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Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 1 of 23

Enhancing prevention of heart disease and cancer using nutraceuticals derived from Washington Agriculture

A. Specific Aims Heart disease and cancer as the 1st and 2nd causes of death in the state not only are the major health threats to Washingtonians, but also accounted for a significant share of Washington State’s multibillion dollar Medicaid payments. The goal of this project is to explore a partial solution to the health threat and increasing healthcare cost problems by proving a cost effective, practical, sustainable, and win-win alternative. Building upon the fact that the most effective approach for combating disease is prevention, our concept is developing a strategy and associated technology to incorporate nutraceuticals that have proven cancer and cardiovascular disease prevention benefits to some common food items to increase the intake of these important nutrients by the citizens of Washington State thus ultimately improving their health. Our specific approach is to produce -3 polyunsaturated fatty acids (-3 PUFAs) and extract antioxidants. We can incorporate these essential fatty acids and phytochemicals into commonly consumed foods for convenient delivery in a typical diet. The essence of our efforts is to develop the necessary science and critical technology that constitutes a delivery platform for these xenobiotics to make the implementation of this approach possible. The major research objectives are: (1) exploring strategies to produce multiple PUFAs using agricultural by-products at minimum cost, (2) investigating novel separation processes to obtain antioxidants from low value by-products of fruit and vegetable processing, (3) investigating processes to preserve the nutritional values of these products, (4) developing food processing technologies to incorporate these nutraceuticals into various food items that are popular to targeted high risk populations with suboptimal dietary habits, (5) examining the stability and availabilities of these nutraceuticals during and after food processing as well as their release, dissolution and bioavailability, and (6) assessing the multiple economic benefits of this overall approach to the state. B. Background, Significance, and Relevance to LSDF Program Goals Health and health-care problems to be addressed and their significance in Washington State This project addresses problems related to heart disease and cancer and their prevention. In 2003 there were 222,000 population reported cases (PRC) of cancer in the state of Washington, including 23,000 cases of breast cancer, 8,000 colon cancer, 9,000 lung cancers, 20,000 prostate and 163,000 cases of other types. Total direct costs for caring for these patients amounted to almost $1 billion (DeVol and Bedroussian, 2007). In the US as a whole there were 10.6 million cases of cancer costing $48.1 billion dollars in direct care costs. Heart disease was even more prevalent and costly. In Washington State there were 302,000 population reported cases of the disease with direct care costs of $1.05 billion. For the US as a whole there were 19.2 million cases of heart disease with direct care costs of $64.7 billion dollars. These were only part of the total costs. Lost work days on the part of patients and caregivers had an economic impact of $5.7 billion dollars in Washington State for cancer, and $1.65 billion for heart disease. Thus, the direct and indirect costs from these two diseases totaled over $9 billion in 2003 in the State of Washington. By 2023 projections are that there will be 390,000 cases of cancer and 487,000 cases of heart disease in Washington State with total direct and indirect costs of almost $35 billion. According to the Centers for Disease Control and Prevention (CDC), the costs of heart disease and stroke in the U.S. is projected to be $448 billion in 2008, whereas cancer costs the nation an estimated $89 billion annually in direct medical costs (http://www.cdc.gov). The cost figure would be much higher if lost productivity from death and disability are counted (http://www.cdc.gov). Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 2 of 23

Current strategies for addressing the problems Although chronic diseases such as heart disease and cancer are among the most common and costly health problems, they are also among the most preventable (http://www.cdc.gov/nccdphp/). At the national level, the CDC’s National Center for Chronic Disease Prevention and Health Promotion is at the forefront of the nation's efforts to prevent and control chronic diseases. Promoting healthy behaviors through education and advocating early detections through screening are among the most important strategies of the center. According to CDC, “adopting healthy behaviors such as eating nutritious foods, being physically active and avoiding tobacco use can prevent or control the devastating effects of these diseases”. Approaches other than that being proposed for alleviating the problem Recognizing the importance of health diet to the health of citizens, the government has made great effort in providing information and promoting health education. A good example is the establishment of the Center for Nutrition Policy and Promotion (http://www.mypyramid.gov/global_nav/about.html), an organization of the U.S. Department of Agriculture to improve the nutrition and well-being of Americans. This center advances and promotes dietary guidance for all Americans. The Guidelines provide authoritative advice for people regarding good dietary habits which can promote health and reduce risk for major chronic diseases. They serve as the basis for Federal food and nutrition education programs. Although these guidelines and recommendations are well established, their implementation in American’s daily dietary practice still remains the major challenge to reach the optimal benefit as it has been difficult to enable people to change their food consumption patterns to meet the levels required for protective health benefits. For example, in 2005 only 25 percent of adults Washington State consumed the recommended 5 or more servings a day of fruits and vegetables that provide these nutrients (http://apps.nccd.cdc.gov). Moreover, economic analysis has demonstrated that individuals under invest in self-protection when it comes to health (Wu, 2003; Byrne, et. al., 2001; Finkelstein, et. al., 2006), a situation that is exacerbated by current policies for financing government subsidized healthcare. In fact, recent analysis shows that using tax dollars collected primarily from higher income groups to subsidize health care for noninfectious diseases that could be avoided by self-protective behavior (for lower income people cannot afford the care themselves) will lower investment in self-protection for both higher income and lower income groups (Rosenman, 2008). As a result, innovative interventions that lower the cost are needed to promote self-protecting behavior. The economic modeling in Rosenman (2008) shows those policies promoting nutraceuticals may be just the sort of intervention needed to increase self- protecting behavior among both higher and lower income groups. The rationale of the proposed research leads to a possible solution for these problems Our research provides another avenue to complement the dietary guidelines by supplementing some of the important nutritional elements contained in the healthy food to the popular food items so that a person can obtain them without eating the foods that are not in their regular diet habit. We will focus on (-3 PUFAs) that are contained in fish and antioxidants contained in fruits and vegetables as they are among the most important categories.  -3 polyunsaturated fatty acids – beneficial health effects Polyunsaturated fatty acids (PUFAs) are essential components of eukaryotes, well known are their properties of flexibility, fluidity and selective permeability to membranes. Eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) are the two important -3 PUFAs. Nutritional and clinical studies have shown that -3 PUFAs are important nutraceuticals for enhancing human health and preventing human diseases (Nettleton, 1995). The inclusion of supplementary DHA in infant formulas is strongly recommended by the World Health Organization, and such products have Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 3 of 23 been made available on the infant formula market. In September 8, 2004, the U.S. Food and Drug Administration gave "qualified health claim" status to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ω−3 fatty acids, stating that "supportive but not conclusive research shows that consumption of EPA and DHA ω−3 fatty acids may reduce the risk of coronary heart disease"(United States FDA, 2004). People with certain circulatory problems, such as varicose veins, benefit from fish oil. Fish oil stimulates blood circulation, increases the breakdown of fibrin, a compound involved in clot and scar formation, and additionally has been shown to reduce blood pressure (Morris et al, 1993; Mori et al, 1993). There is strong scientific evidence, that ω−3 fatty acids significantly reduce blood triglyceride levels (Harris, 1997; Sanders, et al, 1997), and regular intake reduces the risk of secondary and primary heart attacks (Bucher et al, 2002; Burr et al, 1994). Some benefits have also been reported in conditions such as rheumatoid arthritis (Fortin et al, 1995; Kremer et al, 1985) and cardiac arrhythmias after PUFA consumption (Christensen et al, 1996). Sources of  -3 PUFA The conventional source of -3 PUFAs is mostly from fish oil. The