Progress 04/01/03 to 03/31/09
Outputs
OUTPUTS: We have been able to modify a number of fats and oils to produce structured lipids suitable for application as trans-free margarines, use as human milk fat substitute in infant formula or caprine milk infant formula analogs. A review of the enzymatic approach to biodiesel production was published to enable other researchers understand what has been done and where the enzyme and biodiesel technology is going. Georgia-grown crops such as blueberries, onions, blackberries, and muscadine grapes have been characterized to report the phenolics, flavonoids (mainly anthocyanins), and anitoxidant capacities of the fractions. Studies on the cancer preventing abilities of these crops were performed using colon cancer, liver cancer cell lines and rats as animal model. Overall, blueberry and muscadine fractions were effective on preventing cancer cell growth, and DNA damage in the liver.The effect of processing on the biological activities of the phenolic compounds were also reported. The results of this Hatch project were diseminated through presentations at scientific conferences in USA and abroad and through publications in refereed journals. PARTICIPANTS: Joan G. Fisher, Gerard Krewer,were UGA collaborators on the flavonoids work. Colleagues from Taiwan (Jei-Fu Shaw's lab) helped with biodiesel work and supplied Candida rugosa Isoform 1 enzyme. Young Park from Fort Valley collaborated on the caprine infant formula analogs work. Various graduate students and visiting scientists contributed to this work. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The solid fat content and crystal properties of trans-free structured lipids were optimized by blending with other fats and emulsifiers. Obtaining the desired crystal polymorphism of fats intended for margarine application is a delicate process that may not be achieved by physical blending of fats and oils or even by enzymatic interesterification. However, the our results have shown some light on the best ways to obtain desirable crystals and desirable modified fats for specific applications. We tested the hypothesis that blueberies would reduce DNA damage and lipid peroxidation and increase phase II enzyme activities in vivo using male Sprague-Dawley rats. Our results indicated that supplementation with 1% bluberry extract (anthocyanin) was able to decrease oxidative DNA damage in the liver. Our results add to the existing knowledge that there is an association between diets rich in fruits and vegetables and reduced chronic diesease risks. We could not have been able to do this without Federal grants from CSREES/USDA/NRI, Hatch and State support, and the postdoctoral and graduate students who worked on the various projects.
Publications
- Kim, B.H., Sandock, K.D., Robertson, T.P., Lewis, S.J. and Akoh, C.C. 2008. Dietary effects of sesame oil-based structured lipids and phytosteryl esters on blood lipid profiles and cardiovascular parameters in spontaneously hypertensive rats. Lipids 43:55-64.
- Lee, J.H., Akoh, C.C., and Lee, K.T. 2008. Physical properties of trans-free bakery shortening produced by lipase-catalyzed interesterification. J. Am. Oil Chem. Soc. 85:1-11.
- Shaw, J.F., Chang, S.W., Lin, S.C., Wu, T.T., Ju, H.Y., Akoh, C.C., Chang, R.H., and Shieh, C.J. 2008. Continuous enzymatic synthesis of biodiesel with Novozym 435. Energy Fuels 22:840-844.
- Lee, J.H., Akoh, C.C., Himmelsbach, D.S., and Lee, K.T. 2008. Preparation of interesterified plastic fats from fats and oils free of trans fatty acid. J. Agric. Food Chem. 56:4039-4046.
- Kim, B.H., Lumor, S.E., and Akoh, C.C. 2008. Trans-free margarines prepared with canola oil/palm stearin/palm kernel oil-based structured lipids. J. Agric. Food Chem. 56:8195-8205.
- Akoh, C.C., Chang, S.W., Lee, G.C., and Shaw, J.F. 2008. Biocatalysis for the production of industrial products and functional foods from agricultural produce. J. Agric. Food Chem. 56:10445-10451.
- Lumor, S.E., Kim, B.H., and Akoh, C.C. 2008. Optimization of solid fat content and crystal properties of a trans-free structured lipid by blending with palm midfraction. J. Agric. Food Chem. 56:9294-9298.
