Source: UNIVERSITY OF GEORGIA submitted to NRP
ENZYME ASSISTED MODIFICATION OF LIPIDS FOR HEALTH AND FUNCTIONALITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1006325
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2015
Project End Date
Mar 31, 2020
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Food Science & Technology
Non Technical Summary
Consumption of too much fat in the diet is linked to heart disease, cancer and obesity. However, an increase in the consumption of polyunsaturated fatty acids (PUFAs) of the n-3 series from fish and n-9 fatty acids (FAs) from certain vegetable oils has been linked to reduced risk of coronary heart disease, which is still the leading cause of death in the Western World. The food industry and researchers are looking for ways to provide the consumer with healthier and functional fats and are focusing attention on the development of structured lipids (SLs), human milk fat analogues, and foods containing n-3 and n-9 FAs.. It is possible to modify natural fats and oils with lipases using the appropriate reaction conditions. Enzymes are specific and selective in their reactions. Enzyme assisted modification of lipids will lead to lipids with specific functionality in foods and positive health outcome.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011119100030%
5011899100020%
5021899101020%
7241899100015%
5015010100015%
Knowledge Area
724 - Healthy Lifestyle; 501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
1119 - Deciduous tree fruits, general/other; 1899 - Oilseed and oil crops, general/other; 5010 - Food;

Field Of Science
1010 - Nutrition and metabolism; 1000 - Biochemistry and biophysics;
Goals / Objectives
The main goal is to use enzymes to modify lipids to add value and impact functional and healthful properties. Enzyme-assisted modification of lipids will result in functional and healthful lipids which can be used to formulate food products to benefit infants and adult nutrition.Objectives:1) To produce healthful and functional lipids using enzymes2) To use lipases to produce human milk fat analogues3) To use encapsulation technology to protect and deliver modified lipids4) To characterize the modified lipids and formulated food products5) To study the in vitro digestion of enzymatically modified lipids
Project Methods
Preparation of FFAs from DHASCO® and ARASCO®. They will be converted to FFAs according to Vázquez and Akoh (2011) with modifications. One hundred and fifty grams of oil will be saponified using a mixture of KOH (34.5 g), distilled water (66 mL), 96% ethanol (396 mL), and butylated hydroxytoluene (0.03 g), by placing the reagents in a 1 L stirred batch reactor with a circulating water bath at 60 °C, for 1 h. The reaction will then be stopped by adding 120 mL of distilled water. The mixture will be acidified by adding 6 M HCl and adjusting to pH 2 to release the FFAs. Hexane used for extraction will be removed using a rotovapor at 40 °C.Enzymatic Synthesis. Lipase-catalyzed acidolysis of substrates will be performed in stirred batch bioreactor. Palm stearin 200-400 g will be mixed with ARA-FFA + DHA-FFA (ARA:DHA ratio will be kept between 1:1 and 2:1) at different molar ratios that range from 2-8 and 10 wt% immobilized Lipozyme TLIM lipase (sn-1,3 specific) and mixed at 200 rpm for 24 h at 60 ºC. Substrates could be tripalmitin, gamma linolenic acid (GLA), high stearidonic acid soybean oil (SDA), Krill oil, anhydrous milk fat, olive oil and canola oil at various combinations can also be used as substrates. Reactions involving TAG and another TAG (enzymatic interesterification) will also be explored to produce healthful SL and infant formula fat analogs. All reactions will be performed in triplicate.Analysis of Products. After enzymatic reaction, the resulting product will be analyzed using silica gel G TLC plates. A mixture of petroleum ether: diethyl ether: acetic acid (70:30:0.5, v/v/v) will be used to separate the TAG (Nagachinta and Akoh, 2012, 2013a,b) from other reaction products. The TAG band will be identified using triolein as standard and visualized under UV light after spraying the plates with 0.2% 2, 7-dichlorofluorescein in methanol. The TAG band will be recovered into test tube for conversion to fatty acid methyl esters (FAME) and positional analysis. TPurification. Short-path distillation (KDL-4 unit, UIC Inc.) will be used to remove FFAs from the SL under the following conditions: holding temperature: 60 °C; feeding rate: ~100 mL/h; heating oil temperature: 185 °C; coolant temperature: 15-20 °C; and vacuum: <100 mTorr. After short-path distillation, the FFA content will be determined according to AOCS Official Method Ac 5-41 (2009).Pancreatic Lipase Catalyzed sn-2 Positional Analysis. The pancreatic lipase hydrolysis of TAG will be as described by Luddy et al. (1964) and modified by Pina-Rodriguez and Akoh (2009). The bands corresponding to 2-MAG will be collected and converted into FAME for FA composition analysis.Fatty Acid Composition Analysis. The fatty acid composition of DHASCO, ARASCO, palm