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United States Department of Agriculture

Agricultural Research Service



2005 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? What does it matter?
Developing new byproducts as outlets for the 5 million tons of waste residue generated annually by the Florida citrus processing industry is critical to industry health. With low profit margins from juice sales and increased competition from foreign imports, the revenue obtained by production of waste stream byproducts is becoming increasingly important for the Florida Citrus Industry to remain profitable. Currently, most citrus processing waste is dried to make citrus pulp pellets for use as cattle feed. Unfortunately, citrus pulp pellets have not had sufficient value to cover production and transportation costs for over six years. The cost and environmental impact of putting this waste in landfills eliminates this as an option if the citrus industry is to remain profitable and survive. The citrus waste stream contains numerous phytochemicals with demonstrated human health benefits, and large quantities of carbohydrates in the form of simple sugars, pectin and other polysaccharides. The development of new by-products from the citrus processing waste stream via integrated and economical new processes offers a tremendous opportunity to increase crop value. The investigators will characterize the structure and functional properties of the carbohydrate fraction. They will develop new and use existing procedures including chemical, physical or enzymatic processes to modify and produce polysaccharides, value added polymers, or resins with unique functional properties, either isolated or while still contained in the bulk waste stream residue. These new materials will be tested for use in industrial applications requiring metal chelating and ion-exchange materials, as additives to modify building and construction materials, as paper additives, and other non-food related applications. Economically viable and environmentally friendly processes will be stressed. Processing citrus peel waste into fuel ethanol, while recovering valuable co-products such as pectin, flavonoids (antioxidants) and limonene is also under investigation.

This work is relevant to citrus growers and citrus juice processors who will benefit by realizing increased value for their products. The impact of this project for the citrus industry is increased crop value, more competitive U.S. citrus products, and a reduction in biomass pollution and disposal problems. Other customers of the research are industrial manufacturers of building and construction materials, paper additives, and industries requiring materials for waste water remediation that are environmentally friendly. It is also possible that the applications developed by this project to produce new by-products from the citrus waste stream could be applied to other fruit and vegetable processing waste streams rich in polysaccharides, such as those produced by the apple and sugar beet industry. This project is 100% devoted to addressing Component 2 (New Processes, New Uses, and Value-Added Foods and Biobased Products) of NP 306, Quality and Utilization of Agricultural Products. We seek to enhance the value and competitiveness of the U.S. citrus industry by developing new by-products and industrial applications for existing and newly developed citrus by-products through the use of economical, environmentally benign, and efficient processing methods.

2.List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY 2005) 1. Demonstrate feasibility of ultrafiltration or an alternative separation system to isolate soluble sugars, pectin, or other carbohydrates from solid residues and initiate technology transfer. 2. Characterize pectins isolated from fresh peel and initiate technology transfer. 3. Determination of Pectin Methylesterase (PME) structural modification of pectin and accumulation of PME modified pectins.

4. Scale up on promising pectin modifications with chemicals to provide testing material to industry.

5. Further chemical modification of citrus peel materials and testing of new products.

Year 2 (FY 2006) 1. Further modifications on pectin, using enzymes, acid, base, and crosslinking of resins. 2. Complete studies on disaggregation using proteases and surface-active agents and continue technology transfer.

3. Further structural modification of pectin with PME, accumulation of PME modified pectins, and determination of their functional properties and technology transfer.

4. Scale up of pectin modifications, provide test materials to industry partners.

5. Continue modification of citrus peel and testing and initiate technology transfer.

Year 3 (FY 2007) 1. Continue with resin crosslinking studies and continue technology transfer.

2. Incorporate disaggregation improvements into isolation process and continue technology transfer.

3. Continue determination of pectin structural modifications and functional properties, continue scale up production of modified pectin materials, and provide test materials to industry partners.

4. Optimize modification of citrus peel and testing and initiate technology transfer

Year 4 (FY 2008) 1. Finish technology transfer with ion exchange, polymer, and resin product for industry

2. Finish technology transfer of processes and additive by-products for building products industry

3. Finish determination of functional properties of PME modified pectins and technology transfer

4. Complete physical properties testing of new products and finish technology transfer

4a.What was the single most significant accomplishment this past year?
Fractionation of pectin: A method was developed for preparation and fractionation of pectin into several weight classes (low, medium, and high degree of polymerization) of polygalacturonic acid. These fractions were incorporated into coatings and tested for their effect on decay organisms on strawberries and lychee fruits. The medium-range size class was found to inhibit the growth of decay organisms and to elicit the production of ethylene when applied to strawberries. This would play a role in the development of chemical-free treatments for fruit to prevent spoilage.

4b.List other significant accomplishments, if any.
Production of fuel ethanol from citrus peel waste on a commercial scale: In the commercial scale up for converting citrus waste carbohydrates to ethanol for use as a biofuel, feasibility for a process at the 100 gallon scale was demonstrated. The process involves enzyme hydrolysis of the complex carbohydrates to simple sugars, which are then fermented to ethanol. Conversion of citrus peel waste to ethanol would provide a more economic by-product than the current situation where peel is converted to low cost cattle feed. This would provide a local source of fuel ethanol, reduce dependence on foreign oil for balance of trade and fuel security and offer an octane enhancer replacement for methyl tertiary butyl ether (MTBE), which is being phased out in the U.S.