- Lumor, S.E. and Akoh, C.C. 2008. Esterification and hydrolytic activities of Candida rugosa lipase isoform1 (LIP 1) immobilized on Celite 545, Duolite A7, and Sephadex G-25. J. Agric. Food Chem. 56:10396-10398.
- Dulebohn, R.V., Yi, W., Srivastava, A., Akoh, C.C., Krewer, G., and Fischer, J.G. 2008. Effects of blueberry (Vacicinium ashei) on DNA damage, lipid peroxidation, and phase II enzyme activities in rats. J. Agric. Food Chem. 56:11700-11706.
- Maduko, C.O., Park, Y.W., and Akoh, C.C. 2008. Characterization and oxidative stability of structured lipids: infant milk fat analog. J. Am. Oil Chem. Soc. 85:197-204.
- Vu, P.L., Shin, J.A., Lee, Y.J., Nam, H.Y., Lee, J.H., Akoh, C.C., and Lee, K.T. 2008. Development and characterization of structured lipids containing capric and conjugated linoleic acids as functional dietary lipid molecules. Intl. J. Food Sci. Nutr. 59:95-104.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Total polyphenols (TPP), total anthocyanin (TACY), Trolox-equivalent antioxidant capacity (TEAC), phenolic acids, and individual anthocyanins of blueberry extract stored in glass bottles and ability of blueberry extract to inhibit cell proliferation were studied. Extract was stored at -20, 6, 23, and 35C. Recovery of TPP, TACY and TEAC in blueberry extract after pressing and heating were ~25, ~29, and ~69%, respectively for both Tifblue and Powderblue cultivars. Losses due to storage were less when compared with initial loss due to processing. At -20C, no statistically significant loss of TPP, TACY and TEAC was observed up to 30 days. At 6C storage, there was a significant loss observed from 15 to 30 days. There was retention of more than 40% of ellagic and quercetin after 60 days at 35C. Anthocyanins were not detected after 60 days of storage at 35C. Significant retention (P < 0.05) was obtained for malvidin (42.8 and 25.8%) and peonidin (74.0 and 79.5%) after 60 days
storage at 23C in glass bottles for Tifblue and Powderblue, respectively. Our study confirmed that anthocyanin fractions from highbush blueberry cultivars increased apoptosis using DNA fragmentation and caspase-3 activity. Tripalmitin was interesterified with vegetable oil blends and incorporated into skim caprine milk to yield goat milk-based infant formula analog. The formulated caprine milk with a molar ratio of 1:1 and a 12-h incubation was similar in fatty acid composition of human milk. We also synthesized infant milk fat analogs resembling human milk fat by interesterifying tripalmitin, coconut oil, safflower oil, and soybean oil using response surface methodology (RSM). Trans-free structured lipids were synthesized using lipases. Response surface methodology was used to study the effects of Lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl
stearate gave the best result. Stearic acid was incorporated into canola oil, mainly at the sn-1, 3 positions for the lipase reaction and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta prime crystal forms for tub margarine, although these may be suitable for light margarine formulation. The effect of a series of emulsifier preparations on the crystal forming habits and morphology of the blends was also investigated. The emulsifiers used were sucrose stearate (SE), sorbitan stearate (ST), distilled monoglycerides (DMG) and blends of the three in the ratios (SE:ST:DMG) of 1:1:1, 1:2:3, 2:3:1 and 3:1:2. Samples treated with DMG and the 1:2:3 blend showed the best improvements. DMG seemed to be more suitable than the other emulsifiers for regulating crystal types and morphology. Other structured lipids were produced and characterized. We fed spontaneously hypertensive rats sesame oil-based structured lipids and phytosteryl esters and
observed their effect on cardiovascular function.
