Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): 1. Develop processes to fractionate sorghum and corn/sorghum oils into new commercially-viable coproducts. 2. Develop processes to fractionate grain-derived brans into new commercially-viable coproducts. 2a: Develop processes to fractionate grain-derived brans into new commercially-viable coproducts such as lipid-based coproducts and for other industrial uses such as extrusion or producing energy or fuel. 2b: Develop commercially-viable, value-added carbohydrate based co- products from sorghum brans and the brans derived from other grains during their biorefinery process. 3. Develop processes to fractionate biorefinery-derived celluloses and hemicelluloses into new commercially-viable coproducts. 3a: Develop commercially-viable, value-added hemicellulose based co- products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 3b: Develop commercially-viable, value-added cellulose based co-products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 4. Develop technologies that enhance biodiesel quality so as to enable greater market supply and demand for biodiesel fuels and >B5 blends in particular. 4a: Improve the low temperature operability of biodiesel by chemical modification of the branched-chain fatty acids. 4b: Develop technologies that significantly reduce quality-related limitations to market growth of biodiesel produced from trap and float greases. 4c: Further develop direct (in situ) biodiesel production so as to enable its commercial deployment. 5. Develop technologies that enable the commercial production of new products and coproducts at lipid-based biorefineries. 5a: Enable the commercial production of alkyl-branched from agricultural products and food-wastes. 5b: Enable the commercial production of aryl-branched fatty acids produced from a combination of lipids and natural antimicrobials possessing phenol functionalities. Approach (from AD-416): In conjunction with CRADA partners and other collaborators, develop technologies that identify new biorefinery coproducts, evaluate their applications and estimate their profitability and marketability. The approach will focus on development processes to produce several types of new coproducts. First, processes will be developed to extract and fractionate sorghum oil from sorghum kernels and sorghum bran. Processes will also be developed to extract and fractionated cellulose-rich and hemicellulose-rich fractions from sorghum kernels, sorghum bran, sorghum bagasse, and biomass sorghum. Other processes will be developed to improve the biofuel value of biodiesel by blending biodiesel with modified fatty acid derivatives to enhance its low temperature performance, reduce the levels of impurities that block fuel lines, economically convert trap grease and float grease to biodiesel, and improve the in situ process to make biodiesel directly from oil-rich low value agricultural products. In addition to biodiesel applications, other processes will be developed to produce branched fatty acids with unique functional (including improved lubricity) and biological properties (including antimicrobial and antioxidant properties). Considerable progress was made on all objectives, all of which fall under National Program 306 ⿿ Product Quality and Uses, Component 3 - Biorefining. Objective 1: A GREET (Greenhouse Gases, Regulated Emissions, and Energy Use in Transport) process and cost model was constructed to optimize and evaluate the fractionation of distillers milo oil. The model showed that the cost of extracting sorghum wax from intact sorghum kernels or from sorghum bran was probably not economically feasible. However, if distillers milo oil (or distillers oil from ethanol plants that utilize a blend of corn and sorghum) could be obtained at a cost similar to the current cost of distillers corn oil (~$0.35/lb), then it could potentially be possible to economically fractionate sorghum wax from these distillers oils, with yields ranging from 2 to 10 lbs of sorghum wax per 100 pounds of distillers oil. Objective 2a: The concept of using sorghum bran as a feedstock for combustion or fast pyrolysis was considered. However, our fractionation and analytical studies (Hums et al, 2018B) revealed that sorghum bran has a relatively high nutritive value (~10% protein, ~10% crude fat, and ~40% starch) and more profit can be obtained from sorghum brans by selling them for animal feed applications or for fractionating all or some of its components, such as sorghum wax, for nutraceutical or industrial application than for using them as feedstocks for combustion or fast pyrolysis. Objective 2b: Arabinoxylan (AX) from sorghum bran was isolated and tested to determine antioxidant properties. AX from sorghum bran had an improvement of 8% in oxygen radical scavenging assays over corn bran AX (cAX). Further characterization of the AX fractions showed that sorghum bran AX had a lower mass quantity of both ferulic and p-coumaric acid (0. 532 mg/100 g AX) compared to cAX (1.66 mg/100 g AX). The better scavenging efficiency for sorghum bran AX may be reflected in the presence of other compounds aside from the hydroxy cinnamic acids located within the AX structure. Objective 3a: Sorghum bagasse and biomass arabinoxylan (AX) was isolated similarly to sorghum bran AX and utilized to determine relevant antioxidant properties. Sorghum biomass AX scavenging efficiency underperformed compared to sorghum bran AX, but sorghum bagasse AX had a 25% improvement in scavenging efficiency. The overall hydroxy cinnamic acids content of the sorghum bagasse AX was 13.15 mg/100 g AX, which indicates the greater presence of phenolic acids in the AX fraction can improve the overall antioxidant capacity. Prior work has also indicated that sorghum bagasse AX utilized to prepare films had poor quality due to extreme brittleness even with plasticizer addition. Conversely, sorghum bran AX films had superior quality in terms of strength and moisture barrier properties at low relative humidity. AX isolation and recovery from different sorghum fractions has shown that integration into biorefining processes is possible by determining end use applications. Sorghum bran AX has more useful functionality for biomaterial products, while AX from sorghum bagasse are more suited for food based applications as an additive. Objective 3b: Cellulose rich fraction (CRF) from sorghum bran (SBR), sorghum bagasse (SBA) and sorghum biomass (SBI) were isolated, characterized and their functionalities (water holding capacity and dietary fiber) were studied. They can hold water at 22.76 to 35.27 times their dry weight at room temperature, which is a very good property for their application in food products. All three CRF isolated from SBR, SBA and SBI sources were very rich in insoluble dietary fiber (IDF) containing 89.12, 97.01 and 87.89 % (w/w), respectively. The above two properties qualify them as good ingredients in many food products, which include: a. Bakery products, e. g. biscuits, buns, rolls, muffins, sweet breads, wheat rolls, cookies, brownies, cakes, bakery mixes, pie dough, pizza dough, pita bread, pie filling, tortillas, crackers, snack food; b. Dairy products, e.g. cream cheese, ricotta cheese, processed cheese, cheese sauces, sour cream, dips, ice cream, puddings, custards, whipped toppings, yogurt, yogurt drinks, smoothies; c. Meats, e.g. ground meat, ground meat patty, sausages, hot dogs, meat fillings, hamburger; and d. Dressing, e.g. mayo spread, salad dressings, dips, sauces, salsa, barbecue sauce, tomato sauce. This technology has been transferred to our CRADA partner. Objective 4a: Cold flow improvers, in the form of additive mixtures of branched chain alkyl esters in fatty acid methyl esters (biodiesels), only slightly improved the low temperature properties (i.e. cloud point, pour point, kinematic viscosity) of biodiesels made from lard, tallow and sewage scum grease. Results of the study indicated that process analysis was not necessary for this data set. Objective 4b: We continued to identify sulfur-bearing species in biodiesel produced from ⿿brown⿿ greases such as ⿿trap⿿ grease and float greases using modified state-of-the art analytical instrumentation and protocols. These brown greases have been collected from local municipal underground grease traps and waste water treatment plants at various times of the year. Techno-economic analysis will commence when the previous milestones are satisfactorily accomplished. Those milestones remain in the optimization phase. Objective 4c: We have improved the i.s.t. of the lipids in post- fermentation sorghum stillage (DDGS) by increasing conversion of the lipids in sorghum DDGS to biodiesel from approximately 30% to greater than 70% as a result of feedstock pretreatment. Co-product meals are being collected for evaluation. There is no collaborator pilot plant for this project. The designated collaborator provided insufficient substrate for this study. Therefore, we had to continue the work internally with ARS scientists who could provide adequate substrate. If necessary, techno-economic analysis will commence when the previous milestones are satisfactorily accomplished. Those milestones remain in the optimization phase. Objective 5a: A scale-up process designed according to the laboratory size data was successfully developed to produce the targeted alkyl- branched fatty acids (i.e., isostearic acid). This process can produce a large volume of the materials per batch and only involves the starting refined oil feedstock, solid catalyst and cocatalyst, and a small amount of water. The solid catalyst can be easily removed by filtration and can be recycled and reused for at least up to 20 times. Efforts were made to integrate the technology to end users (e.g., Arizona Chemical currently known as Kraton) and products were sent for evaluation. However, from industry feedback, even though this process can utilize existing capital to produce the products, it is still not economically feasible compared to existing plants where isostearic acids are produced from less refined oils. Objective 5b: A scale-up process to produce the aryl-branched fatty acids (i.e., phenolic-branched-chain fatty acids) was successfully developed by utilizing a similar reactor design concept as to the isostearic acid process. The solid catalyst can also be recycled and reused for at least 10 times to reduce cost and waste. This process also involves phenolic reagents, which are used in excess and can be very expensive; therefore, research efforts were made to recycle these materials. The products are currently being evaluated by an industrial partner (Colgate Palmolive Inc.) in their antimicrobial applications.