Combined use of evaporative and multi-angle light scattering detectors to determine molecular weight and charge of pectin molecules: A new analysis methodology for the study of pectins and other polysaccharides from peel was developed using an evaporative light scattering detector coupled with multi-angle light scattering detector. This new methodology permits molecular weight determination of large molecules under gradient conditions to determine molecular charge and size relationships for extracted pectins. The functional properties of pectins are mostly dependent on the charge and size characteristics of these molecules. Understanding the ionic charge/size properties is essential in the development on new products since these properties control many of the functional properties of pectins in industrial applications.

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

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
Methodology was developed and improved for quantification of individual galacturonic acid oliogmers separated from pectinaceous material. Latest developments allow separation of pectic fragments containing more than 70 galacturonic acid units. This procedure facilitates mapping of pectin structure and modifications made to pectin with treatment with enzymes and chemicals. It allows for determining functional properties related to introduced structural features.

A method was developed for preparative fractionation of polygalacturonic acid fragments into several weight classes (low, medium, and high degree of polymerization) of polygalacturonic acid.

Three of the four isozymes of PME present in citrus fruit peel have been prepared as monocomponent preparations and characterized for their physical and biochemical properties. Conditions have been described that favor activity of one isoform over the others which will allow for their use in tailoring the structural and functional properties of modified pectin or fruit peel fragments for industrial utilization.

Assay procedure for determination of the major component of citrus peel, pectin, was developed. This will assist in quantitation of this important component as new products and processes are developed for its modification and release from peel. This technique could be of value in all future products involving pectin in citrus peel.

Demonstrated non-calcium sensitive pectins could be isolated from washed orange peel in yields representing approximately 15% on a dry weight basis. This is approximately half of the total available pectin present in the peel. These pectins were extracted under relatively mild reaction conditions which simplify the process and aids in technology transfer.

A scalable process for the conversion of citrus waste sugars and polysaccharides into ethanol for use as a biofuel was developed and demonstrated at the 100 gallon process level. Limonene is also recovered during the process which is another valuable by-product with an already established market and helps make the process economically feasible in addition to reducing concerns about volatile organic emissions from citrus processing plants.

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?
CRADA with local industry partner continues who is interested in the commercial development to convert waste citrus carbohydrates into ethanol for use as a biofuel. It is expected the technology will become available towards the end of FY 2006.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Luzio, G.A. Extraction of Pectin for Industrial and Non-industrial Uses at Cargill Juice North America, 100 East 6th Street, Frostproof, FL, November, 2004.

Review Publications
Cameron, R.G., Grohmann, K. 2005. Separation, detection and quantification of galacturonic acid oligomers with a degree of polymerization greater than 50. Journal of Liquid Chromatography and Related Technologies. 28:559-570.

Cameron, R.G., Luzio, G.A., Grohmann, K. 2004. Characterization of esterified blocks in pectin homogalacturonan regions after de-esterification with the thermally tolerant pectin methylesterase from citrus fruit. Proceedings of Florida State Horticultural Society. 117:410-415.

Cameron, R.G., Savary, B.J., Hotchkiss, A.T., Fishman, M.L. 2005. Isolation, characterization and pectin modifying properties of a thermally tolerant pectin methylesterase from valencia oranges. Journal of Agricultural and Food Chemistry. 53:2255-2260.

Luzio, G.A. 2004. Determination of galacturonic acid content of pectin using a microtiter plate assay. Proceedings of Florida State Horticultural Society. 117:416-421.

Cameron, R.G., Grohmann, K. 2004. Mapping demethylated block size and distribution in pectin from citrus processing waste. Subtropical Technology Conference Proceedings. 55:43.

Wilkins, M.R. 2004. Citrus peel ethanol: florida's biofuel for the future. Subtropical Technology Conference Proceedings. 55:40-41.

Savary, B.J., Nunez, A., Cameron, R.G. Mass spectroscopy profiling of tryptic-peptides for identifying pectin methylesterases isolated from valencia orange [(citrus sinensis (l.) osbeck] fruit. Abstract 229TH NATIONAL MEETING, AMERICAN CHEMICAL SOCIETY, SAN DIEGO, CA., March 13-17, 2005. Cell 118.

Luzio, G.A. 2004. Investigation of galacturonic acid colorimetric assay of pectin in citrus peel. Subtropical Technology Conference Proceedings. 55:42.

Cameron, R.G., Luzio, G.A., Grohmann, K., Savary, B.J. 2005. Mode of action of two commercial pectolytic endo-polygalacturonases. National Meeting of Institute of Food Technologists/Food Expo. Paper No. 71B-3.

Last Modified: 4/20/2014
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