PARTICIPANTS: Joan Fischer and Gerard Krewer were collaborators from the university of Georgia. Byung Hee Kim, Stephen Lumor, and Catherine Maduko were graduate students. The USDA/ARS/ERRC helped with the NMR and structural analysis of the structured lipids. Young Park was a colloborator from Fort Valley University.
Impacts
Our results suggest that heating and storage conditions were significantly affecting the phenolic compounds and their biological activity. Frozen and low temperature storage is suggested for blueberry extract in order to retain the bioactive components.We found that prolonged storage of blueberry extract at room temperature significantly reduces the phenolic compounds and their biological activities. However, no difference in total polyphenols, total anthocyanins, and antioxidant capacity as measured by TEAC was observed among the two cultivars (Powderblue and Tifblue) studied. Infant formula analogs to replace human milk fat were successfully synthesized using lipases. The placement of significant amount of palmitic acid at the sn-2 position of the triacylglycerols will increase calcium absorption by infants and yet provide the needed energy for growth and development. Trans-free structured lipids were successfully prepared and this may serve as a healthy alternative
to hydrogenated fats used in margarine formulations. It is important to obtain the desirable crystal structure for structured lipids intended to replace hydrogenated fats in margarine and shortening applications. Consumption of structured lipids with phytosteryl esters may have beneficial effects on some cardiovascular parameters in rats.
Publications
- Maduko, C.O., Akoh, C.C. and Park, Y.W. 2007. Enzymatic interesterification of tripalmitin with vegetable oil blends for formulation of caprine milk infant formula analogs. J. Dairy Sci. 90:594-601. Karaali, A., Sahin, N. and Akoh, C.C. 2007. Marine oils in infant formulae. Inform 18:17-19.
- Vu, P.L., Park, R.K., Lee, Y.J., Kim, Y.M., Nam, H.Y., Lee, J.H., Akoh, C.C. and Lee, K.T. 2007. Two-step production of oil enrinched in conjugated linoleic acids and diacylglycerol. J. Am. Oil Chem. Soc. 84:123-128.
- Maduko, C.O., Akoh, C.C. and Park, Y.W. 2007. Enzymatic production of infant milk fat analogs containing palmitic acid: optimization of reactions by response surface methodology. J. Dairy Sci. 90:2147-2154.
- Srivastava, A., Akoh, C.C., Yi, W., Fischer, J. and Krewer, G. 2007. Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles. J. Agric. Food Chem. 55:2705-2713.
- Lee, J.H., Yu, F., Vu, P.L., Choi, M.S., Akoh, C.C. and Lee, K.T. 2007. Compositional study on rice bran oil after lipase-catalyzed glycerolysis and solvent fractionations. J. Food Sci. 72:C163-C-167.
- Srivastava, A., Akoh, C.C., Fischer, J. and Krewer, G. 2007. Effect of anthocyanins fractions from selected cultivars of Georgia-grown blueberries on apoptosis and phase-II enzymes. J. Agric. Food Chem. 55:3180-3185.
- Lee, J.H., Akoh, C.C., and Lee, K.T. 2007. Physicochemical and volatiles characterization of trans-free solid fats produced by lipase-catalyzed interesterification. J. Food Sci. 72:E368-374.
- Kim, B.H., Sandock, K.D., Robertson, T.P., Lewis, S.J. and Akoh, C.C. 2007. Dietary effects of structured lipids and phytosteryl esters on cardiovascular function in spontaneously hypertensive rats. J. Cardiovasc. Pharmacol. 50:176-186.
- Akoh, C.C., Chang, S.W., Lee, G.C., and Shaw, J.F. 2007. Enzymatic approach to biodiesel production. J. Agric. Food Chem. 55:8995-9005.
- Lumor, S.E., Jones, K.C., Ashby, R., Strahan, G., Kim, B.H., Lee, G.C., Shaw, J.F., Kays, S.E., Chang, S.W., Foglia, T.A. and Akoh, C.C. 2007. Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application. J. Agric. Food Chem. 55:10692-10702.