stearin, phospholipids, and acidolysis products will be analyzed on a 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA) with a flame ionization detector (FID). A Supelco SP-2560 column (100 m x 250 μm, 0.20 μm film) will be used for FA separation. Injection of 1 μL of sample will be made at a split ratio of 20:1. Helium will be the carrier gas at the flow rate of 1.1 mL/min and at a constant pressure (45.0 mL/min). The relative FAME content will be calculated using the online computer. The average and standard deviation of triplicate analyses will be reported.TAG Molecular Species Analysis. Reversed phase HPLC, with a Sedex 85 evaporative light scanning detector (ELSD), will be used to analyze the TAG molecular species of SL, on an Ultrasphere C18, 250 mm, 4.6 mm, 5 μm particle size column following the methods described by Pande and Akoh (2012) and Teichert and Akoh, (2011b, c). The column temperature will be kept at 30 °C.Encapsulation of SLs and their Properties Microencapsualtion will be by spray-drying (Kosaraju et al., 2009; Nagachinta and Akoh, 2013c). Encapsulation efficiencies will be calculated as: Encapsulation efficiency (%) = 100 - (% free oil/% total oil) x 100. Other encapsulating materials and technologies will be explored also.Free oil (solvent extractable) on the surface of microcapsules will be estimated after extraction with petroleum ether, solvent evaporation and drying. The free oil will be expressed as % (w/w) of microcapsules. Total oil will be estimated as reported by Kosaraju et al. (2009). Particle size distribution of the emulsions will be measured using a particle size analyzer. Shelf-life and oxidative stability of the encapsulated SL will be measured using the Oxidative Stability Instrument, OSI (Omnion, Rockland, MA) following the AOCS (2009) Method Cd 12b-92. Natural antioxidants will be evaluated for their ability to delay oxidation of the SLs and infant formula emulsions. All samples will be analyzed in triplicate and average values reported.Production of Prototype Infant Formulas and their In Vitro Digestion Prototype, control (physical blend), and infant formula will be prepared using the protocol and ingredients reported by Koletzko et al. (2005), modified and reported by Pina-Rodriguez and Akoh (2010). Vitamin premix (with vitamin C as antioxidant) will also be added. The prototype infant formula will include encapsulated SL or non-encapsulated SL as the complementary fat. The prototype's formulation will meet the recommended considerations for the composition of infant formula proposed by the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) (Koletzko et al., 2005).In Vitro Model Digestion and Absorption Studies using Formulated Prototype Infant Formulas: The main goal of the in vitro trial is to investigate digestion and absorption of a selected SL in formulated prototype infant formulas (encapsulated and non-encapsulated) and compare them to a physical blend and a commercial infant formula. Simulated gastric juice, pancreatic juice, bile salts, and serum (the recipient of intestinal absorption) will be prepared. Simulated digestive organs of young infants (in vitro) will be used to follow the digestion and absorption of the prototype infant formulas.Modification of current GI models to simulate infant digestive systems. We will modify the current stomach and intestinal models to allow accurate simulation of fat digestion in infant digestive system. We will validate the model performance by measuring the contraction forces, pH, and concentration of digested products as compared with literature values from in vivo trials.In vitro gastric fat digestion of infant formula and physical blend. 50 mL of gastric juice will be preloaded in the HGS and the entire stomach reactor will be warmed up to 37 ºC before the infant formula is loaded.In vitro duodenal digestion of infant formula and physical blend. The emptied digesta from HGS (GD samples) will be further digested in DIM. The GD samples will be kept in a beaker at 37 ºC in a water bath. A peristaltic pump will pump the sample to DIM through the dialysis membrane at a velocity of 0.016 m/min, or 0.214 mL/s, representative of that induced by peristalsis in the lumen side of the small intestine (Tharakan et al., 2010). Samples will be measured for solids content, pH, emulsion size distribution and microscopy analysis. The samples will be then frozen for future analysis of lipids. The digesta samples after duodenal digestion will be collected and pooled, kept frozen at -20 ºC for further analysis. Similar trials will be conducted for materials B, C and D.Note: If the HGS and DIM models prove to be difficult to accomplish, we will use the simple in vitro static digestion (test tube or beaker), as an alternative approach (Hur et al., 2013; Minekus et al., 2014), to assess the digestion of the modified lipids and infant formula.Extraction of lipids and GC measurements oflipid profiles. The GD and DD samples from in vitro gastric and intestinal digestion trials will be analyzed for lipid profile.