purification of DHA/EPA from low-grade fish oil is difficult and costly. In addition, the declining marine fish stocks are further subjected to seasonal and climatic variations (Gill and Valivety, 1997), and may not meet the increasing demands of DHA/EPA. Moreover, it is reported that fish, like humans, are not capable of synthesizing PUFA de novo. Much of their PUFA is derived from the primary producer in the oceanic environment: the microalgae or algae-like microorganisms. Polyphenols and health benefits Polyphenols are a group of secondary plant metabolites derived from D-glucose found in plants, characterized by the presence of more than one phenol unit or building block per molecule. Research indicates that polyphenols may reduce the risk of cardiovascular disease and cancer as antioxidants. Polyphenols have also been investigated as a source of additional health benefit in organic produce (Roupe et al. 2006), Grape pomace, for example, contains such polyphenolic compounds (Pohl et al., 2006; Yoon et al., 2007). These polyphenols can be used as food preservatives and dietary supplements because they have antioxidant activities and the ability to scavenge free radicals from the body, thus potentially reducing their negative impacts on human health. Well-known epidemiological studies indicate the protective effects of red wine consumption in reducing cardiovascular diseases. The effects are strongly correlated to the presence of polyphenols in wine. These polyphenols are also contained within grape wastes such as the pomace being studied in this project proposal. Proposed research components and integration This multi-disciplinary project consists of six components each of which corresponds to a specific objective described earlier. In the first part of our study, we will develop a cost effective technology to produce -3 PUFAs (DHA&EPA) using microalgae. The algae will be fed with organic carbon from agricultural by-products such as cull potatoes which accounts for about 10% of total potato production that does not meet the standard of processers thus has a much lower value. The second component will focus on investigating Figure 1. The structure of this multi-disciplinary project a more cost effective process for extracting Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 4 of 23 antioxidants from fruit processing by-products, such as grape and apple pomace. In the third component, processing technology will be developed for protecting antioxidants from loss of activity. The fourth research part will develop processes that incorporating these protected -3 PUFAs and antioxidants into food products that are popular among the general public, especially groups with high risk for heart disease and cancer. In the fifth research part, we will assess the activities of these ingredients to provide feedback to the production and processing research activities to make sure that they are optimized for efficacy. In the sixth research component, we will conduct a comprehensive economic analysis on the cost effectiveness of the approach and identify main cost barriers to be further addressed. The integration of these components is illustrated in Figure 1. We envision making this approach a reality in two phases. This project is the first phase that will be completed within three years during which the critical scientific knowledge will be generated and key technology developed and tested. The second phase will be additional 2-3 years during which pilot tests and commercialization will be the focus. Relevance to LSDF program goals This project responds to the primary strategic goals of LSDF – “promoting life sciences competitiveness, improving health and health care for Washington’s citizens, and fostering economic growth.” The proposed project fits well with the LSDF program in all three criteria: Scientific and Technical Merit, Importance to Health and Health Care and Future economic return. From a scientific and technical perspective, the project is innovative in that (1) the overall concept proposes a new approach for providing essential fatty acids and antioxidants in the diet to enhance prevention of heart disease and cancer; (2) each specific component individually fills related scientific and technological gaps, and (3) the integration of these components establishes a research framework with strong potential to achieve novel and important results in the form of new food products. This project will fill important gaps in our knowledge of micronutrient composition, the effect of processing on micronutrient content, and the potential anti-cancer activity properties of processed fruit products. The few very recent studies that exist provide limited information on profiles of phytochemicals for processed foods. Unfortunately, there are no well-controlled studies on how processing methods affect the level and biological activity of these nutritive compounds in fruit products. Furthermore, reported studies generally do not test products reflective of commercial practice, making any data that may be available of limited use to growers and processors. More specifically, the scientific contributions of this project will include (1) discovery of mechanisms and strategies for high rate of cell growth and -3 PUFAs production; (2) assessing and developing new technologies to extract antioxidants from fruit processing by-products; (3) generating new knowledge on chemical behavior of nutraceuticals under various conditions of food processing, storage and delivery, (4) obtaining new information on stability and rate and extent of absorption after delivery, and (5) testing economic models for economic benefits from developing a nutraceutical industry for improving health of the citizens and reducing health care costs of the state. In addition, this project builds upon solid research experience of the team members with supporting preliminary results. Thus, it is both feasible highly likely to be executed successfully. The project directly addresses the No. 1 and No. 2 cause of death in the state by improving heart disease and cancer prevention. It builds on the agriculture strength of Washington State and the unique research capacity of Washington State University and has the potential to make a substantial, beneficial and measurable contribution to improving health and health care in Washington. Moreover, the project develops strategies to accomplish these health and health-care benefits in a more cost-effective manner as it capitalizes on the food system. Besides heart disease and cancer, these nutraceutically enhanced common food items will Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 5 of 23 improve general public health, resulting in a broad impact to a great number of Washington States citizens. The proposed project will benefit Washington’s economy in a multiple ways. First, the availability of these nutraceuticals will reduce the chance of disease and associated costs; Secondly, converting and processing by-products to nutraceuticals and high value foods will enable farmers to benefit economically; Third, the conversion and processing research will create intellectual property that presents attractive licensing opportunities and new business opportunities, creating new companies and jobs and attracting investment capital to Washington. This project will also produce great positive impact and is thus strongly supported by the Washington industry (see the attached letters). The current emphasis on functional foods is driving much of the development of new and high value products from produce. According to Nutrition Business Journal, 2006 total antioxidant sales in the U.S. were $3 billion, representing 4.5% growth from the year before. Last year, the majority of growth came from sales of herbal/botanical antioxidants, which shot up 43% in food, drug and mass market channels. The state of Washington is a leading producer of tree fruits and small fruits that are rich in polyphenols. A vast majority of the fruits such as apple are discarded due to sun scald. Juice and wine manufacturing industry also produces apple and grape pomace which is not utilized effectively. There exits opportunities to extract phytochemicals from discarded and low value raw material and incorporate these essential phytochemicals into commonly consumed foods for delivery in the diet. Tuning our agricultural and food processing industry for the growing nutraceutical market not only can benefit the health of the citizens of the State, but also offers an exciting opportunity for growth and development of the industries. In addition, this proposing project for producing essential nutraceuticals with cheap feedstock could provide great benefits to the citizens of the State for job creation, and rural development for income increase to farmers. For instance, more than 55,000 tons of grape waste pomace is annually produced, costing growers and processors about $2 million per year to properly dispose of the pomace in the State of Washington (Washington grape report, 2005). Research is ongoing for industrial application of the pomace; including use as an animal feed and in the production of yeast and grape dyes, however these are all low value-added products compared to polyphenols and their use as a nutraceutical. Importance of LSDF funding to accomplish the work being proposed The LSDF funding is critical to the research work proposed in two major aspects: First, the success of proposed work requires a comprehensive scope that needs multidisciplinary expertise. As a result, the funding amount requested is beyond most federal grant programs. Second, it strengthens future life sciences research of this group of WSU