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Progress 01/01/06 to 12/31/06
Outputs
We found that phenolic compounds in Georgia-grown blueberries and muscadine grapes can inhibit HepG2 cell proliferation and induce programmed cell death (apoptosis). We evaluated the absorption of anthocyanins fractions from blueberries using Caco-2 human intestinal cell monolayers. We found that anthocyanins can be transported through the Caco-2 cell monolayers. We showed that having more free hydroxyl groups and less methoxyl groups can decrease the bioavailability of anthocyanins. Various structured lipids were prepared with lipases to simulate human milk fat substitutes and to contain omega-3 polyunsaturated fatty acids (EPA and DHA). Phytosteryl esters and sesame oil-based structured lipids were also synthesized and characterized.
Impacts
The ability to modify natural fats and oils by adjusting their fatty acid composition with the introduction of new and beneficial fatty acids has implications in the development of functional and nutraceutical lipids. Milk fat substitutes suitable for infant formula formulations were successfully synthesize with potential to help with infant development and nutrition. A healthy cold-spreadable butter substitute was also prepared with vegetable oil. Phytosteryl esters and structured lipids may help lower cholesterol and the study is on-going. The ability of anthocyanins to be transported across intestinal monolayer is a significant finding with implication in cancer prevention and their bioavailability.
Publications
- Yi, W., Akoh, C.C., Fischer, J. and Krewer, G. 2006. Effects of phenolic compounds in blueberries and muscadine grapes on HepG2 cell viability and apoptosis. Food Res. Int. 39:628-638.
- Sahin, N., Akoh, C.C. and Karaali, A. 2006. Human milk fat substitutes containing omega-3 fatty acids. J. Agric. Food Chem. 54:3717-3722.
- Yi, W., Akoh, C.C., Fischer, J. and Krewer, G. 2006. Absorption of anthocyanins from blueberry extracts by Caco-2 human intestinal cell monolayers. J. Agric. Food Chem. 54:5651-5658.
- Kim, B.H. and Akoh, C.C. 2006. Characteristics of structured lipid prepared by lipase-catalyzed acidolysis of roasted sesame oil and caprylic acid in a bench-scale packed bed reactor. J. Agric. Food Chem. 54:5132-5141.
- Kim, B.H. and Akoh, C.C. 2006. Modeling and optimization of lipase-catalyzed synthesis of phytosteryl esters of oleic acid by response surface methodology. Food Chem. 102:336-342.
- Srivastava, A., Akoh, C.C., Chang, S.W., Lee, G.C. and Shaw, J.F. 2006. Candida rugosa lipase LIP1-catalyzed transesterification to produce human milk fat substitute. J. Agric. Food Chem. 54:5175-5181.
- Kim, B.H. and Akoh, C.C. 2006. A healthy cold-spreadable butter. Inform 17:547-548.
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Progress 01/01/05 to 12/31/05
Outputs
Lipases were used to modify palm olein to incorporate caprylic acid and the physical properties of the structured lipid (SL) product determined. Caprylic acid was also incorporated into canola oil and used to prepare butterfat-vegetable oil blend spread. Sensory evaluation, physical and chemical properties of the spreads were measured. Our results indicate that SL can counterbalance the hypercholesterolemic attributes of butterfat and improve its cold-spreadability without any adverse effect on the flavor attributes of the spread. SLs enriched with gamma-linolenic (GLA) and linoleic acids or stearic acid were prepared with palm kernel oil/palm olein blend. SLs resembling human milk fat substitutes for possible use in infant formulas were prepared using hazelnut oil and tripalmitin and/or GLA. We found that phenolic compounds in blueberries and muscadine grapes can induce apoptosis and inhibit colon cancer cell proliferations with anthocyanin having the greatest
effect.