Impacts
(N/A)

Publications

• Moreau, R.A., Harron, A.F., Hoyt, J.L., Powell, M.J., Hums, M.E. 2018. Analysis of wax esters in seven commercial waxes using C30 reverse phase HPLC. Journal of Liquid Chromatography and Related Technologies. 41(10) :604-611.
• Mendez-Encinas, M.A., Carvajal-Millan, E., Yadav, M.P., Kale, M., López- Franco, Y., Rascon-Chu, A., Lizardi-Mendoza, J., Brown-Bojorquez, F., Silva-Campa, E., Pedroza-Montero, M. 2019. Partial removal of protein associated with arabinoxylans: impact on the viscoelasticity, crosslinking content and microstructure of the gels formed. Journal of Applied Polymer Science. 47300:1-10.
• Li, J., Yadav, M.P., Zhu, Y., Li, J. 2019. Effect of different hydrocolloids with gluten proteins, starch and dough microstructure. Journal of Cereal Science. 87:85-90.
• Zhang, J., Yadav, M.P., Li, J. 2019. Biodegradability and biodegradation pathway of di-(2-ethylhexyl) phthalate by Burkholderia pyrrocinia B1213*. Chemosphere. 225:443-450.
• Stoklosa, R.J., Latona, R.J., Yadav, M.P., Bonnaillie, L. 2019. Evaluation of arabinoxylan isolated from sorghum bran, biomass, and bagasse for film formation. Carbohydrate Polymers. 213:382-392.
• Bhinder, S., Kaur, A., Singh, B., Kaur, M., Kumari, S., Singh, N., Yadav, M.P. 2019. Effect of infrared roasting on antioxidant activity, phenolic composition and maillard reaction products of tartary buckwheat varieties. Food Chemistry. 285:240-251.
• Hums, M.E., Moreau, R.A., Powell, M.J., Hoyt, J.L. 2018. Extraction of surface wax from whole grain sorghum. Journal of the American Oil Chemists' Society. 95:845-852.
• Hums, M.E., Moreau, R.A., Yadav, M.P., Powell, M.J., Simon, S. 2018. Comparison of bench-scale decortication devices to fractionate bran from sorghum. Cereal Chemistry. 95:720-733.
• Zhang, J., Uknalis, J., Moreau, R.A., Lew, H.N. 2019. Development of magnesium oxide-zeolite catalysts for isomerization of fatty acids. Catalysis Letters. 149:303-312.
• Lew, H.N., Wagner, K., Zhang, J., Nunez, A., Fan, X., Moreau, R.A. 2018. New classes of antimicrobials: poly-phenolic branch-chained fatty acids. Natural and Bio-Based Antimicrobials for Food Application. ACS Symposium Series: American Chemical Society: Washington DC, 2018. 209-221.
• Jones, P.J., Shamloo, M., Mackay, D.S., Rideout, T.C., Myrie, S., Plat, J., Roullet, J., Baer, D.J., Calkins, K., Davis, H. Duell, B., Ginsberg, H., Gylling, H., Jenkins, D., Lütjohann, D., Moghadasian, M., Moreau, R.A., Mymin, D., Ostlund, R., Ras, R., Reparaz, J., Trautwein, E., Turley, S., Vanmierlo, T., Weingärtner, O. 2018. Progress and perspectives in plant sterol and plant stanol research. Nutrition Reviews. 76:725-745.
• Wyatt, V.T., Boakye, P.G., Jones, K.C., Latona, N.P., Liu, C., Strahan, G. D., Zhang, J., Besong, S.A., Lumor, S.E. 2019. Synthesis of absorbent polymer films made from fatty acid methyl esters, glycerol, and glutaric acid: thermal, mechanical, and porosity analysis. Journal of Applied Polymer Science. 1-15.
• Suri, K., Singh, B., Kaur, A., Yadav, M.P., Singh, N. 2019. Impact of infrared and dry air roasting on the oxidative stability, fatty acid composition, Maillard reaction products and other chemical properties of black cumin (Nigella sativa L.) seed oil. Food Chemistry. 295:537-547.
• Kaur, A., Yadav, M.P., Singh, B., Bhinder, S., Simon, S., Singh, N. 2019. Isolation and characterization of arabinoxylans from wheat bran and study of their contribution to wheat flour dough rheology. Carbohydrate Polymers. 221:166-173.
• Liu, Y., Yadav, M.P., Yin, L. 2017. Enzymatically catalyzed corn fiber gum- bovine serum albumin conjugates: their interfacial adsorption behaviors in oil-in-water emulsions. Food Hydrocolloids. 77:986-994.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 1. Develop processes to fractionate sorghum and corn/sorghum oils into new commercially-viable coproducts. 2. Develop processes to fractionate grain-derived brans into new commercially-viable coproducts. 2a: Develop processes to fractionate grain-derived brans into new commercially-viable coproducts such as lipid-based coproducts and for other industrial uses such as extrusion or producing energy or fuel. 2b: Develop commercially-viable, value-added carbohydrate based co- products from sorghum brans and the brans derived from other grains during their biorefinery process. 3. Develop processes to fractionate biorefinery-derived celluloses and hemicelluloses into new commercially-viable coproducts. 3a: Develop commercially-viable, value-added hemicellulose based co- products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 3b: Develop commercially-viable, value-added cellulose based co-products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 4. Develop technologies that enhance biodiesel quality so as to enable greater market supply and demand for biodiesel fuels and >B5 blends in particular. 4a: Improve the low temperature operability of biodiesel by chemical modification of the branched-chain fatty acids. 4b: Develop technologies that significantly reduce quality-related limitations to market growth of biodiesel produced from trap and float greases. 4c: Further develop direct (in situ) biodiesel production so as to enable its commercial deployment. 5. Develop technologies that enable the commercial production of new products and coproducts at lipid-based biorefineries. 5a: Enable the commercial production of alkyl-branched from agricultural products and food-wastes. 5b: Enable the commercial production of aryl-branched fatty acids produced from a combination of lipids and natural antimicrobials possessing phenol functionalities. Approach (from AD-416): In conjunction with CRADA partners and other collaborators, develop technologies that identify new biorefinery coproducts, evaluate their applications and estimate their profitability and marketability. The approach will focus on development processes to produce several types of new coproducts. First, processes will be developed to extract and fractionate sorghum oil from sorghum kernels and sorghum bran. Processes will also be developed to extract and fractionated cellulose-rich and hemicellulose-rich fractions from sorghum kernels, sorghum bran, sorghum bagasse, and biomass sorghum. Other processes will be developed to improve the biofuel value of biodiesel by blending biodiesel with modified fatty acid derivatives to enhance its low temperature performance, reduce the levels of impurities that block fuel lines, economically convert trap grease and float grease to biodiesel, and improve the in situ process to make biodiesel directly from oil-rich low value agricultural products. In addition to biodiesel applications, other processes will be developed to produce branched fatty acids with unique functional (including improved lubricity) and biological properties (including antimicrobial and antioxidant properties). Progress was made on all objectives, all of which fall under National Program 306 � Quality and Utilization of Agricultural Products, Component 3 Biorefining. Addressing Problem Statement 3.B. Technologies that reduce risks and increase profitability in existing industrial biorefineries. Objective 1: Concerning the development of physical and centrifugation methods to fractionate Distillers Milo Oil, samples of this oil (from fuel ethanol plants fermenting only milo) and Distillers Milo/Corn Oil (from fuel ethanol plants fermenting mixtures of milo and corn at defined ratios) were dissolved in hexane, ethanol, and methanol. After incubating at various temperatures, the solutions were centrifuged and/or filtered to separate the solids and dissolved materials. The optimal combination of solvent and temperature was identified to efficiently separate the oil (triacylglcyerols) and waxes in Distillers Milo Oil. The composition of fractionated wax was then thoroughly characterized using both high performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The waxes fractionated from Distillers Milo Oil were found to be a mixture of surface waxes (mainly C28 and C30 alkanes, alcohols, aldehydes and acids) that originated from the surface of the sorghum kernel and nonpolar waxes (50-60 carbons) that were either produced or extracted during fermentation, ethanol distillation, or drying of the Distillers Milo Oil and Distillers Dried Grains and Solubles. Objective 2: Sorghum bran (a fraction consisting of the outer layers