scientists. Due to the lack of such large-sized grant programs, the investigators have never had an opportunity to put such an integrated project together. Consequently, although each individual PI has a very well established program and is competitive within a specific area, collectively, there has little prior record of collaboration among the team members. Funding this project will provide the opportunity for the research team to synergize their strength centered on the delivery of the project goal. The opportunity of working collaboratively will not only make their research effort more productive, but will also greatly enhance collective competitiveness at a national level. C. Preliminary Studies Significant amount of related work has been done within the laboratory (or group) of each individual PI. In the area of -3 PUFAs production (Research Component #1), DHA production by culturing marine algae Schizochytrium was studied by Dr. Zhanyou Chi in Dr. Chen’s lab. A Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 6 of 23 two-stage culture technology was developed first to increase cell number then cell size by manipulating the culture conditions in the two distinct stages. A culture procedure with high cell density and effective yields and productivities within a relatively short fermentation period (5- day) produced 140 g/L algae biomass, consisting of 40-50% fat with nearly 50% and 40% being PUFA and DHA, respectively (Chi et al, 2007a; 2007b; 2008). Objective#2, based on the hypothesis that low temperature extraction with economic yields and efficiency is possible if particular parameters are altered, such as allowing for enhanced polarity and removal efficiency created by adding a water soluble compound, preliminary results obtained by Dr. Tianxi Zhang have demonstrated that by utilizing a moderated ethanol/water mixture as the solvent and controlling its ratio about 6.5 g of polyphenols can be extracted from 100 g of grape seeds using a 50% ethanol solution at 35oC. The 50% ethanol solution achieves the maximum of polyphenol extraction in comparison with various ethanol concentrations of 10-95%. The difference of ethanol concentrations suggests change of solution polarity that affects the extraction efficiency. Objective#3, Dr. Sablani has considerable experience in production of shelf stable nutraceutical powders (Sablani et al., 2007 and 2008). Dr. Sablani and coworkers have explored the application of glass-transition concept to evaluate physicochemical stability of nutraceutical powders. It was demonstrated that by proper controlling of glass-transition temperature, a shelf- stable fortified powder can be produced (Sablani et al., 2007). Microencapsulation of dried active sugar-rich powder in appropriate concentration of carrier/wall material can improve physical stability (i.e. stickiness) during storage (Sablani et al., 2008). Objective#4, Dr. Tang’s group documented that the novel Refractance Window (RW) drying technology developed by MCD Technologies in Tacoma, WA retains high level of anti- oxidant activities in green asparagus (Nindo et al., 2003, 2007). Dr. Tang’s group has also documented in a previous quality study that this drying method results in high retention of β- carotene in carrots and vitamin C in strawberries with bright natural colors which are highly desirable for use as ingredients in other food products (Abonyi, et al., 2002). Over the past seven years, Dr. Tang’s group has successfully used extrusion technology to produce breakfast type cereals and snack foods using proteins from legumes, starch from potato and dietary fiber from apple and wheat. Objective#5, Dr. Neal Davies has studied the effect of various drying methods on anti- oxidant levels in raspberries and blueberries which could ultimately result in freeze-dried fruit at half the cost to consumers. We have expertise in previously validating assays for several polyphenols according to International Harmonization Criteria. (Yanez et al. 2005ab; Yanez et al. 2007ab; Remsberg et al. 2008) Objective#6, Dr. Rosenman recently conducted an analysis showing that using tax dollars collected primarily from higher income groups to subsidize health care for non-infectious diseases that could be avoided by self-protective behavior (for lower income people cannot afford the care themselves) will lower investment in self-protection for both higher income and lower income groups (Rosenman, 2008). As a result, innovative interventions that lower the cost are needed to promote self-protecting behavior. Additionally, Dr. Rosenman’s economic modeling indicates that policies promoting nutraceuticals may be just the sort of intervention needed to increase self-protecting behavior among both higher and lower income groups. D. Research Design and Methods Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 7 of 23

Objective 1. Produce Omega-3 from agricultural by-products–develop a highly productive process for producing both EPA and DHA. 1.1. Background EPA synthesis -3 PUFA biosynthesis consists of a series of biochemical reactions, and it can be divided into two steps. To date, all the enzymes that are involved in the EPA biosynthesis have been identified from various lower eukaryotic species (Pereira et al., 2004). Two pathways have been involved in EPA biosynthesis. The omega-3 pathway leading to EPA production is most common to eukaryotes, and a few lower eukaryotes can use an alternative pathway, using elongase and desaturase. In addition, in some lower eukaryotes, the omega-6 intermediates may be converted into ω-3 fatty acids through a ω-3 desaturase, leading to EPA production. On the basis of the previously defined EPA biosynthetic gene clusters, EPA biosynthetic gene clusters have significant matches to numerous multifunctional enzyme complexes involved in such processes as polyketide antibiotic synthesis (Pfeifer & Khosla, 2001), eukaryotic fatty acid synthesis (Beaudoin et al., 2000; Parker-Barnes et al., 2000) and heterocyst glycolipid synthesis (Campbell et al., 1997). DHA/EPA production by microalgae - cultivation systems Only a few species have demonstrated production potentials on an industrial scale (Alonso and Maroto, 2000), mainly due to the low specific growth rates and low cell density of the algae, as they could only grow, in many cases, in photoautotrophic conditions. Heterotrophic culturing is an alternative to the photoautotrophic culture of microalgae. This culture mode eliminates light limitation and therefore, offers the possibility of greatly increasing cell density. Furthermore, the cost of harvesting and purification can be reduced due to the high cell density obtained. Consequently, intensive research into the production capabilities of these microalgae led researchers and commercial industries to focus on and develop heterotrophic algal production processes for DHA. EPA production from heterotrophic algal culture The perfusion-cell bleeding culture allowed a high biomass productivity and EPA productivity, 175 mg/ l/ day, which is the highest ever reported (Wen & Chen, 2001). However, compared with DHA production by Schizochytrium, which could produce DHA with a rate of 0.5 g/L/hr, the productivity of EPA is too low to be industrialized. It should be noted that EPA can be produced from some strains of Schizochytrium sp. such as Schizochytrium limacinum SR 21 with a small amount, whereas other strains, such as Schizochytrium ATCC 20890, can produce 18.9% of total lipids as EPA, and 43.5% as DHA (Barclay, et al, 1997). If these strains with high EPA content could be cultured in a high cell density mode, like DHA producers did, it would be feasible for the EPA production to be industrialized. Engineering algae for the expression of PUFAs such as EPA and DHA may require expression of several separate enzymes to achieve synthesis. 1.2. Research aims Only the research on EPA production improvement will be conducted in this work, since the DHA production process has been developed in our previous work, and its scaling up study is undergoing. The research aims of this component include three parts. The first part is to scientifically identify the EPA synthesis pathway in Schizochitrium; The second part is to use the pathway information to optimize the culture conditions that enhance the EPA synthesis according to such a pathway; The third part is to technically develop a heterotrophic culture process to produce high DHA and/or EPA content algae with high productivity. 1.3. Experimental design and procedures Task 1.1. Determining which pathway for EPA synthesis in Schizochitrium Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 8 of 23