Progress 07/01/15 to 03/31/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project director and the students participated in professional meetings such as IFT, AOCS, ACS, and World Congress on Oils & Fats (Sydney, Australia) to present results of their findings and to enrich their knowledge by listening to other presentations. How have the results been disseminated to communities of interest?Through publications, seminars, and presentations at professional meetings. What do you plan to do during the next reporting period to accomplish the goals?This is the final report.

Impacts
What was accomplished under these goals? Human milk fat (HMF) is an important source of nutrients and energy for infants. Triacylglycerols (TAGs) account for about 98% of HMF and have a unique molecular structure. HMF is highly enriched in palmitic acid at the sn-2 position of the glycerol backbone (more than 70%) and in unsaturated fatty acids at the sn-1,3 position. The specific TAG structure in HMF plays a valuable function in infant growth. Sn-2 palmitate (mainly 1,3-dioleoyl-2-palmitoyl-glycerol) is one of the structured TAGs that is commonly supplemented into infant formula in order to enable it to present a similar structure to HMF. We reviewed the development of the lipase-catalyzed synthesis of sn-2 palmitate over the last 25 years are with a focus on the reaction schemes in a laboratory setting. Particular attention was also paid to the commercialized sn-1,3 regioselective lipases that are used in structured TAGs synthesis, to general methods of TAG analysis, and to successfully developed sn-2 palmitate products on the market. Prospects for the lipase-catalyzed synthesis of sn-2 palmitate were discussed. The normal development and maintenance of central neural functions are highly correlated with the amount of docosahexaenoic acid (DHA; w-3 fatty acid) accumulated in the brain. DHA incorporated at the sn-2 position of lipids is well absorbed by intestinal mucosa and utilized efficiently in vivo. However, modern consumers have a reduced direct intake of DHA and increased intake of saturated fats or w-6 fatty acid oils, resulting in behavioral and neurophysiological deficits. To provide an understanding of the integrated beneficial effects of DHA on the human brain, our review introduced the positional difference (sn-2 and sn-1,3 positions) of DHA on a glycerol skeleton in natural fats and oils, and further discussed the possible functional mechanism regarding DHA supplementation and the gut-brain axis. The multiple bidirectional routes in this axis offer a novel insight into the interaction between DHA supplementation, the gut microbiota, and brain health. To achieve high sn-2 DHA in diets, it is suggested that sn-2 DHA lipids be enzymatically produced in more efficient and economical ways by improving the specific activities of lipases and optimizing the purification procedures. These types of diets will benefit individuals with strong needs for sn-2 w-3 lipids such as infants, children, and pregnant and lactating women. Two collaborative review articles were published.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jin, J., Jin, Q., Wang, X. and Akoh, C.C. 2020. High sn-2 docosahexaenoic acid lipids for brain benefits and their enzymatic synthesis: A review. Eng. 6:424-431.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wei, W., Sun, C., Wang, X., Jin, Q., Xu, X., Akoh, C.C., and Wang, X. 2020. Lipase-catalyzed synthesis of sn-2 palmitate: A review. Eng. 6:406-414.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Akoh, C.C. and Pande, G. 2020. Structured Lipids and Health. In Baileys Industrial Oil and Fat Products. (Shahidi, ed.) 7th Edition. John Wiley & Sons, Limited, West Sussex, UK, p. 1-17.