of the kernel and representing ~10% of the mass of the kernel), compared to other typical cereal grains, has a unique composition because it contains high percentages of starch and protein. This allows sorghum bran to be potentially utilized for applications ranging from food products to composite materials such as bio-based plastics. For this work, we fractionated the sorghum grain by using a rice polisher to remove sorghum bran. To begin to study the extrusion applications of sorghum bran, a 1 kg sample was sent to collaborators at the ARS-Western Regional Research Center, where they utilized the material to prepare bio-based plastic material and compared the performance to materials prepared from other similar feedstock sources. The study of the prebiotic properties (the ability to increase the proportions of �good� bacteria in the human colon) of sorghum bran was conducted via a growth study of 80 lactobacillus strains under both aerobic and anaerobic conditions. Unfortunately, the oligosaccharides from sorghum bran did not initiate the growth of these health-promoting bacteria. When sorghum bran was separated into a cellulose-rich fraction and a hemicellulose-rich fraction, the cellulose-rich fraction had interesting rheological (texture) properties, similar to those of catsup (a well known non-Newtonian shear thinning fluid). Like catsup, solutions of cellulose-rich fraction had a high viscosity but when force was applied (such as shaking the catsup bottle) there was a dramatic decrease in viscosity. These viscosity properties are very desirable for many food applications, as it implies that the material will pump easily and move through the processing equipment with very little difficulty but then, after high shear is removed, will resume the high viscosity needed for the application. The hemicellulose-rich fraction (which contained high levels of arbinoxylans) from sorghum bran, sorghum bagasse, and biomass sorghum were used to make films for food applications. These films were prepared to determine water absorption and mechanical strength properties. The films were found to be quite sensitive to the surrounding relative humidity. The films from sorghum bran exhibited a 40% increase in mass at high relative humidity. An equilibrium condition of about 50% relative humidity appears to be the point where the rate of water absorption from the surroundings is the lowest. The films from each sorghum source have been prepared for mechanical strength testing. Once these analyses are complete the utilization of hemicellulose-rich fraction as a co-product can be directed towards either food or packaging applications. Objective 3: Samples of pure oligosaccharides from the hemicellulose- rich fraction of sorghum bagasse (the fiber-rich material that remains after squeezing the juice from sweet sorghum) and biomass sorghum were generated and purified via a dialysis process. The study of their prebiotic properties was conducted. Unlike those from sorghum bran, the oligosaccharides from sorghum bagasse and biomass sorghum significantly increased the proportions of �good� bacteria in the human colon, demonstrating that they can potentially be useful as prebiotics. Interestingly, the oligosaccharides from the hemicellulose-rich fraction from sorghum bagasse and biomass are not as branched as those from sorghum bran. The hemicellulose-rich fractions from sorghum bagasse and biomass sorghum did not make as good films as those from sorghum bran, which may be due to their less branched structure. The rheological (texture) study of the cellulose-rich fraction isolated from sorghum bagasse and biomass sorghum showed similar promising properties to those described above for the same fraction from sorghum bran. This implies that this material will also pump easily and move through the processing equipment with very little difficulty but then, after high shear is removed, will resume the high viscosity needed for many food applications. Objective 4: Branched chain fatty acid additives were synthesized and are currently being evaluated by a collaborator for engine emissions and power testing. We have successfully identified several sulfur-containing compounds in biodiesel produced from �brown� greases such as �trap� grease and float greases using state-of-the art analytical instrumentation and protocols. These brown greases have been collected from local municipal underground grease traps and waste water treatment plants at various times of the year. In addition, we have successfully determined the efficiency of the in-situ transesterification process (a proprietary process developed at ERRC (Eastern Regional Research Center) that combines oil extraction and formation of biodiesel into one step) of the lipids in sorghum bran and post-fermentation sorghum stillage (material remaining after fermentation). Conversion of sorghum bran to biodiesel has been successful (>95%). The conversion of sorghum stillage to biodiesel using the in-situ method was increased from approximately a 30% yield to greater than 70% yield as a result of feedstock pretreatment and preliminary economic estimates have been conducted. Co-product meals are being collected for evaluation. Objective 5: Isostearic acid and dimer acid were produced for lubricant and polyamide studies. To formulate the isostearic, isooleic acid (which is the precursor of isostearic acid) was blend with two base oils (i.e., polyalphaolefin (PAO-6) and high oleic sunflower oil). Lubricant properties (slipperiness) were evaluated by measuring the four-ball anti- wear friction and wear of the neat isooleic acid, oleic acid, and blends. Data on the physical properties including cloud and pour points, oxidation stability, kinematic viscosity and viscosity index were also collected. Results showed that blends (0 � 10 %, w/w) of isooleic and oleic acid in PAO-6 displayed the following similar trends with increasing concentration: mildly decreasing kinematic viscosity 40 and 100-degree C, increasing viscosity index number, lower coefficient of friction, and no change in wear. Blends (0 � 10 %, w/w) of isooleic and oleic acid in sunflower oil displayed the following similar trends: decreasing oxidation stability with increasing concentration, and constant pour point and cloud point with increasing concentration. Dimers, which are the major low value byproduct of isostearic acid production were successfully isolated by molecular distillation. Detailed characterization by mass spectroscopy showed that they are mixtures of different types of dimer products. Presently, methods for converting dimers into polyamide resins are being evaluated and will be followed by application studies. Studies were begun to evaluate the cytotoxicity of the phenolic-branched-chain fatty acid products using the pathogen-free chicken embryo method. The embryos were examined for any signs of internal or external abnormal development; all appeared to have developed normally. In other experiments it was demonstrated for the first time that heterogeneous catalysts such as zeolites and other types of acid catalysts, could be used to modify the structures of fatty acids in their natural state when they are naturally bound in common plant oils. In addition to modifying the oils by causing branching of their fatty acids it may also be possible to use catalysts to modify the oils in other ways, by attaching other molecules such as phenolics, which may give them new physical and biological properties such as antioxidant and antimicrobial properties. Accomplishments 01 Catalytic modification of plant oils. Chemists at the Eastern Regional Research Center (ERRC) in Wyndmoor, Pennsylvania, demonstrated for the first time that heterogeneous catalysts such as zeolites and other types of acid catalysts, could be used to modify the structures of fatty acids in their natural state when they are naturally bound in common plant oils. Plant oils are comprised of natural compounds called triacylglcyerols. Previously, it was thought that only fatty acids in the free form or as fatty acid methyl esters could be modified by heterogeneous catalysts. The team demonstrated that oleic acid and other unsaturated fatty acids in sunflower oil could be modified by producing �branches� within the fatty acids. This newly modified type of plant oil has a lower melting point and may possess other valuable physical properties.