Although separate biosynthetic pathway for DHA and EPA in algal strains have been predicted (Pereira et al., 2004) true biosynthetic pathway for the compounds has not been illustrated yet. Whole genome sequence of the algae strain can provide insight into the pathway involved in EPA production. In this research, whole genome sequence of Schizotrichium (ATCC 20890) will be completed first by Roche Diagnostics Corporation (Indianapolis, IN), a company providing genome sequencing service. Then, computer based analysis of whole genome sequence data will be employed for the identification EPA biosynthetic cluster. The results will provide information on the possible involvement of biosynthetic genes responsible for EPA production. Genetic characterization of possible regulators, desaturases, elongases, dehydrogenases will help to determine the functional role of these genes related to EPA production. We will use the information to optimize culture conditions that would enhance the expression of these genes. The regulators will be focused which regulate the biosynthesis of EPA. The regulators can be expressed in the presence of particular nutrient source which would be easier and more efficient after elucidating the regulators in the gene cluster. Additionally, the promoter region of the regulators or the essential gene for EPA biosynthesis, such as desaturase and elongase could be in association with particular carbon, nitrogen or other elemental sources. Task 1.2. The optimization of cultural conditions for the algal EPA production by the Schizochytrium ATCC20890 will be optimized in terms of carbon source concentration, nitrogen sources and its concentration, linseed oil, and various salt concentrations, as well as the temperature, pH, and dissolved oxygen in the culture. The experimental design includes three major components: (1) selection of significant factors influencing cell reproduction by a Plackett-Burman design; (2) Optimization of the significant factors by a central composite design; and (3) verification of optimal conditions. The Plackett-Burman (PB) design will be used as a tool for screening for the most important factors from a variety of candidates. The algae cell density will be the responses (Chi et al, 2007b). . Then, a central composite design (CCD) will be used to optimize the levels of these variables. Based on the central composite design results, a second-order polynomial model will be calculated by nonlinear regression with Design-Expert software. The optimal value for each variable will be obtained with this model for the highest cell density. The verification experiments will be conducted according to the optimal value of the predicted model. Task 1.3. Developing a high yield EPA production process culture As the optimal culture conditions for algal growth and EPA formation are different, the optimal culture condition for EPA production will be studied according to the similar process used in our preliminary work for DHA production. The dry cell weight, EPA yield and EPA content will be used as responses in these statistical experiments. Based on the data obtained, the model predicts the optimal conditions for EPA production will be established, and the optimal culture conditions identified. All of the optimization experiments will be conducted in shake-flask cultures. Once the optimal conditions are obtained, a strategy for shifting fed-batch culture conditions will be tested in a 5-L fermentor in order to achieve high EPA production levels. Dissolved oxygen and pH effects will be investigated to produce higher cell density in the first stage, and provide better culture conditions for higher EPA production than in the flask. Further investigation will be conducted to determine the optimal feeding strategy, since high carbon source concentration and low nitrogen source concentration are superior conditions for lipid production for algae. A variety of carbon to nitrogen ratios will be investigated, to determine the most optimal C/N ratio. Objective 2. Extracting polyphenols from fruit processing wastes 2.1. Technical background and research aims Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 9 of 23

Polyphenol extraction is presently accomplished using organic solvents or supercritical fluid processing, or water extraction at high temperature (Pinelo et al, 2005; Palenzuela et al, 2004; Shrikhande et al, 2003). The supercritical fluid extraction requires expensive equipment due to high pressure of the process. Within the organic solvent approach, particular polar solvents have been used to extract the polyphenols. Of these solvents, methanol stands out as the most selective for polyphenol extraction (1); however, it along with many of the other solvents is easily flammable and quite volatile with the remaining alternatives other than water being undesirable because of their toxicity. The current water extraction techniques use pure water at high temperature (~100oC) and with a long contact time of several hours to days (Shrikhande et al, 2003), which requires high energy input and low efficiency of the extraction. 2.2. Research aims The research aim of this component is to develop a cost-effective and environmentally benign process of extracting antioxidants from agricultural by-products. We will use low temperature aqueous extraction of the polyphenols through the innovative insertion of water soluble species, such as polymers. The working hypothesis is that low temperature extraction with high yields and efficiency is possible because of the innovative additives within the aqueous solution. It is believed that although water alone can act as an extractor, its yield will be quite low. Enhanced polarity created by mixing water with poly (ethylene glycol) (PEG) will improve the yield and selectivity. PEG is being considered because of its high affinity interaction with polyphenols, and its polar properties as well as its low-toxicity and non-flammability. The compounds tested for the extraction will meet FDA safety requirements suitable for foods. This research component includes three specific tasks: (1) enhancing polyphenol extraction with the addition of water-soluble species; (2) designing an innovative polyphenol extraction process; and (3) integrating the extraction and purification processes and demonstrating a pilot study. 2.3 Research tasks and methodology Task 2.1. Enhancement of polyphenol extraction with the addition of water-soluble species This part of the research will focus on selection of the best polar polymers to enhance polyphenol extraction. Experiments will be performed by varying the molecular weights and concentration of PEG to ascertain their effect on the optimization of the extraction process. Additionally, since the PEG solutions may be reused after the downstream polyphenol isolation, studies will conducted to determine the effect of using recycled PEG on the process; either through the stir tank reactor method or through the fluidized immobilized polymer containing method. The extraction efficiency and process selectivity will be evaluated by product yield and purity. The total polyphenolic compounds and specific polyphenols in pomace from different origins (such as red or white grapes) will be analyzed by Folin-Ciocalteau reagent and HPLC methods (Rice, 1976), respectively. The pomace with the highest content of polyphenolic compounds will be used in the extraction experiments. Task 2.2. Design of a polyphenol extraction process and comparison of two reactor scenarios Two types of extractors, a stirred tank extractor and a fluidized extractor, will be investigated. The stir extractor is the traditional and widely used reactor with flexible operation and ease of scale up. The fluidized extractor, though, has the advantages of low energy consumption and as has been seen to offer a system whereby the immobilized polymer can be incorporated. Regardless of the type of extractor reactor used, the polyphenol inside the grape pomace will be transferred into aqueous solution and thus mass transfer becomes one of the most important factors for enhancing extraction efficiency. The focus of this part of the research then will be determining the stir rate and configurations of both the stir and fluidized extractors for optimum mass transfer and polyphenol solubility. The processes will be compared for performance, cost, Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 10 of 23 reliability and effect on downstream isolation with the better of the two being optimized for pilot study. Task 2.3. Process integration and a pilot study for demonstration The results obtained from the above studies will be integrated into a process of polyphenol extraction and purification. The selected extractor system will be scaled up to a pilot study (e.g. 25 liters) for demonstration. The re-optimization of a continuous process through discerning a relationship between polyphenol content and biological activity, and associated economic analysis are of main interest from an industrial point of view and will be completed within this task. The final antioxidant product in a powder form will be isolated and purified by an adsorbent resin to meet food purity requirements. The products obtained will be compared with commercial products on purity, activity, and cost. The process cost for each reactor will also be evaluated by economic analysis. Objective 3. Microencapsulation for storage and processing 3.1. Technical background Microencapsulation (ME) is defined as ‘the technology of packaging solid, liquid and gaseous materials in small capsules that release their contents at controlled rates over prolonged periods of time’ (Champagne and Fustier, 2007). Industrial applications for this technique are increasing because the encapsulated materials can be protected from moisture, heat, oxygen or other extreme conditions, thus enhancing their stability and maintaining viability (Jimenez et al., 2004). The industrial production of nutraceuticals will require the addition of functional ingredients to control its release and preservation properties. Adding nutraceuticals to functional foods presents many challenges, particularly with respect to their stability during processing and storage and the need to prevent undesirable interactions with the carrier food matrix. In this respect, the ME of nutraceuticals could help to address some of these problems. A spray-drying process is most commonly used method to encapsulate food ingredients such as flavors, lipids, carotenoids and bioactive molecules. However, many factors during spray-drying microencapsulation process must be optimized due to the complexity of the heat and mass transfer phenomena that take place during the microcapsule formation. Fats, starches, dextrins, alginates, protein and lipid materials can be employed as encapsulating materials. In spite of the recent developments of the spray-drying technique, the process remains far from completely being controlled. There are no clear guidelines for making the choice of encapsulation materials. Further study is needed of the various types of molecular interactions: water/wall, water/core, and wall/core (Gharsallaoui and Roudaut, 2007). 