Tracy Kahn (UC Riverside) initiate the evaluation of metabolic profiling as a tool for selecting new cultivars.

FY06, FY07 - Extract plant materials and evaluate extracts through bioassay screen protocols, identify extracts of interest, isolate and characterize biologically active components in extracts, assess toxicity of biologically active compounds, initiate synthetic procedures for structure/activity studies, begin accumulation of biologically active materials or the development of synthetic methods to provide adequate amounts for animal testing. Human cholesterol/inflammation study - Recruit study subjects, begin study, collect samples, analyze samples, report results, develop follow-up studies. Animal metabolism/toxicity study - Provide limonoids, obtain animals, begin study, record weight data, sacrifice animals, analyze tissues and fluids, report results, develop follow-up studies.

FY06, FY07 - Formulated methodology for isolation of pure limonoid glucosides will be applied and altered to maximize yields, methodology alteration to accommodate raw material character (i.e., molasses vs. seed extracts) will be developed, methodology will be maximized for efficiency, pilot scale-up will be formulated, methodology adjustment or development will begin to accommodate accumulation of new biologically active compounds identified in objective 1. Patent(s) will be submitted for lab-proven technology.

FY06, FY07 - Characterization and engineering of LGTs - Engineer LGT for improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Characterization of LDLHs - In collaboration with Jose Chapparro (USDA) complete the DNA and protein sequence analysis of hydrolases derived from various citrus cultivars. Evaluate the activity of hyrdolases derived from different cultivars and clone and express as recombinants for further characterization those enzymes identified with promising properties will be for additional characterization. Metabolic Profiling - Continue the evaluation of metabolic profiling of limonoid aglycones and glucosides over the growing season and draw a conclusion to the usefulness of the method.

FY08, FY09 - Report structure/activity results, establish bioactivity criteria and develop protocols for animal model tests of biologically-active compounds, develop analytical methodology to detect metabolites, conduct animal tests, identify metabolites, characterize pharmacokinetics, evaluate potential for human application, formulate plans for human tests. Human cholesterol/inflammation study – Begin planning for further studies. Animal metabolism/toxicity study – Begin planning for further studies.

FY08, FY09 - Formulation of pilot-scale scale-up, CRADA partner search, pilot scale-up, pilot scale process validation, patent licensing, industrial scale application.

FY08, FY09 - Characterization and engineering of LGTs - Optimize enzyme activity and generate additional transgenic plants and seek to patent the engineered enzyme. Characterization of LDLHs - Engineer hydrolase for improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Metabolic Profiling - Release a metabolic profiling of limonoids methods to be used by breeders and researchers.

4a.List the single most significant research accomplishment during FY 2006.
Cholesterol Lowering Potential of Limonoid Glucosides. Limonoid glucosides, found in citrus processing by-products, offer potential as cholesterol lowering agents to improve human health. Researchers in the Processed Foods Research Unit, Albany, CA are collaborating with the Western Regional Nutrition Research Center, Davis, CA to perform a human clinical feeding study funded by a trust agreement with an industrial partner. Large amounts of pure limonoid glucosides are being extracted using a patent pending method. The human study will begin by the end of fiscal year 2006 and will be the most comprehensive evaluation of limonoid glucosides ever. Validation of the cholesterol lowering properties should provide an economic incentive for the citrus processing industry to utilize their by-products as sources of valuable health promoting compounds. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.

4b.List other significant research accomplishment(s), if any.
Potential Antioxidant Activity of Limonoids. Biological activity of many naturally occurring compounds has been correlated with their ability to act as antioxidants. Recurring reports of citrus limonoid antioxidant activity are present in the literature. Researchers in the Processed Foods Research Unit, Albany, CA found unequivocal evidence that citrus limonoids do not act as antioxidants. This information will correct existing literature and support future development of more accurate analytical profiles for determination of the antioxidant activity of naturally occurring compounds. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.

4c.List significant activities that support special target populations.

4d.Progress report.