Impacts
(N/A)

Publications

• Marquez-Escalante, J.A., Carvajal-Millan, E., Yadav, M.P., Kale, M., Rascon-Chu, A., Gardea, A.A., Valenzuela-Soto, E., L�pez-Franco, Y., Lizardi-Mendoza, J., Faulds, C.B. 2018. Rheology and microstructure of gels based on wheat arabinoxylans enzymatically modified in arabinose and xylose. Journal of the Science of Food and Agriculture. 98:914-922.
• Deng, C., Liu, Y., Li, J., Yadav, M.P., Yin, L. 2018. Diverse rheological properties, mechanical characteristics and microstructures of corn fiber gum/soy protein isolate hydrogels prepared by laccase and heat treatment. Food Hydrocolloids. 76:113-122.
• Nwokocha, L.M., Williams, P.A., Yadav, M.P. 2018. Physicochemical characterisation of the galactomannan from delonix regia seed. Food Hydrocolloids. 78:132-139.
• Yadav, M.P., Hicks, K.B. 2018. Isolation, characterization and functionalities of bio-fiber gums isolated from grain processing by- products, agricultural residues and energy crops. Food Hydrocolloids. 78:120-127.
• Qiu, S., Wang, Y., Chen, H., Liu, Y., Yadav, M.P., Yin, L. 2018. Reduction of biogenic amines in sufu by ethanol addition during ripening stage. Food Chemistry. 239:1244-1252.
• Kale, M., Yadav, M.P., Chau, H.K., Hotchkiss, A.T. 2018. Molecular and functional properties of a xylanase hydrolysate of corn bran arabinoxylan. Carbohydrate Polymers. 181:119-123.
• Liu, Y., Selig, M.J., Yadav, M.P., Yin, L., Abbaspourrad, A. 2018. Transglutaminase-treated conjugation of sodium caseinate and corn fiber gum hydrolysate: Interfacial and dilatational properties. Carbohydrate Polymers. 187:26-34.
• Moreau, R.A., Nystrom, L., Whitaker, B.D., Moser, J.K., Baer, D.J., Gebauer, S.K., Hicks, K.B. 2018. Phystosterols and their derivatives: structural diversity, distribution, metabolism, analysis, and health promoting uses. Progress in Lipid Research. 70:35-61.
• Yan, Z., Wagner, K., Fan, X., Nunez, A., Moreau, R.A., Lew, H.N. 2018. Bio- based phenolic-branched-chain fatty acid isomers synthesized from vegetable oils and natural monophenols using modified h+-ferrierite zeolite. Industrial Crops and Products. 114:115-122.
• Sarker, M.I., Latona, R.J., Moreau, R.A., Micheroni, D., Jones, K.C., Lin, W., Lew, H.N. 2017. Convenient and environmentally friendly production of isostearic acid with protonic forms of ammonium cationic zeolites. European Journal of Lipid Science and Technology. 119(1700262):1-8.
• Sarker, M.I., Lew, H.N., Moreau, R.A. 2018. Comparison of various phosphine additives in zeolite based catalytic isomerization of oleic acid. European Journal of Lipid Science and Technology. 120 (1800070):1-8.
• Hughes, M., Jones, K.C., Hums, M.E., Cairncross, R.A., Wyatt, V.T. 2018. Identification of sulfur-containing impurities in biodiesel produced from brown grease. Journal of the American Oil Chemists' Society. 95:407-420.
• Wyatt, V.T., Jones, K.C., Johnston, D., Moreau, R.A. 2018. Production of biodiesel via the in situ transesterification of grain sorghum bran and DDGS. Journal of the American Oil Chemists' Society. 95:743-752.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 1. Develop processes to fractionate sorghum and corn/sorghum oils into new commercially-viable coproducts. 2. Develop processes to fractionate grain-derived brans into new commercially-viable coproducts. 2a: Develop processes to fractionate grain-derived brans into new commercially-viable coproducts such as lipid-based coproducts and for other industrial uses such as extrusion or producing energy or fuel. 2b: Develop commercially-viable, value-added carbohydrate based co- products from sorghum brans and the brans derived from other grains during their biorefinery process. 3. Develop processes to fractionate biorefinery-derived celluloses and hemicelluloses into new commercially-viable coproducts. 3a: Develop commercially-viable, value-added hemicellulose based co- products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 3b: Develop commercially-viable, value-added cellulose based co-products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 4. Develop technologies that enhance biodiesel quality so as to enable greater market supply and demand for biodiesel fuels and >B5 blends in particular. 4a: Improve the low temperature operability of biodiesel by chemical modification of the branched-chain fatty acids. 4b: Develop technologies that significantly reduce quality-related limitations to market growth of biodiesel produced from trap and float greases. 4c: Further develop direct (in situ) biodiesel production so as to enable its commercial deployment. 5. Develop technologies that enable the commercial production of new products and coproducts at lipid-based biorefineries. 5a: Enable the commercial production of alkyl-branched from agricultural products and food-wastes. 5b: Enable the commercial production of aryl-branched fatty acids produced from a combination of lipids and natural antimicrobials possessing phenol functionalities. Approach (from AD-416): In conjunction with CRADA partners and other collaborators, develop technologies that identify new biorefinery coproducts, evaluate their applications and estimate their profitability and marketability. The approach will focus on development processes to produce several types of new coproducts. First, processes will be developed to extract and fractionate sorghum oil from sorghum kernels and sorghum bran. Processes will also be developed to extract and fractionated cellulose-rich and hemicellulose-rich fractions from sorghum kernels, sorghum bran, sorghum bagasse, and biomass sorghum. Other processes will be developed to improve the biofuel value of biodiesel by blending biodiesel with modified fatty acid derivatives to enhance its low temperature performance, reduce the levels of impurities that block fuel lines, economically convert trap grease and float grease to biodiesel, and improve the in situ process to make biodiesel directly from oil-rich low value agricultural products. In addition to biodiesel applications, other processes will be developed to produce branched fatty acids with unique functional (including improved lubricity) and biological properties (including antimicrobial and antioxidant properties). Progress was made on all objectives, all of which fall under National Program 213 � Biorefining, Component 1 Biochemical Conversion. Addressing Problem Statement 2. Technologies that reduce risks and increase profitability in existing industrial biorefineries. Component 2 (Biodiesel) addressing Problem Statement 2.1, to improve the engine performance of biodiesel, and Problem Statement 2.2, development of new technologies that reduce risks and increase profitability in existing industrial biorefineries for converting lipids. Objective 1: Distillers Milo Oil and blends of Distillers Corn Oil/ Distillers Milo Oil were obtained from industrial sources. We developed a countercurrent method with ethanol that allowed us to fractionate these oils into two fractions, one is sorghum wax and the other is a mixture of other conventional oil components (triglycerides, free fatty acids, and sterols). Using our new HPLC-MS analytical method, the wax fraction was shown to have a purity of 80-90%. We have begun to conduct experiments to extract and fractionate sorghum oil and sorghum wax with supercritical CO2. Objective 2: Samples of several sorghum brans were analyzed for protein and other water soluble components. The major water soluble component was protein, at a level of about 6.5%. The sugar composition (relative mole %) of the arabinoxylan (AX) from sorghum bran was mainly xylose (42%) and arabinose (35%), with less than 10% each of glucuronic acid, glucose, galactose, and galacturonic acid. Its molecular characterization was completed and the manuscript is in preparation. Its emulsion stability was characterized and published. The results indicate that the arabinoxylan from sorghum bran is similar, but not identical to, the well-characterized arabinoxylans of corn and other grains. The sugar composition (relative mole %) of Cellulose-Rich Fraction (CRF) from sorghum bran was glucose (55%), arabinose (23%), xylose (17%), and galactose (6%). The glycosyl linkage composition of CRF from sorghum bran was predominately 1, 4 linked glucose (66%) and less than 10% each of 11 other linkages. Its water binding properties were characterized and the results were published. The composition and linkages of the Cellulose-Rich Fraction from sorghum bran indicate that it is a unique carbohydrate polymer with more of the properties of cellulose (55% glucose) than of hemicellulose (40% arabinose + xylose). Objective 3: The sugar composition (relative mole %) of the arabinoxylan (AX) from sorghum bagasse was xylose (58%), arabinose (18%), and less than 10% each of glucose, galactose, galacturonic acid, and glucuronic acid. The results indicate that the composition of the arabinoxylan from sorghum bagasse is similar, but not identical to, the well-characterized arabinoxylans of corn and other grains. The sugar composition (relative mole %) of the arabinoxylan (AX) from biomass sorghum was xylose (64%), arabinose (17%), and less than 10% each of glucose, galactose, galacturonic acid, and glucuronic acid. Their molecular characterization was completed and the manuscript is in preparation. Their emulsion stability was studied and the results are published. The results indicate that the composition of the arabinoxylan from biomass sorghum is similar, but not identical to, the well- characterized arabinoxylans of corn and other grains. The sugar composition (relative mole %) of Cellulose-Rich Fraction (CRF) from sorghum bagasse was xylose (51%), glucose (39%), and less than 10% each of arabinose and galactose. The predominant glycosyl linkages of CRF from sorghum bagasse were 1, 4 linked glucose (69%) and 1, 4 linked xylose (22%) and less than 10% each of 10 other linkages. The composition and linkages of the Cellulose-Rich Fraction from sorghum bagasse indicate that it is a unique carbohydrate polymer with more of the hemicellulose (60% arabinose + xylose) properties than of cellulose (39% glucose) properties. The sugar composition (relative mole %) of Cellulose-Rich Fraction (CRF) from biomass sorghum was glucose (53%), xylose (38%), and less than 10% each of and arabinose and galactose. The glycosyl linkage of CRF from biomass sorghum was predominantly 1, 4 linked glucose (64%), 1, 4 linked xylose (27%), and less than 10% each of six other linkages. The composition and linkages of the Cellulose-Rich Fraction from biomass sorghum indicate that it is a unique carbohydrate polymer with about half of the properties of cellulose (53% glucose) and half of the properties of hemicellulose (45% arabinose + xylose). Objective 4: We have continued to develop new methods to synthesize and purify a group of iso-oleic acid derivatives for use as fuel additives. We found that the first step in making the iso-oleic acid at the large scale level is achievable. The reaction conditions gave high yields of the desired products and high conversions of the starting fatty acids. The next step in making the ester derivatives from iso-oleic acid is currently being evaluated. Once the ester products are made, they will be sent to collaborators to evaluate as fuel additives. We have developed two strategies to remove sulfur species from biodiesel made from trap grease and waste water treatment scum. Distillation protocols have been developed that lower the concentration of sulfur- bearing species in biodiesel below the industry mandated 15 PPM in some samples. However, some of the sulfur-bearing species remain difficult to remove. Attempts to remove the remaining sulfur-bearing species by adsorption onto silica have also proven successful. Using gas chromatography with a sulfur detector and mass spectrometry we have successfully begun to characterize some of the sulfur-bearing species. We have adapted the ERRC in situ transesterification method to successfully make biodiesel from sorghum bran and from sorghum distillers dried grains and solubles. The chemical composition of this new type of biodiesel was analyzed and the fatty acid methyl esters were found to be similar to those of biodiesels made from other feedstocks and processes. We have confirmed that the remaining coproduct meal contains little or no residual oil, fatty acid methyl esters or other lipids. Objective 5: The economic impact of the new skeletal isomerization process for producing the isostearic acid was successfully evaluated using the SuperPro Designer computer software. The model predicted that the overall production cost of the isomerization process is competitive compared to the current technology. The process of our economic model was based on 10 million pounds of isostearic acid annually produced or 10% of the global market consumption. The results predicted that the unit production cost of the isostearic acid using our new process is competitive with the current technology. We have successfully worked with engineers to construct a batch mode 1 Liter size reactor which can run reactions at high pressures and high temperatures. With this batch reactor, a tenfold increase in the production of isostearic acid was achieved with an environmentally friendly and economically feasible solid catalyst. The process with this reactor was found to be reproducible. Furthermore, the distribution of the products and conversion were found to be very similar to the numbers from the small scale production. A significant amount of time was spent in the scale up of reaction conditions to make, purify and characterize the phenolic branched-chain lipids and their physical properties of the products are now being evaluated. The phenolic branched-chain lipids were found to be potent antimicrobials against Gram-positive bacteria including Listeria innocua, Bacillus subtilis, and Enterococcus faecium. Compared with the minimum inhibitory concentrations in the literature, our phenolic lipid compounds are much stronger than the common additives used by the industry. Unfortunately, preliminary results show that the compounds are less effective against Gram-negative bacteria and they do not have good antioxidant properties. Accomplishments 01 Using sorghum wax to produce Distillers Milo Oil. Ethanol plants occasionally use sorghum (milo) as a feedstock, and in the process produce Distillers Milo Oil. ARS researchers at Wyndmoor, Pennsylvania have evaluated the chemical composition of Distillers Milo Oil and have determined that these oils are like Distillers Corn Oil and that both could potentially be used for biodiesel and animal feed applications. Also, Distillers Milo Oil contains significantly higher levels of wax (1-2%), which can be recovered as wax. This sorghum wax has similar physical properties to commercial imported carnauba wax. Ethanol plants that are fermenting sorghum to produce Distillers Milo Oil can also produce sorghum wax as an additional new valuable coproduct.