3.2. Research aims The overall research goal of this part of the study is to microencapsulate the polyphenols extracted from biomass such as apple and grape pomace using spray drying technology and determine the functionality and physicochemical stability of the encapsulated powder. Specifically, the research topics include optimization of microencapsulation process parameters including types of wall/carrier material, in feed solid concentration and spray drying temperatures, and evaluation of influence of storage conditions such as temperature, relative humidity and time on the anti-oxidant stability of encapsulated polyphenols.. 3.2. Research tasks and methodology Task 3.1. Material selection The criteria for selecting a wall/carrier material for encapsulating are mainly based on the physico-chemical properties such as solubility; molecular weight; glass/melting transition; crystallinity; diffusibility; film forming and emulsifying properties. The wall system is designed to protect core material from factors that may cause its deterioration, to prevent a premature interaction between the core material and other ingredients, to limit Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 11 of 23 diffusion of gases, and also to allow controlled or sustained release under desired conditions (Shahidi & Han, 1993). Modified starch, whey protein concentrate and maltodextrin (10–30 DE) will be investigated as wall materials for encapsulation of polyphenols. Task 3.2. Preparation of the emulsion and microencapsulated powder The hydrated solution of wall material with concentration of 20, 30, and 40% (w/w) will be prepared using distilled water. Coarse emulsions will be prepared by blending polyphenols and the prepared wall solution using a rotor-stator blender. The feed mixtures will be dried in a spray drier (APV, Denmark) equipped with wheel/nozzle atomizer. The dryer will be operated at different combination of air inlet/outlet temperatures (inlet temperature 180 to 200oC and outlet temperature 60 to 80oC) and air humidity 65 to 70%. The spray dry powders collected will be stored in dark glass bottles with tight caps at -20oC until further analysis. Task 3.3. Characterization of microencapsulated powder A JEOL (model 6400, Akishima, Tokyo, Japan) scanning electron microscope will be used to investigate the outer and internal microstructure of spray dried encapsulated powders. The stability of polyphenols during storage will be influenced by type wall/carrier material due to physicochemical interaction between wall/carrier and polyphenols. This interaction will be studied using molecular mobility and glass- transition concepts. The glass-transition temperature of encapsulated powders will be determined using analytical technique of differential scanning calorimetry (Q2000, TA Instrument, New Castle, DE). Task 3.4. Stability of polyphenols and antioxidant activity Encapsulated powders will be analyzed for content of polyphenols and its anti-oxidant activity using the method described in sections 5.1 and 5.2. The encapsulated and unencapsulated powders packed in brown bottles with screw caps will be placed at 4oC and 25oC and relative humidity varying from 10 to 85% to determine the influence of storage conditions on polyphenols antioxidant activity. Degradation of antioxidant activity will be followed for 6 months of storage and it will be analyzed weekly. 3.4 Data analysis Response surface methodology will be used to study the influence of types of wall materials (modified starch+maltodextrin and whey protein concentrate+maltodextrins), feed concentration, inlet and outlet air temperatures. The purpose of this analysis will be to identify combination of process condition to optimize retention of antioxidant activity in encapsulated powder. The stability data will be correlated with glass-transition temperature to develop a stability map of encapsulated powders. This model will be useful in evaluating the shelf life of encapsulated powder. Objective 4. Processing technologies for conversion into foods 4.1. Technical background There has been a conventional belief that processed fruits and vegetables deplete antioxidants in foods. This idea is derived from research focused only on selected and unstable antioxidants like vitamin C (Miller et al., 1995). Literature also demonstrated the thermal degradation of enzymatic or chemical oxidation of vitamin C during blanching, dehydration or heating with few a paucity of data on polyphenols and other compounds contributing to anti-oxidant activities (Lathrop & Leung, 1980; Van den Broeck et al., 1998). Foods contain a mix of phytochemicals (natural anti-oxidants) and other bioactive compounds that contribute to overall anti-oxidant activities. Eberhardt et al. (2000) found less than 0.4 % of total anti-oxidant activities from vitamin C compared to other phytochemicals that contribute most of the activities. Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 12 of 23

Many investigators have showed retention of anti-oxidant activities in processed fruits and vegetables (Eriksson & Na, 1995). Very limited data are available on the effect of heat processing on omega-3 fatty acids. Lytle et al., (1992) reported that air, light and heat rapidly oxidized and chemically modified omega-3 fatty acids. However, the investigators also demonstrated stability by preventing oxidation in an animal diet with 16% refined fish oil and 4% corn oil. Alamed et al., (2006) reports that the presence of ETDA (chelating agent acts as antioxidant) in oil-in-water emulsions containing omega-3 fatty acid reduces lipid oxidation. Our recent studies also reveal that thermal processing salmon fillets in sealed containers (in anaerobic conditions) does not cause measurable losses of omega-3 fatty acids (Kong et al., 2008). In order to maximize retention of bioactive elements in foods, we choose two main food processing technologies to convert or incorporate ingredients into convenient foods for direct human consumption. These two technologies are novel drying and extrusion cooking, both have a broad range applications for agricultural products produced in the state of Washington. High quality, shelf stability, and the need to retain antioxidant activity of foods or extracts used as nutritional supplements have become an increasingly important consideration in the design and operation of dryers. MCD Technologies Inc. (Tacoma, WA) has recently developed, patented and commercialized a novel Refractance Window® drying technology. In the operation of a RW dryer, liquid or semi-liquid foods are applied in a thin film onto a plastic belt that moves over a hot water flume. The thermal energy is transferred from the hot water through the belt to remove moisture, the temperature of the product is brought to below the glass transition temperature over a cooling section, and the dried product is removed from the belt with a knife scraper. Due to the favorable conditions and short duration, this technology has demonstrated potential to produce dehydrated foods and human nutritional supplements with very high quality (Nindo et al., 2007). In addition, this technology has better energy efficiency than hot air, freeze drying and spray drying (Nindo et al., 2004). Extrusion technology has gained popularity over the last two decades in food industry because of its versatility, lower processing costs, high productivity and improved product quality. Extrusion cooking is the mostly used technology in production of crisp breads, breakfast cereals, biscuits and puffed snack food products. It combines several unit operations, including mixing, kneading, shearing and cooking under high temperature (~150oC), short time (30-90s) and high pressure (up to 17MPa), and high pressure puffing, and offers a palette of processing conditions (temperatures, moisture, shear and screw speed) from which new food products can be created. Most importantly, the processes in the extrusion take place in an anaerobic condition. In conventional processes, chemical and enzymatic oxidations were mostly responsible for degradation of anti-oxidants in food matrices. Extrusion kinetics of enzymatic and chemical oxidations depends on processing conditions such as oxygen availability, time, temperature, pH and water activity. Grela et al., (1999) reported lower content of polyenoic acids and natural antioxidants (alpha-tocopherol, gamma-tocopherol, beta-carotene and lutine) during moistening and extrusion conditions. Athar et al. (2006) recommended short term high temperature cooking of extruded snacks allowing retention of higher level of vitamin B. Research aims Our knowledge of the degradation kinetics of antioxidant and omega-3 fatty acids during processing of drying, extrusion, and storage is of paramount importance. In order to design processes for improved retention of antioxidant activities in final products, there is a need to understand the changes of -3 PUFAs and antioxidant activities during those processes and consequent storage. Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 13 of 23