Impacts
Synthesis of structured lipids (SLs) enriched with essential fatty acids and other fatty acids to improve their use in foods and for infant formula was achieved and may have health benefits. The ability to arrest cancer cell proliferation by specific fractions of blueberries and muscadine grapes is a positive step towards our understanding of the mecahnisms and possible treatments for colon and liver cancer. Overall, these studies have the potential to improve human health and provide healthier foods to the consumer.
Publications
- Lumor, S.E. and Akoh, C.C. 2005. Incorporation of gamma-linolenic and linoleic acids into a palm kernel oil/palm olein blend. Eur. J. Lipid Sci. Technol. 107:447-454.
- Yi, W., Fischer, J., Krewer, G. and Akoh, C.C. 2005. Phenolic compounds from blueberries can inhibit colon cancer cell proliferation and induce apoptosis. J. Agric. Food Chem. 53:7320-7329.
- Yi, W., Fischer, J. and Akoh, C.C. 2005. Study of anticancer activities of muscadine grape phenolics in vitro. J. Agric. Food Chem. 53:8004-8812.
- Lai, O.M., Low, C. T. and Akoh, C. C. 2005. Lipase-catalyzed acidolysis of palm olein and caprylic acid in a continuous bench-scale packed bed reactor. Food Chem. 92:527-533.
- Kim, B.H. and Akoh, C.C. 2005. Chemical and physical properties of butterfat-vegetable oil blend spread prepared with enzymatically transesterified canola oil and caprylic acid. J. Agric. Food Chem. 53:4954-4961.
- Agyare, K.K., Addo, K., Xiong, Y.L. and Akoh, C.C. 2005. Effect of structured lipid on alveograph characteristics, baking and textural qualities of soft wheat flour. J. Cereal Sci. 42:323-330.
- Kim, B.H., Shewfelt, R.L., Lee, H. and Akoh, C.C. 2005. Sensory evaluation of butterfat-vegetable oil blend spread prepared with structured lipid containing canola oil and caprylic acid. J. Food Sci. 70:S406-412.
- Sahin, N., Akoh, C.C. and Karaali, A. 2005. Enzymatic production of human milk fat substitutes containing gamma-linolenic acid: optimization of reactions by response surface methodology. J. Am. Oil Chem. Soc. 82:549-557.
- Sahin, N., Akoh, C.C. and Karaali, A. 2005. Lipase-catalyzed acidolysis of tripalmitin with hazelnut oil fatty acids and stearic acid to produce human milk fat substitutes. J. Agric. Food Chem. 53:5779-5783.
- Lumor, S.E. and Akoh, C.C. 2005. Enzymatic incorporation of stearic acid into a blend of palm olein and palm kernel oil: optimization by response surface methodology. J. Am. Oil Chem. Soc. 82:421-426.
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Progress 01/01/04 to 12/31/04
Outputs
Immobilized lipase was used to modify a high n-6 polyunsaturated fatty acid triacyglycerol containing 40.3 percent arachidonic acid (ARASCO) with a high n-3 polyunsaturated fatty acid triacylglycerol containing 33.1 percent docosahexaenoic acid (DHASCO). The ratio of n-6/n-3 in the modified lipid product was 2:1. We also used response surface methodology to study the incorporation of stearic acid into a blend of palm kernel oil (PKO) and palm oil (PO) catalyzed by Lipozyme RM IM. Parameters studied include incubation time, temperature, and substrate molar ratio. All parameters studied had positive effect on incorporation of stearic acid with substrate molar ratio having the greatest effect. Three Georgia-grown rabbiteye blueberries and isolated fractions of phenolic acids, tannins, flavonols, and anthocyanins were evaluated for their ability to affect cancer cell proliferation and apoptosis. In the 3 cancer cell lines (2 colorectal adenocarcinoma cell lines and 1
hepatocellular carcinoma cell line) studied, anthocyanins fraction had the greatest antiproliferation effect and apoptosis.