Impacts
(N/A)

Publications

• Harron, A.F., Powell, M.J., Nunez, A., Moreau, R.A. 2017. Analysis of sorghum wax and carnauba wax by reversed phase liquid chromatography mass spectrometry. Industrial Crops and Products. 98:116-129.
• Qiu, S., Yadav, M.P., Yin, L. 2017. Characterization and functionalities study of hemicellulose and cellulose components isolated from sorghum bran, bagasse and biomass. Food Chemistry. 230:225-233.
• Jin, Q., Li, X., Cai, Z., Yadav, M.P., Zhang, H., Zhang, F. 2017. A comparison of corn fiber gum, hydrophobically modified starch, gum arabic and soybean soluble polysaccharide: interfacial dynamics, viscoelastic response at oil/water interfaces and emulsion stabilization mechanisms. Food Hydrocolloids. 70:329-344.
• Jin, Q., Cai, Z., Li, X., Yadav, M.P., Zhang, H. 2017. Comparative viscoelasticity studies: Corn fiber gum versus commercial polysaccharide emulsifiers in bulk and at air/liquid interfaces. Food Hydrocolloids. 64:85-98.
• Lew, H.N., Latona, R.J., Wagner, K., Nunez, A., Ashby, R.D., Dunn, R.O. 2016. Synthesis and low temperature characterization of iso-oleic ester derivatives. European Journal of Lipid Science and Technology. 118:1915- 1925.
• Yadav, M.P., Kale, M., Hicks, K.B., Hanah, K. 2017. Isolation, characterization and the functional properties of cellulosic arabinoxylan fiber isolated from agricultural processing by-products, agricultural residues and energy crops. Food Hydrocolloids. 63:545-551.
• Kale, M., Yadav, M.P., Hanah, K. 2016. Suppression of psyllium husk suspension viscosity by addition of water soluble polysaccharides. Journal of Food Science. 81(10):E2476-E2483.
• Liu, Y., Yadav, M.P., Chau, H.K., Yin, L. 2017. Peroxidase-mediated formation of corn fiber gum-bovine serum albumin conjugates: molecular and structural characterization. Carbohydrate Polymers. 166:114-122.
• Gashua, I.B., Williams, P.A., Yadav, M.P., Baldwin, T.C. 2016. Characterisation and molecular association of Nigerian and Sudanese Acacia gum exudates. Food Hydrocolloids. 51:405-413.
• Nwokocha, L.M., Senan, C., Williams, P.A., Yadav, M.P. 2017. Characterisation and solution properties of a galactomannan from bauhinia monandra seeds. International Journal of Biological Macromolecules. 101:904-909.
• Labourel, A., Crouch, L.I., Br�sb, J.L., Jackson, A., Rogowski, A., Grav, J., Yadav, M.P., Henrissat, B., Fontes, C.M., Gilbert, H.J., Najmudin, S., Basle, A., Cuskin, F. 2016. The mechanism by which arabinoxylanases can recognize highly decorated xylans. Journal of Biological Chemistry. 291(42) :22149-22159.
• Johnston, D., Moreau, R.A. 2017. A comparison between corn and grain sorghum fermentation rates, distillers dried grains with solubles composition, and lipid profiles. Bioresource Technology. 226:118-124.
• Singh, A., Geveke, D.J., Yadav, M.P. 2016. Improvement of rheological, thermal and functional properties of tapioca starch using gum arabic. LWT - Food Science and Technology. 80:155-162.
• Mendez-Encinas, M.A., Carvajal-Millan, E., Yadav, M.P., Valenzuela-Soto, E. M., Figueroa-Soto, C.G., Tortoledo-Ortiz, O., Garcia-Sanchez, G. 2016. Gels of ferulated arabinoxylans extracted from distillers dried grains with solubles: rheology, structural parameters and microstructure. In: Masuelli, M., and Renard, D. editors. Advances in Physicochemical Properties of Biopolymers. Part 1. Bentham Science, Emirate of Sharjah, United Arab Emirates. p. 202-214.
• Malhotra, B., Kharkwal, H., Yadav, M.P. 2017. Polymers targeting habitual diseases. In: Kharkwal, H., Srinivas J., editors. Natural Polymers for Drug Delivery. Oxfordshire, UK: CABI. p. 171-182.
• Malhotra, B., Kharkwal, H., Yadav, M.P. 2017. Cellulose based polymeric systems in drug delivery. In: Kharkwal, H., Srinvas, J., editors, Natural Polymers for Drug Delivery. Oxfordshire, UK: CABI. p. 10-21.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 1. Develop processes to fractionate sorghum and corn/sorghum oils into new commercially-viable coproducts. 2. Develop processes to fractionate grain-derived brans into new commercially-viable coproducts. 2a: Develop processes to fractionate grain-derived brans into new commercially-viable coproducts such as lipid-based coproducts and for other industrial uses such as extrusion or producing energy or fuel. 2b: Develop commercially-viable, value-added carbohydrate based co- products from sorghum brans and the brans derived from other grains during their biorefinery process. 3. Develop processes to fractionate biorefinery-derived celluloses and hemicelluloses into new commercially-viable coproducts. 3a: Develop commercially-viable, value-added hemicellulose based co- products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 3b: Develop commercially-viable, value-added cellulose based co-products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 4. Develop technologies that enhance biodiesel quality so as to enable greater market supply and demand for biodiesel fuels and >B5 blends in particular. 4a: Improve the low temperature operability of biodiesel by chemical modification of the branched-chain fatty acids. 4b: Develop technologies that significantly reduce quality-related limitations to market growth of biodiesel produced from trap and float greases. 4c: Further develop direct (in situ) biodiesel production so as to enable its commercial deployment. 5. Develop technologies that enable the commercial production of new products and coproducts at lipid-based biorefineries. 5a: Enable the commercial production of alkyl-branched from agricultural products and food-wastes. 5b: Enable the commercial production of aryl-branched fatty acids produced from a combination of lipids and natural antimicrobials possessing phenol functionalities. Approach (from AD-416): In conjunction with CRADA partners and other collaborators, develop technologies that identify new biorefinery coproducts, evaluate their applications and estimate their profitability and marketability. The approach will focus on development processes to produce several types of new coproducts. First, processes will be developed to extract and fractionate sorghum oil from sorghum kernels and sorghum bran. Processes will also be developed to extract and fractionated cellulose-rich and hemicellulose-rich fractions from sorghum kernels, sorghum bran, sorghum bagasse, and biomass sorghum. Other processes will be developed to improve the biofuel value of biodiesel by blending biodiesel with modified fatty acid derivatives to enhance its low temperature performance, reduce the levels of impurities that block fuel lines, economically convert trap grease and float grease to biodiesel, and improve the in situ process to make biodiesel directly from oil-rich low value agricultural products. In addition to biodiesel applications, other processes will be developed to produce branched fatty acids with unique functional (including improved lubricity) and biological properties (including antimicrobial and antioxidant properties). Objective 1: The new HPLC (high performance liquid chromatography) method which we developed in the previous year has been further improved to analyze and compare sorghum waxes and other commercial waxes (carnauba wax, candelilla wax, sunflower wax, rice bran wax and beeswax). We have also used the new method to quantitatively analyze the levels of sorghum wax in sorghum oil obtained by solvent extraction and in distillers milo (sorghum) oil obtained from commercial sorghum ethanol plants. Because some corn ethanol plants also ferment blends of corn and sorghum, our method can be used to quantify the amount of sorghum wax in blends which contain both distillers corn oil and distillers milo oil. Objective 2: We have continued to study the chemical properties of sorghum brans, obtained by decortication using a scarifier. We have found that the bran from sorghum contains compounds that are different than those from corn and other grains and we are in the process of identifying these compounds. These