In the proposed project, we will study the kinetic changes of -3 PUFAs and antioxidant activities during those processes and develop unique and economical viable processes for production of consumer favored products with highest retention of health promoting functional activities for high risk population groups and the general population. 4.2. Research tasks and methodology Task 4.1. Investigate drying technology for processing nutraceuticals In conventional drying methods, dried foods are fully exposed to oxygen, which is prone to inducing loss of antioxidant activities and oxidation of -3 PUFAs. In Refractance Window drying, thermal energy is supplied through the belt from heated water. It is our hypothesis that water vapor from product surface serves as a protective level against oxygen, thus significant reduces oxidation of the food products. We will study kinetic changes in anti-oxidant activities and -3 PUFAs during Refractance Window drying, and select processing parameters to produce high quality ingredients with high level of anti-oxidants and natural color. In experiments, we will conduct experiments using the pilot-scale unit (5 kg/hr capacity) at MCD Technologies in Tacoma, WA (see attached support letter). We will evaluate the influence of commercial drum drying, freeze drying and other drying methods, using pilot scale unit in WSU Food Processing Pilot Plant, to process similar raw materials to 4-5% moisture content for comparison of the impact on retention of antioxidant activities and oxidation of -3 PUFAs to allow processors make appropriate decisions regarding the choice of drying technology in commercial production. Task 4.2. Extrusion cooking for incorporating nutraceuticals into food We will study the effect of operating parameters on the retention of anti-oxidant activities of the extruded products, and obtain system parameters for further scaling-up the technology. Experiments for food grade products will be conducted using a Clextral EVOL HT32-H twin-screw extruder at USDA ARS Western Regional Research Center (WRRC), Albany, CA. Other extrusion tests will be conducted in WSU Wood Labs. Based on our experience with previous projects, we will use external heating to maintain barrel temperature in five consecutive sections from 80oC (inlet) to 140oC (outlet), chose 500 rpm for the twine screws, 16-18% moisture content (wet basis) for the conditioned ingredients, and a final product moisture content of 6-7% for shelf-stability. Triplicate tests will conducted on a 5kg/h pilot scale extrusion unit, the samples will be sealed in nitrogen flushed air-tight containers for storage before analyses Objective 5. Nutraceutical activity and benefits evaluation 5.1. Technical background and research aims Consumption of fruits and vegetables has been strongly associated with reduced risk of some of the leading causes of deaths – heart disease, cancer and stroke – in industrialized nations. Thus, fruits and vegetables, which contain significant amounts of bioactive phytochemicals, may provide desirable health benefits beyond basic nutrition. Phytochemicals are natural products with diverse biological activities. Several thousand phytochemicals have been identified in fruits and vegetables, but the majority remains uncharacterized both in terms of chemical structure and biological activity Among the most prominent phytochemicals with demonstrated health- promoting effects are polyphenols (antioxidants that can prevent diseases such as cancer and coronary heart disease), lignans (phytoestrogens that have been implicated in regulating cholesterol levels, reducing the risk of certain cancers, and maintaining proper bone density post- menopause), and essential vitamins. The goal is to determine important nutritive properties of produce and processed fruit products important to Washington agriculture and the potential that these foods may have for inhibiting cancer cell development or fat deposition using in vitro tests. Naturally derived polyphenols and -3 PUFAs have pharmacological action, acting on molecular targets that are Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 14 of 23 integral in the progression of adipogenesis and cancer. It is hypothesized that these natural products can be integrated into food that when consumed can provide phytopreventative benefits in disease. To test this central hypothesis, we propose two broadly integrated tasks. 5.2. Research tasks and methodology Controlled-atmosphere stored samples will be collected and obtained from members of the tram in aims 1 through 4. Samples for each crop will be obtained, with multiple samples (5-6 per crop) representing fruit pomace and algae. The intent of this study is to determine the nutritional quality of specific products that will ultimately become available to consumers. Task 5.1. development and validation of analytical methodology Antioxidant Analysis: This task includes method validation of (high-performance liquid chromatography) HPLC, HPLC-Mass spectrometry (MS) and Gas chromatography. Mass spectrometry methods to characterize the content uniformity and purity and stability and quantify the “nutrient density” (concentration on a dry weight basis) of several phytochemicals (most importantly flavonoids, and -3 PUFAs) and the antioxidant activity of processed products made from them. Anti-Oxidant Capacity Determination This assay relies in the capacity of the anti-oxidants found in the sample to inhibit the oxidation of ABTS (2,2’-azino-di-(3-ethylbenzthiazoline sulphonate)) to ABTS·+ by metmyoglobin. as previously described.(Remsberg et al. 2008) Task 5.2. Analyses of Antioxidant Activities in Final Products Measurement of total anthocyanin, polyphenolics and anti-oxidant activity: The analysis of total antioxidant activity will be done using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. In this test, the scavenging of DPPH radicals will be monitored by observing the decrease in absorbance at 520nm which occurs due to reduction by antioxidants (Gordon, 2001). Anthocyanins (for example, those present in grape pomace) have typical absorbance maxima at 520nm (Skrede et al., 2000). Measurements will be made at pH 1 and 4.5 following the pH differential method of Skrede and Wrolstad (2002). The scavenging activity will be measured continuously for up to 30 minutes or until equilibrium is attained. The data will be reported as EC50 (i.e. the concentration of antioxidant required for 50% scavenging of DPPH radicals in a specified time period). In case of aloe vera, absorbance will be measured at 517 nm to compare with data reported by Hu et al. (2003). As for mushrooms, the TAA will be determined by the Trolox equivalent antioxidant capacity (TEAC) assay (Nindo et al., 2003; Murcia et al., 2002). The absorbance will be measured at 734nm until stable condition is reached. The data collected will be used to compare the kinetics of pigment and antioxidant loss of the samples after drying by the three different methods and after storage for 7 weeks. Task 5.3. Disease prevention benefit study. To study the in vitro anti-oxidant activity, adipogenesis and cytotoxicity of these agents alone and in combination, focusing on proliferating colon cancer (colorectal adenocarcinoma (HCT-116)) and breast cancer [estrogen receptor negative breast adenocarcinoma (MDA-MB-231)] cells. To reduce the production of fat (adipogenesis) in an in vitro cell culture (3T3-L1 preadipocyte cells) screening model is used. Anti-Cancer Activity: Either colon cancer (HCT-116) or breast cancer (MDA-MB-231) cells will be seeding into 96 well plates and incubated at 37ºC in a 5% CO2 atmosphere for 48 hrs. Then, the cells will be treated with plant extracts at different concentrations and incubated at 37ºC for 72 hrs. as previously described.( Remsberg et al. 2008) Adipogenesis assay: A line of pre-adipocyte cells (3T3-L1) will be used for determination of inhibition of adipogenic activity. Cells will be cultivated by seeded into 96 well plates and incubated at 37ºC in a 5% CO2 atmosphere for 48 hrs. After two days post confluency, the 3T3- L1 cells will be treated with extracts at different concentrations in the presences of an induction Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 15 of 23 medium (IMBX, insulin and dexamethasone solutions) plus DMEM (10% FBS). The treated cells will be incubated at 37ºC for 72 hrs. Then the cells will be treated again with plant extracts at the same concentrations and with the insulin medium solution plus DMEM (10% FBS) solution and incubated at 37ºC for 48 hrs. Further cell differentiation is stimulated by re- incubation in the same medium containing insulin. On day 9, once the desired degree of differentiation is attained (80%), lipid accumulation can be quantified using a staining technique (Oil Red O). 5.3. Data analysis One-way analysis of variance, or analysis of variance based on ranks, with the Dunnett post hoc comparison with P-value of  0.05 considered significant. Data will be presented as mean  standard deviation (SigmaStat version 2.0; SPSS Science, Chicago, IL). Objective 6. Economic Benefit Analysis 6.1. Technical background Although the preventive value of antioxidants and -3 PUFAs is well-documented it has been difficult to get people to change their food consumption to meet the levels required for protective effects. In 2005 only 25 percent of adults Washington State consumed the recommended 5 or more servings a day of fruits and vegetables that provide these nutrients. Getting people to consume these nutrients should lower the incidence of cancer and heart disease and their related direct and indirect costs. Besides the costs savings from improved health two other economic benefits are envisioned. First, converting food processing by-products to nutraceuticals increases the value of farm products which should benefit farmers. Second, the conversion and processing will create new business opportunities. 