5.Describe the major accomplishments to date and their predicted or actual impact.
Biological Activity of Citrus Limonoid Glucosides. It has been shown that limonoid glucosides in citrus are active as anti-tumor agents, possess anti-infective properties and act as anti-feedants for insects. Researchers in the Processed Foods Research Unit, Albany, CA, in collaboration with researchers in the Western Regional Nutrition Research Center, Davis, CA discovered for the first time that citrus limonoids commonly present in orange juice are bioavailable to humans up to 24 hours after ingestion. This research supports citrus as an essential component in the human diet. Correlation of the biological activity of citrus limonoids to improving human health also provides incentives to the citrus industry to isolate these compounds from citrus processing by-products. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.

Manufacture of Limonoid Glucosides from Citrus Processing By-Products. Scientists in the Processed Foods Research Unit, Albany, CA, developed an improved method for the isolation of pure limonoid glucosides and metal salts of limonoid glucosides (patent application submitted). This method utilizes selective preparative chromatographic methods to purify limonoid glucosides present in citrus processing by-products including citrus molasses, peel wash, pulp wash and seeds. This process is currently the only method for the manufacture of pure limonoid glucosides from citrus sources. These compounds have been shown to have significant anti-tumor activity in in vitro human cancer cell line tests and have been shown to be bioavailable when fed to humans. They have high potential as nutraceuticals and chemopreventatives for the improvement of human health and the patent pending method is available for utilization by the citrus industry. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.

Method to Evaluate Citrus Juice Quality. A juice quality method for measuring the juices' potential of forming delayed-bitterness was developed by scientists in the Processed Foods Research Unit, Albany, CA. Freeze damage or physical damage to citrus fruit, including juicing, initiates the formation of the bitter dilactone limonoids from non-bitter monolactone precursors. This phenomenon is referred to as delayed bitterness and is a major problem for both fresh fruit and juice producers worldwide. We developed a rapid and sensitive method for the quantification of limonin and nomilin precursors to the development of bitterness. This method provides a valuable tool for citrus growers and juice producers to evaluate the susceptibility of a fruit or juice to delayed bitterness. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.

Extraction Methods for Analysis of Citrus Leaf Proteins. Human encroachment on land traditionally used for citrus cultivation, environmental changes, emerging diseases and pests, in addition to changing consumer preferences have driven both researchers and commercial producers of citrus to search for methodologies to reduce the time and financial costs in generating new citrus varieties. A proteomics approach using two-dimensional electrophoresis analysis in combination with mass spectrometry has the potential to be a powerful tool in the selection and evaluation of new varieties. However, realization of the full potential of two-dimensional electrophoresis (2-DE) separation of citrus proteins is dependent on good sample preparation. Researchers in the Processed Foods Research Unit, Albany, CA, have developed general procedures for the extraction of Citrus leaf proteins for analysis by 2-DE. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
The process for the isolation and purification of limonoids from citrus by-products has been established through this research. A patent for the isolation of limonoid glucosides from citrus by-products exists and an application for a second patent has been submitted. An Australian company is currently isolating a mixture of limonoid glucosides from a citrus processing by-product. This company has expressed interest in the isolation of pure limonoid glucosides from the same material and may be interested in licensing the newest patent. This technology transfer should lead to the first commercialization of limonoid glucosides obtained from citrus processing by-products.

Collaboration research involving WRRC/ARS and WHNRC/ARS has established the bioavailability of limonoids in humans. This research has led to an externally funded research program to examine the potential cholesterol-lowering effects of citrus limonoids in humans that will get underway in 2006. Technology transfer from these types of studies can be expected to have significant influence on the role of limonoids in human health and nutrition and in the degree of interest in the reclamation of limonoids from citrus processing by-products over the next two to three years.

Review Publications
A.P. Breksa III, G.D. Manners, 2006. Evaluation of the Antioxidant Capacity of Limonin, Nomilin and Limonin Glucoside. Journal of Agricultural and Food Chemistry. V54:3827-3831.

Manners, G.D., Breksa III, A.P. 2005. Manufacture of limonoid compounds. Patent Application, No. 11/289/240, June 1, 2006.

Last Modified: 10/22/2014
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