compounds can form problematic precipitates during both aqueous and organic solvent extraction so it is important that they be identified and evaluated for potential applications. Objective 3: The proximate composition of the arabinoxylan and cellulose rich fractions isolated from sorghum bran was determined and the emulsifying and rheological properties of arabinoxylan were studied. The proximate composition of arabinoxylans isolated from sorghum biomass and sorghum bagasse was determined and their emulsifying and rheological properties were studied. The proximate composition of the cellulose rich fraction isolated from sorghum biomass and sorghum bagasse was determined. A manuscript on these results has been written and submitted to a journal for publication. Objective 4: In collaboration with an ARS scientist from the National Center for Agricultural Utilization Research, introduction of esters with a bulky alkyl alcohol group (isopropyl, 2-butyl, and 2-ethylhexyl) to the �headgroup� of the skeletal unsaturated branched-chain fatty acid (iso- oleic) materials were investigated. These ester alcohols were chosen because of their bulky and branched-chain alkyl groups which can disrupt the crystal nucleation and growth mechanisms at low temperatures to reduce the melting point of these fats. These ester derivative fats are liquid at room temperature with enhanced fluidity. The results showed that these ester fats demonstrated improved cloud points (temperature where solid crystals become visible). The results also showed that they performed much better than the original parent fatty acids and saturated fats. This research demonstrated that the crystal structures can be significantly altered by simply chemically modifying the headgroup or tailgroup of esters. These findings are important as these iso-oleate ester fats can potentially replace solid fats which are often problematic at low temperatures. They can also potentially improve the cold flow properties in biodiesel when they are mixed with biodiesel, due to their capability to disrupt the crystal growth mechanism. When biodiesel is produced from trap grease and float grease it often contains levels of sulfur that exceed the 15 ppm U.S. limits. Distillation by Wiped-Film Evaporation has been employed to fractionate the sulfur containing molecules into 3 primary temperature ranges that are designed to isolate low molecular weight and highly volatile impurities (low cut) from the high molecular weight and less volatile impurities (high cut). This process produces clean biodiesel in the middle cut that meets American Society for Testing and Materials (ASTM) specifications for all parameters except for sulfur concentration. To find economical and environmentally friendly ways to strategically remove the sulfur, those species must be identified. In collaboration with scientists from Drexel University, significant progress has been made in the quantification, isolation and identification of sulfur species in trap grease and float grease biodiesels by use of solid phase extraction, total sulfur analysis, and GC-MS. The U.S. biodiesel industry currently generates about 2 billion gallons of biodiesel per year. The production goal by 2020 is 4 billion gallons per year. To make this a reality, additional feedstocks must be identified. While devoid of sugar, post-fermentation solids from sorghum and other grains contain up to 10% oil and is a potential feedstock for making biodiesel. Previous studies, primarily conducted by ARS scientists, have shown that the in-situ transesterification (I.S.T.) method can be used to make biodiesel from oil-bearing solids such as flaked soybeans and corn-derived DDGs. In this project, it has been proven that I.S.T. can be used to produce biodiesel from sorghum bran and sorghum distillers dried grains and solubles (DDGD). HPLC is used to quantify biodiesel yields and to quantify the amount of unreacted free fatty acids that remain in the reaction mixture. Objective 5: To ensure accurate assessment of the potential utility of the isostearic acid products as biolubricants, the three byproducts (saturated linear-chain fatty acids, lactones, and dimer fatty acids) should be should be removed from the crude isostearic acid mixture. First, the mixture was recrystallized to remove the saturated acids (stearic and palmitic acids) at low temperature (-15 degrees C) in the presence of solvents. The recrystallized products were transferred to an additional funnel attached to a wiped film molecular distillation device. The products were slowly added to the evaporator to separate the dimer products. Although these two purification steps (recrystallization and distillation) can successfully remove the saturated acids and dimer acids, they are not sufficient to remove the lactones. Ongoing research efforts will be invested to reduce the lactones to an acceptable level. ARS scientists developed a systematic regeneration technique for the spent zeolite to activate them so that one sample of zeolite can be used for many cycles of isomerization reaction to produce isostearic acid. This invented method will help to reduce the overall cost of this technology when it will be used in the commercial scale production. With this method, we mainly use heat treatment to activate the fresh zeolite and regenerate the spent zeolites instead of doing acid treatment which generates an abundance of acid waste which is costly to handle at industrial scale. It has been found that after using in 10 cycles of isomerization reaction, the zeolite catalyst is still capable of yielding high selectivity of isostearic acid. Phenolic branched-chain lipids made from the ARS technology are a complex mixture of products. Therefore, it is important to develop methodology to purify the products efficiently in order to determine their effectiveness in killing bacteria. A highly efficient wiped film distillation technique has been developed to distill the products. The results showed that up to 95% purity of these phenolic branched-chain lipids could be obtained. Most importantly, regardless of which phenolics (i.e., thymol, carvacrol, and creosote which have strong antimicrobial properties, which all posses unpleasant characteristic odors) after coupling onto the fatty acids, these compounds which mimic the existing phenolics no longer have the unpleasant odors. Using this distillation technique, a significant amount of products can be obtained which have been evaluated as antimicrobial agents. The preliminary results show that the phenolic branched-chain lipids have potential to kill bacteria. Accomplishments 01 Development of the first successful HPLC method to analyze sorghum wax and commercial waxes. ARS researchers in Wyndmoor, Pennsylvania developed a new HPLC (high performance liquid chromatography) method to quantitatively analyze sorghum wax. This new method is valuable because it provides an accurate method to quantify sorghum wax in sorghum oil, distillers milo oil, and in sorghum grain processing fractions such as bran and distillers dried grains and solubles (DDGS). It will also be very useful for the analysis of commercial waxes such as carnauba wax, candelilla wax, sunflower wax, rice bran wax and beeswax. It is the first successful HPLC method for waxes. Gas chromatography has traditionally been used for analysis of waxes but it has drawbacks and inaccuracies because it uses very high temperatures and the wax components can break down during analysis. This new method employs an evaporative light scattering detector for quantification and LC-mass spectrometry for chemical structural analysis. Previous attempts to develop HPLC methods for waxes have mainly focused on C8 and C18 HPLC columns, but this new method uses a C30 column and uses methanol and chloroform as solvents, which are the best solvents to solubilize all of the wax components. It is anticipated that this new method will become widely used for commercial wax analysis.