6.2. Research aims Aim 6.1 Estimate the health effects of that will come about from the increased consumption of these nutraceuticals and to measure the anticipated direct and indirect cost savings to the people and government of Washington State. Aim 6.2 Evaluate the economic impact of a the decreased healthcare needs, the diminishment of lost workdays, the increased demand for farm products and the creation of a new industry on the state economy. The analysis of the economic benefits from increasing consumption of nutraceuticals will be two separate but related projects which in the end can be combined for an overall picture of the economic benefits. 6.3 Research tasks and methodology Task 6.1 Decreased incidence and direct and indirect cost savings. The first task will be to collect and review evidence-based analyses of the preventive value of antioxidants and -3 PUFAs by conducting a meta-analysis of available studies, and then translating the results of that meta-analysis into direct and indirect cost savings from the expected decreased incidence of cancer and heart disease. The meta-analysis, when complete, will provide statistical estimates of the preventive value of the nutraceuticals. We will review results from a broad literature of medical and science studies. The literature includes many different types of studies. Some studies employ weak research designs that do not allow us to draw conclusions about the effectiveness of the nutrients. Others use strong designs that effectively isolate the effects of increased consumption of antioxidants and -3 PUFAs. Because we use two types of criteria to include or exclude studies: methodological criteria and those related to outcome measures. Methodological Criteria First, any program we include must have data from an evaluation that examines outcomes from a group that participates in a particular program in comparison to an Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 16 of 23 equivalent group that does not participate in the program. We do not consider a design following a single group’s results over time to be strong enough to include in our analysis. Preferably the groups come from random assignment, but at the very least the evaluation must show that any comparison group is indeed comparable to the treatment group on pre-existing variables that may influence outcome measures (such as age, gender, race and smoking). If a study finds pre- existing differences between groups, the study authors must control for these differences in their analysis. If there are disparities between the groups, the authors must statistically control for these differences in their analysis. Outcome Criteria Second, the studies we include must measure health outcomes, particularly with respect to cancers and heart disease. In other words, to be included in our analysis, a study must measure its outcomes in a methodologically sound manner, but those outcomes must also include one of our outcomes of interest. Once the expected decreases in disease incidence is estimated these changes will be applied to cost estimates, again harvested from a meta-analysis of available studies, of the direct and indirect costs of the diseases. All estimates will be subject to sensitivity analyses to specify a range of cost savings that can reasonably be expected. Task 6.2 Evaluate the economic impact of a the decreased healthcare needs, the diminishment of lost workdays, the increased demand for farm products and the creation of a new industry on the state economy. We will use input-output analysis (Steinacker, 2005), in particular, the IMPLAN (Impact Analysis for Planning) modeling system, to generate estimates of these multiple and simultaneous changes. IMPLAN was developed jointly by the USDA Forest Service, the Federal Emergency Management Agency, the Bureau of Land Management and the University of Minnesota, and is now owned and disseminated by the Minnesota IMPLAN Group (MIG). The results will be checked with alternative economic impact models such as regression-based models (Goldstein and Drucker, 2006), the REMI input-output model (Crihfield and Campbell, Jr., 1991) and MMBF-based input-output modeling (Cox and Munn, 2001). The mechanics of input-output analysis are straightforward (Dorsett and Weiler, 1982). Within a particular economic region, there are n different (mutually exclusive and collectively exhaustive) industries. Consumption comes from two sources: household consumption of final goods and services (Y) and intermediate consumption by firms (X) representing those firms’ supply chains. Thus, we have X = AX + Y where X is an nx1 matrix of production; Y is an nx1 matrix of household consumption; and A is an nxn “absorption” matrix identifying the average interrelationships across firms’ supply chains; that is, how much of one firm’s output is purchased by other firms to facilitate their production. Each year, MIG gathers data at the national level on inputs and outputs for hundreds of different industrial sections. The data are collected at the 4-6 digit NAICS code level from a variety of different sources, including (but not limited to) the Bureau of Labor Statistics, the Department of Commerce and the Bureau of Economic Analysis. MIG uses this information to compute absorption levels and market shares, among other factors, and subsequently uses these calculations to estimate national, state and county level input-output models for the entire U.S. (Bunting and Jones, 2004). IMPLAN using the MIG data gives us a baseline for the matrix X. When a shock or change to a sector in the local economy is simulated by imposing a change on X we can trace the impact on both the element of X corresponding to that particular industry (i.e., the “direct impact”) and also the effect on other industries in that economy (i.e., the “indirect and induced impacts”). This also allows us to calculate the multipliers for each industry as the ratio of the Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 17 of 23 total impact to the direct impact. For a national economy with k local economies, one can simply redefine X, Y and A to be (k*n)x1, (k*n)x1 and (k*n)x(k*n) matrices, respectively. When using IMPLAN to conduct our analysis, we will use a three step approach. First, in order to obtain a reasonable baseline to interpret our results, we conduct an analysis to examine the overall economic impact of low value agricultural products on the Washington State economy in a base year using the most recently available data. Next, we will estimate the economic impact of an increased demand for these products on the state economy using our previous analysis while we analyze the impact of lowered healthcare needs. Finally, we will alter the model to incorporate a new industry that produces nutraceuticals. As a sensitivity analysis all three steps will be repeated numerous times while changing assumed demands changes for farm products and healthcare needs to generate a range of expected impacts on the economy. E. Challenges Technology to extract the polyphenols and -3 PUFAs is already in place and thus, creation of these naturally derived products should proceed without major problems or obstacles although the process can be further optimized. HPLC-MS and GC-MS characterization studies are routinely performed and validated in our laboratories and no problems are foreseen. Encapsulation of similar xenobiotics has been reported previously. Many studies demonstrate the in vivo benefits of similar natural nutraceuticals. Determining the optimal fermentation, extraction and drying and processing may present challenges. If we encounter difficulties in fermentation (i.e. yield), we may investigate an alternative conditions. We believe the flexibility of using various raw materials combined with flexibility of using various fermentation methods enhances any challenges or risks in this proposal. For polyphenol extraction, the polymer PEG has an extremely high affinity towards tannins (a part of polyphenols), which has been used to reduce the anti-nutritional effects of tannins in animal feed for many years. It could be difficult to break down the complex of PEG- polyphenols because of this strong affinity interaction. Multiple hydrogen bonds (H-bonding) are mainly contributed to this affinity interaction (Arcuri et al, 2003). To solve these problem good polymer candidates will be screened with such a structure to have a selective extraction while remaining medium strength of the affinity interaction. Thus the polyphenol-polymer complex could be easily broken up due to the medium affinity. The biggest challenge facing the economic benefit analysis is the reliability of the previous studies used in the meta-analysis. However, the extensive literatures in both the health effects of antioxidants and -3 PUFAs as well as the wide range of economic analyses which address direct and indirect costs of cancers and heart diseases should mitigate any problems that arise. In fact, a problem may be in limiting the number of studies reviewed rather than finding studies addressing the issues. No problems are foreseen for the regional input-output analysis. F. Timeline and Milestones The timeline to complete the project aims is based on time required in preliminary studies and investigators’ experience. A project plan (PP) for the proposed research on the analysis of nutraceuticals. The PP provides a timeline for the projected milestones on a yearly basis and the major tasks for the Specific Aims. Objectives 1, 2, 3, 4, 5 and 6 are scheduled to be accomplished in three years following the timeline below.