Impacts
Combining both n-6 and n-3 polyunsaturated fatty acids in the appropriate ratio will provide a means of raising n-3 polyunsaturated fatty acid intake in our diets, maximizing the health benefits and minimizing adverse effects of excess n-6 polyunsaturated fatty acids. Using stearic acid to adjust the physical properties of fats without hydrogenation is a good way to avoid trans fatty acids in the diet. The ability of rabbiteye blueberry, especially the anthocyanins fraction, to induce programmed cell death in colon cancer cells is a significant finding with implication in human health.
Publications
- Osborn, H.T. and Akoh, C.C. 2004. Effect of emulsifier type, droplet size, and oil concentration on lipid oxidation in structured lipid-based oil-in-water emulsions. Food Chem. 84(3):451-456
- Yamaguchi, I., Akoh, C.C. and Lai, O.M. 2004. Modification of fish oil by Lipozyme TL IM to produce structured lipid. J. Food Lipids 11(1):65-73.
- Akoh, C.C. 2004. The quest for healthful and functional lipids. Inform 15(7):483-485.
- Shah, A., Akoh, C.C., Toledo, R.T. and Corredig, M. 2004. Isolation of a phospholipid fraction from inedible egg. J. Supercritical Fluids 30(3):303-313.
- Agyare, K.K., Xiong, Y.L., Addo, K. and Akoh, C.C. 2004. Dynamic rheological and thermal properties of soft wheat flour dough containing structured lipid. J. Food Sci. 69:297-302.
- Akoh, C.C., Lee, G.C. and Shaw, J.F. 2004. Protein engineering and applications of Candida rugosa lipase isoforms. Lipids 39(6):513-526.
- Akoh, C.C., Lee, G.C., Liaw, Y.C. Huang, T.H. and Shaw, J.F. 2004. GDSL family of serine esterases/lipases. Prog. Lipid Res. 43(6):534-552.
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Progress 01/01/03 to 12/31/03
Outputs
Fats and oils were modified with lipases to add new fatty acids. Palm oil was modified to incorporate n-3 polyunsaturated fatty acids. Georgia-grown blueberries and muscadine grapes were analyzed for phenolic acids, total antioxidant capacity, and total anthocyanins. For muscadine, we found that ellagic acid, myricetin, quercetin, kaempferol, and trans-resveratrol were the major phenolics. The purple and bronze muscadine leaves, skin, seed, and pulp were analyzed. The seeds and leaves exhibited higher antioxidant capacity than the pulps and the skins.
Impacts
Addition of n-3 polyunsaturated fatty acids to palm oil will expand the use and add value to palm oil. The phenolics, anthocyanins, and antioxidant capacities of Georgia-grown crops are being determined. Our results will benefit the consumers and the farmers in terms of improving their health and marketing the crops as a good source of beneficial phytochemicals.
Publications
- Osborn,H.T.and Akoh,C.C.2004.Effect of emulsifier type, droplet size, and oil concentration on lipid oxidation in structured lipid-based oil-in-water emulsions. Food Chem. 84(3):451-456).
- Osborn-Barnes, H.T.and Akoh, C.C.2003.Copper-catalyzed oxidation of a structured lipid-based emulsion containing alpha-tocopherol and citric acid:influence of pH and NaCl. J.Agric.Food Chem.51(23):6851-6855.
- Osborn-Barnes, H.T. and Akoh, C.C. 2003. Effects of a-tocopherol, beta-carotene, and soy isoflavones on lipid oxidation of structured lipid-based emulsions. J. Agric. Food Chem.51(23):6856-6860.
- Fajardo, A.R., Akoh, C.C. and Lai, O.M. 2003. Lipase-catalyzed incorporation of n-3 polyunsaturated fatty acids into palm oil. J. Am. Oil Chem. Soc. 80(12):1197-1200.
- Pastrana-Bonilla,E.,Akoh, C.C.,Sellappan, S.and Krewer,G.2003. Phenolic content and antioxidant capacity of muscadine grapes. J. Agric. Food Chem.51(18)5497-5503.
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