Impacts
(N/A)

Publications

• Moreau, R.A., Harron, A.F., Powell, M.J., Hoyt, J.L. 2016. A comparison of the levels of oil, carotenoids, and lipolytic enzyme activities in modern lines and hybrids of grain sorghum. Journal of the American Oil Chemists' Society. 93:569-573.
• Lehtonen, M., Teraslahti, S., Xu, C., Yadav, M.P., Lampi, A., Mikkonen, K. S. 2016. Spruce galactoglucomannans inhibit the lipid oxidation in rapeseed oil-in-water emulsions. Food Hydrocolloids Journal. 58:255-266.
• Berlanga-Reyes, C., Carvajal-Millan, E., Hicks, K.B., Yadav, M.P., Rasconchu, A., Lizardi-Mendoza, J., Islas-Rubio, A.R. 2014. Protein/ Arabinoxylans Gels: Effect of mass ratio on the rheological, microstructural and diffusional characteristics. International Journal of Molecular Sciences. 15:19106-19118.
• Cirre, J., Al-Assaf, S., Phillips, G.O., Yadav, M.P., Hicks, K.B. 2013. Improved emulsification performance of corn fiber gum following maturation treatment. Food Hydrocolloids, 35:122-128.
• Zhang, F., Luan, T., Kang, D., Zhang, H., Yadav, M.P. 2014. Viscofying properties of corn fiber gum with various polysaccharides. Food Hydrocolloids Journal. 43:218-227.
• Samala, A., Srinivasan, R., Yadav, M.P. 2014. Comparison of Xylo- oligosaccharides production by autohydrolysis of fibers separated from ground corn flour and DDGS. Journal of Food and Bioproducts Processing. 94:354-364.
• Qiu, S., Yadav, M.P., Chen, H., Liu, Y., Tatsumi, E., Yin, L. 2014. Effects of corn fiber gum (CFG) on the pasting and thermal behaviors of maize starch. Carbohydrate Polymers. 115:246-252.
• Moreau, R.A., Fang, X. 2016. Analysis of alkylresorcinols in wheat germ oil and barley germ oil via HPLC and flourescence detection: Cochromatography with tocols. Cereal Chemistry. 93(3):293-298.
• Qiu, S., Yadav, M.P., Tatsumi, E., Yin, L. 2015. Effects of corn fiber gum with different molecular weights on the gelatinization behaviors of corn and wheat starch. Food Hydrocolloids Journal. 53:180-186.
• Fang, X., Moreau, R.A. 2014. Extraction and demulsification of oil from wheat germ, barley germ, and rice bran using an aqueous enzymatic method. Journal of the American Oil Chemists' Society. 91:1261-1268.
• Yadav, M.P., Hicks, K.B. 2015. Isolation of barley hulls and straws constituents and study of emulsifying properties of their arabinoxylans. Carbohydrate Polymers. 132:529-536.
• Lin, X., Ma, L., Moreau, R.A., Ostlund, Jr, R.E. 2011. Glycosidic bond cleavage is not required for phytosteryl glycoside-induced reduction of cholesterol absorption in mice. Lipids Journal. 46:701-708.
• Lew, H.N., Yee, W.C., Mcaloon, A.J., Haas, M.J. 2014. Techno-economic analysis of an improved process for producing saturated branched-chain fatty acids. Journal of Agricultural Science. 6(10):158-168.
• Wyatt, V.T. 2012. Effects of swelling on the viscoelastic properties of polyester films made from glycerol and glutaric acid. Journal of Applied Polymer Science. 126:1784-1793.
• Wyatt, V.T., Yadav, M.P. 2013. A multivariant study of the absorption properties of poly(glutaric-acid-glycerol) films. Journal of Applied Polymer Science. 130(1):70-77.
• Kokubun, S., Yadav, M.P., Moreau, R.A., Williams, P.A. 2014. Components responsible for the emulsification properties of corn fibre gum. Food Hydrocolloids Journal. 41:164-168.
• Rogowski, A., Briggs, J.A., Mortimer, J.C., Tryfona, T., Terrapon, N., Lowe, E.C., Basle, A., Day, A.M., Zheng, H., Rogers, T.E., Yadav, M.P., Henrissat, B., Martens, E.C., Dupree, P., Gilbert, H.J., Bolam, D.N. 2015. Glycan complexity dictates microbial resource allocation in the large intestine. Nature Communications. 6:1-15. doi: 10.1038/ncomms8481.
• Lew, H.N., Hoh, E., Foglia, T. 2012. Improved synthesis and characterization of saturated branched-chain fatty acid isomers. European Journal of Science and Lipid Technology. 114:213-221.