Year 1 Year 2 Year 3 Objectives Quarter Quarter Quarter 1 2 3 4 1 2 3 4 1 2 3 4 Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 18 of 23

Task 1.1 Task 1.2 Objective 1 Task 1.3 Task 2.1 Task 2.2 Objective 2 Task 2.3 Task 3.1 Task 3.2 Task 3.3 Objective 3 Task 3.4 Task 4.1 Objective 4 Task 4.2 Task 5.1 Objective 5 Task 5.2 Task 6.1 Objective 6 Task 6.2 G. Personnel Dr. Shulin Chen, a Professor of Biological Systems Engineering will serve as the lead Principal Investigator of the project. Dr. Chen will be responsible for the coordination of the entire project and the supervision of Objectives 1 and 2. Dr. Zhanyou Chi, a Post-Doctoral Research Associate will be responsible for the research tasks under Objective 1, and Dr. Tianxi Zhang, also Post- Doctoral Research Associate will be responsible for Management of Objective 2. A graduate student will work on the polyphenol extraction under Objective 2. Dr. Eric Leber from ApresVin will contribute this project in-kind. Shyam Sablani will be responsible for microencapsulation study of polyphenols. He will optimize the spray drying process of encapsulation and determine the degradation kinetics of antioxidant activity of polyphenols during storage. A graduate student will work under Dr Sablani group. Processing research will be carried out by Dr. Juming Tang and his associates in the Food Engineering group of the Department of Biological Systems Engineering using facilities in WSU food engineering laboratories, and pilot-scale facilities in the Food Processing Pilot plant at WSU and in MCD Technologies Inc., Tacoma, WA. Extrusion processing will be conducted at USDA ARS Western Regional Center in Albany, CA. Dr Tang and Dr Sablani will share a full time postdoc position. A graduate student will work with Dr. Tang. Dr. Neal M Davies, Ph.D.,R.Ph., is a pharmaceutical scientist and an expert on phytochemical delivery Dr. Davies has over 20 years of experience working with cell culture animals and works closely with the nutraceutical industry. Dr. Davies will serve as analytical project manager and will be involved in outlining experiments, trouble-shooting, interpretation of data, strategic planning, writing manuscripts, and supervision of graduate students and post- docs. Dr. Davies will also aid in the interpretation of analytical and in vitro studies. A graduate student will work with Dr. Davies for Year 1 and Year 2. Robert Rosenman, Ph.D. is a professor of economics and Associate Director in the School of Economic Sciences. His research focus is in health economics and he has extensive policy analysis experience, including serving on the Board of Directors of the Washington State Institute for Public Policy. Dr. Rosenman is responsible for Objective 6: Economic Benefit Analysis. He has over 25 years experience running economic analyses, teaching and researching economic issues, and supervising graduate students. A graduate student will work with Dr. Rosenman. In addition, Gail Poesy will provide fiscal support at 25% of her time. The research group will develop an internal website for post information and research results. Each component will hold monthly project meeting. The entire team will meet quarterly to discuss progress and problem encountered. Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 19 of 23

H. Budget Justification The total budget of $1,498,920 is requesting for whole project, including personal salaries & wages, benefits, stipend ($924,271), administrative costs ($66,115), materials and supplies ($196,700), subcontract ($31,000), travel ($18,000), tuition costs ($106,517), and facilities costs ($156,317). Dr. Shulin Chen is requesting one month per year to manage and oversee the entire project. Dr. Rosenman and Dr. Sablani are asking one month per year for sub-objects, respectively. Drs. Zhanyou Chi and Tianxi Zhang, each at 75% of time will be reasonable to this project including experimental design, coordinate the team for this project, draft reports, and assist PI to guide a PhD graduate. One postdoc will be shared between Drs. Juming Tang and Shyam Sablani; each has 50% of time. Five PhD graduates will conduct experimental research under individual PIs. Gail Posey will be provided a financial support of $28,122. Goods and services are requested by Dr. Tang of $12,000, Dr. Sablani of $16,500, Dr. Davis of $95,000, Dr Chen of $63,000, and Rosenman of $5,200. These costs will include experiments, analyses, materials and model studies etc. Dr Sablani requests $3,000 for WSU Spectroscopy Center rental. Dr. Juming Tang is asking publication cost of $2,000 in Year 2 and Year 3. The team members will have domestic travel for on-site material collecting, meeting attendances etc. The travel costs are being requested by Dr. Tang of $10,000, Dr. Rosenman of $2,000, and Dr. Zhang of $6,000, for all three years. Dr. Tang is requesting a subcontract with USDA ARS Western regional center with $31,000. WSU will match with $391,182 to support this project, such as university facility cost. I. Facilities and Equipment The Department of Biological Systems Engineering at Washington State University will provide six laboratories and a pilot plant for this research. The first two objectives will be conducted within the Bioprocessing and Bioproduct Engineering Laboratory (BBEL) at WSU. Under the direction of Dr. Shulin Chen, BBEL is designed as first class lab for conducting biomass processing, biofuel and bioproduct research. The laboratory consists of five main sub-labs. The first one is a fermentation and conversion lab which is equipped with fermentors, shakers, incubation and culture devices. The second sub-lab is the analytical lab which is equipped with GCs, GC-MS, HPLC, IC, and VIS/UV Spectrophotometer. The third sub-lab is a molecular biology lab that is equipped with a nucleic acid gel electrophoresis system, a nucleic acid visualization chamber, a protein gel electrophoresis system, a RT PCR machine, an electroporator, a spectrophotometer, a hybridizer, a nucleic acid fixing UV chamber, and other necessary items such as centrifuges and shakers. The fourth sub-lab is an anaerobic digestion lab that is equipped with various reactors, and the fifth sub-lab is a microbiology lab that is equipped for microbial identifications. The sixth laboratory is the Food Engineering Laboratory which is equipped with equipment such modulated differential scanning calorimeter to characterize material properties of encapsulated powder. WSU Food Processing Pilot Plant provides preprocessing and processing equipment, including blanching, mixing, heating, and drying (freeze, hot air, drum and spray), for research under Dr. Juming Tang, Dr. Shyam Sablani and their associates. MCD Technologies Inc., Tacoma, WA provides both pilot scale and full scale systems for research with RW drying technology. USDA ARS Western Regional Center in Albany, CA provides a Clextral EVOL HT32-H twin-screw extruder, and milling and mixing machines for this research. Dr. Davies laboratory has four HPLC systems and one mass spectrometer with UV, photodiodarray, fluorescence and refractive index capabilities and one electrospray ionization mass spectrometer available. Dr. Davies has a separate biosafety level 3 cell facilities with incubators and cell culture hoods. Animal: The Wegner Hall vivarium and staff has resources and Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 20 of 23 room to house, feed, and provide veterinary care to the rodents in any studies. Dr. Davies has 48 mice/rat metabolic cages. Dr. Davies is on the Institutional Animal Care and Use Committee and will obtain animal ethics approval for any requisite animal studies. J. Outcomes and Future Plans The proposed project will bring WSU scientists together from bioprocessing, food engineering, pharmacy, and agricultural economics to focus on strategies to reduce state of Washington health care cost using innovative approach of incorporating nutraceuticals into regular diet of consumers. The short outcomes of the project will include (1) intellectual properties generated from the first 4 components of the project in terms of patents; (2) publications that contributing to the sciences of nutraceutical products; and (3) a competitive research team at WSU working on nutraceutical production, processing and delivery, assessment and economics. The longer terms (with 5 years) outcome of the project will be (1) nutraceutical enriched food products developed by the research team ready to be distributed to the Washington Citizens, (2) a group of new companies that use the technology developed by the research team to produce these food products in Washington, (3) broader employment opportunities created by the group of new companies that convert the agricultural by-products to functional food, (4) health benefits to the citizens who enjoy the food products and (5) health care cost reduction due to the improved health of the citizens. In the short term we will develop assay for polyphenols and fatty acids and evaluate additive and synergistic anti-oxidant, adipogenesis, and cytotoxicity to cancer cells. We anticipate significant intellectual property can be generated from these novel studies and a large number of publications in high impact journal articles. The broader impact is this technology can be applied to other plant based products, with minimal modification to the base platform. The potential for follow-on research funding from the United States Department of Agriculture (USDA) National Institutes of Health (NIH), Department of Defense (DOD), is high based on proof of concept and potential phytopreventative benefits against disease. The nature of this work is multi and cross-disciplinary, translational, and support of this program will increase our scientific competitiveness for other funding agencies, lead to significant intellectual property including patents and publications and perhaps spin off companies and could ultimately lead to human clinical trials. The main results of the project will be a technology that can be used to directly extract polyphenol from fruit and vegetable wastes. The processes have the potential to be applied as a platform extraction technology for obtaining other valuable nutraceuticals from various agricultural wastes and residues. Invention disclosures and possible patent applications will be major outcomes for the ultimate commercialization of the technology through working with the WSU Office of Intellectual Property. Other results of the project will include 12 to 15 peer- review journal publications, along with annual progress reports and a final project report submitted to the funding agency. PhD graduate and undergraduate students will have training opportunities through this funding support. There is growing demand fortified foods i.e. foods enriched with nutraceuticals by today well informed consumers. The state of Washington is a leading producer of organic tree fruits, small fruits, and vegetables those are rich in nutraceuticals. Through this project we will contribute scientific information on the manufacturing and encapsulation polyphenols from locally produced agricultural materials and determine conditions influencing stability of these components during storage and processing. This information will assist companies involved in the production of nutraceuticals and fortified foods enriched with nutraceuticals, which may provide economic incentive to both agriculture and nutraceutical industry. Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 21 of 23

Our future plan is to leverage funding from other sources to conduct the second phase study mainly pilot test and scale-up analysis to commercialize the process developed in the project. Chen, Davies, Rosenman, Sablani, Tang, LSDF08-01, Page 22 of 23

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