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