Progress 10/01/14 to 09/30/15

Outputs
Progress Report Objectives (from AD-416): 1. Develop processes to fractionate sorghum and corn/sorghum oils into new commercially-viable coproducts. 2. Develop processes to fractionate grain-derived brans into new commercially-viable coproducts. 2a: Develop processes to fractionate grain-derived brans into new commercially-viable coproducts such as lipid-based coproducts and for other industrial uses such as extrusion or producing energy or fuel. 2b: Develop commercially-viable, value-added carbohydrate based co- products from sorghum brans and the brans derived from other grains during their biorefinery process. 3. Develop processes to fractionate biorefinery-derived celluloses and hemicelluloses into new commercially-viable coproducts. 3a: Develop commercially-viable, value-added hemicellulose based co- products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 3b: Develop commercially-viable, value-added cellulose based co-products from sorghum biomass, sorghum bagasse and other agricultural based biomasses produced during their biorefining. 4. Develop technologies that enhance biodiesel quality so as to enable greater market supply and demand for biodiesel fuels and >B5 blends in particular. 4a: Improve the low temperature operability of biodiesel by (1) chemical modification of the branched-chain fatty acids and (2) reduce levels of contaminants that block fuel filters (e.g., sterol glucosides and saturated monoglycerides). 4b: Develop technologies that significantly reduce quality-related limitations to market growth of biodiesel produced from trap and float greases. 4c: Further develop direct (in situ) biodiesel production so as to enable its commercial deployment. 5. Develop technologies that enable the commercial production of new products and coproducts at lipid-based biorefineries. 5a: Enable the commercial production of alkyl-branched from agricultural products and food-wastes. 5b: Enable the commercial production of aryl-branched fatty acids produced from a combination of lipids and natural antimicrobials possessing phenol functionalities. Approach (from AD-416): In conjunction with CRADA partners and other collaborators, develop technologies that identify new biorefinery coproducts, evaluate their applications and estimate their profitability and marketability. The approach will focus on development processes to produce several types of new coproducts. First, processes will be developed to extract and fractionate sorghum oil from sorghum kernels and sorghum bran. Processes will also be developed to extract and fractionated cellulose-rich and hemicellulose-rich fractions from sorghum kernels, sorghum bran, sorghum bagasse, and biomass sorghum. Other processes will be developed to improve the biofuel value of biodiesel by blending biodiesel with modified fatty acid derivatives to enhance its low temperature performance, reduce the levels of impurities that block fuel lines, economically convert trap grease and float grease to biodiesel, and improve the in situ process to make biodiesel directly from oil-rich low value agricultural products. In addition to biodiesel applications, other processes will be developed to produce branched fatty acids with unique functional (including improved lubricity) and biological properties (including antimicrobial and antioxidant properties). Objective 1. Numerous samples of modern grain sorghum hybrids have been obtained. Oil, carotenoids, and waxes have been extracted and yields of all three have been compared using several different types of extraction. A new HPLC (high performance liquid chromatography) method to analyze sorghum waxes and carotenoids was developed. Methods were developed to fractionate sorghum oil into fractions that are enriched in waxes and carotenoids. Objective 2. Samples of several sorghum hybrids were decorticated using a pearler and a scarifier and the resulting bran fractions were extracted and compared. Developed a process to produce of arabinoxylans (AX) and cellulose rich fractions (CRF) from brans obtained from sorghum and other grains. Objective 3. Developed a process to produce arabinoxylan (AX) from sorghum biomass, sorghum bagasse and other agricultural based biomasses. Developed a process to produce a cellulose rich fraction (CRF) from sorghum biomass, sorghum bagasse and other agricultural based biomasses. Objective 4. In collaboration with the ARS scientist from National Center for Agricultural Utilization Research (NCAUR), a series of isostearate bulky ester derivatives were synthesized and their low temperature properties were evaluated. Introduction of bulky functional groups to the isostearic acid head is the most direct way to create fatty acids with low melting points with enhanced fluidity. Three new ester derivatives were synthesized from the isostearic acids using the esterification method, which is an efficient method for converting fatty acids to fatty acid esters in the presence of liquid acids (i.e., sulfuric acid, phosphoric acid, hydrochloric acid) and alcohols. This research was completed and the results showed that these synthesized esters have much better low melting points than the original fatty acids. We have made progress in sub objectives. Those include work to reduce the level of contaminants that block fuel filters by the formation of solids at or below the cloud point and to reduce sulfur content in biodiesel below 15 ppm. Solids that form in a diesel fuel can block fuel filters, resulting in engine failure due to fuel starvation. We have recently addressed this problem by attempting to depolymerize lignin into a biodiesel-soluble additive that will reduce the CP of biodiesel. Among potential new feedstocks are the �brown� or �trap� greases collected from commercial food preparation facilities. However, recent attempts to market biodiesel from these feedstocks have failed due to the inability to reduce the sulfur content of the product below 15 ppm. In collaboration with Drexel University, we have been able to successfully reduce the sulfur content to desirable levels. Work to ensure reproducibility and to extend the technology to float greases are currently underway. Objective 5. With the support from a NIFA-AFRI funded project, entitled �Development of Environmentally Friendly and Economically Feasible Engineering Processes for High-Value Biobased Products�, ARS scientists found a zeolite-Lewis base additive combination catalyst system which could efficiently produce the isostearic acid products at excellent yields. Without the Lewis base additives, the resulting products contain greater amounts of unwanted polymeric byproducts. To broaden the utility of the catalytic system, nonfood use feedstocks were used to make these isostearic acid products. However, since these products were much more complex, continued research efforts were needed at the analysis stage in order to efficiently characterize the isostearic acid products. Phenolics such as cinnamaldehyde, thymol and carvacrol have strong antimicrobial properties, but they possess unpleasant characteristic odors which prevent their use in the food industry. The research project focused on producing phenolic fatty acid compounds which mimic the existing phenolics but without the unpleasant odors. These compounds were made from two streams of natural materials. This reaction system showed that it was capable of producing the desired phenolic fatty acid compounds. Analytical methods to characterize the products were developed.

Impacts
(N/A)

Publications

• Dunn, R.O., Lew, H.N., Haas, M.J. 2015. Branched-chain fatty acid methyl esters as cold flow improvers for biodiesel. Journal of the American Oil Chemists' Society. 92(6):853-869. DOI: 10.1007/s11746-015-2643-2.
• Lew, H.N. 2015. Lewis base additives improve the zeolite ferrierite- catalyzed synthesis of isostearic acid. Journal of the American Oil Chemists' Society. 92:613-619.
• Yadav, M.P., Hicks, K.B., Johnston, D., Hotchkiss, A.T., Chau, H.K., Hanah, K. 2015. Production of bio-based fiber gums from the waste streams resulting from the commercial processing of corn bran and oat hulls. Food Hydrocolloids Journal. DOI: 10.1016/j.foodhy.2015.02.017.
• Liu, Y., Qiu, S., Li, J., Chen, H., Tatsumi, E., Yadav, M.P., Yin, L. 2014. Peroxidase mediated conjugation of corn fibeer gum and bovine serum albumin to improve emulsifying properties. Carbohydrate Polymers. 118:70- 78.
• Brooks, W.S., Vaughn, M.E., Berger, G.L., Griffey, C.A., Thomason, W.E., Pitman, R.M., Malla, S., Seago, J.E., Dunaway, D.W., Hokanson, E.G., Behl, H.D., Beahm, B.R., Schmale, D.G., Mcmaster, N., Hardiman, T., Custis, J.T., Starner, D.E., Gulick, S.A., Ashburn, S.R., Jones, E.H., Marshall, D.S., Fountain, M.O., Tuong, T.D., Kurantz, M.J., Moreau, R.A., Hicks, K.B. 2014. Registration of �Atlantic� winter barley. Journal of Plant Registrations. 8:231-236.
• Moreau, R.A. 2015. Composition of plant sterols and stanols in supplemented food products. Journal of AOAC International. 98:670-685.
• Wyatt, V.T. 2014. The effects of solvent polarity and pKa on the absorption of solvents into poly(glutaric acid-glycerol) films. Journal of Applied Polymer Science. 131(13):40434-40440.
• Kale, M.S., Yadav, M.P., Hicks, K.B., Hanah, K. 2015. Concentration and shear rate dependence of solution viscosity for arabinoxylans from different sources. Food Hydrocolloids Journal. 47:178-183.