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project director and the students attended professional meetings such as IFT, AOCS, Euro Fed Lipids, China AOCS Section, and ISBAB to present results of their findings and to enrich their knowledge by listening to other presentations. One PhD dissertation was produced. How have the results been disseminated to communities of interest?Through publications, seminars, and presentations at professional meetings. What do you plan to do during the next reporting period to accomplish the goals?Will continue our research on enzymatic production of functional and healthful lipids and lipid analysis.

Impacts
What was accomplished under these goals? Menhaden oil was enzymatically modified with caprylic (C8:0) and/or stearic acid (C18:0) to produce structured lipids (SL) with high amounts of polyunsaturated fatty acids (PUFA), low level of saturation, and melting point of 25-35°C. Lipases from Candida antarctica, Lipozyme 435, and Rhizomucor miehei, LipozymeRM IM (Novozymes North America, Inc., Franklinton, NC, USA), were compared for all reactions. The physicochemical properties of the produced SL suggest that some of them may be useful in formulating food products such as margarines and spreads that deliver beneficial n-3 PUFA. Problem with using n-3 PUFA in foods is how to control oxidation and stability. To find solution to this problem, oleogels/organogels were produced using different oleogelators/organogelators. One organogel was formed using a phytosterol blend of β-sitosterol and γ-oryzanol (molar ratios of 2:1, 1:1, and 1:2 at 4, 6, and 8% (w/w) in oil), and another was formed with a blend of sucrose stearate (HLB value: 2) and ascorbyl palmitate (SSAP) (molar ratios of 3:1, 1:1, and 1:3 at 8, 10, and 12% (w/w) in oil). All 1:1 molar ratio blends exhibited β-primepolymorphic form with short spacing peaks at 4.20, 3.97, and 3.71 Å. These oleogels were evaluated as alternatives to shortening in the preparation of yellow cake in terms of batter and cake physicochemical properties. All organogels improved the oxidative stability of the menhaden oil, SL, and some could be substituted for shortening in the yellow cake. The organogels have the potential for use as nutraceuticals or health beneficial low saturated fat alternatives to saturated and/or trans-fats. Encapsulation of the oleogels reduced leaching and further improved oxidative stability of the SLs. Fish oil oxidative stability was also studied in a high fat fish oil-in-water emulsions stabilized with sodium caseinate and phosphatidylcholine (PC) as emulsifiers. In another study, fish oil-loaded elctrosprayed capsules were stabilized with seaweed and commercial natural antioxidants. Mango kernel fat (MKF)-based fats characterized as high levels of 1,3-distearoyl-2-oleoyl-glycerol (StOSt), including physical blend (PB), interesterified blend (IB) and its counterpart non-interesterified blend (Non-IB), were used to manufacture "dark chocolates" by optimizing tempering process. Significant improvements on heat and fat bloom stabilities of PB- and IB-chocolates wereattributedto their optimal triacylglycerol compositions (StOSt=55.7-60.9%, 1-palmitoyl-2-oleoyl-3-stearoylglycerol=21.1-23.8%, and 1,3-dipalmitoyl-2-oleoylglycerol=8.2-11.1%) and plate-like growth at 20 °C. One collaborative review article on improving heat and fat bloom stabilities of dark chocolate using mango kernel fat was published. Another invited collaborative review article on novel antioxidants from seaweed was also published.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Willett, S. A. and Akoh, C.C. 2019. Physicochemical characterization of organogels prepared from menhaden oil or structured lipid with phytosterol blend or sucrose stearate/ascorbyl palmitate blend. Food Funct. 10:180-190.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jin, J., Akoh, C.C., Jin, Q. and Wang, X. 2019. Improving heat and fat bloom stabilities of dark chocolate by addition of mango kernel fat-based chocolate fats. J. Food Eng. 246:33-41.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jin, J., Jin, Q., Akoh, C.C. and Wang, X. 2019. Mango kernel fat fractions as potential healthy food ingredients: A review. Crit. Rev. Food Sci. Nutr. 59:1794-1801.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jacobsen, C., Sorensen, A-D.M., Holdt S.L., Akoh, C.C. and Hermund, D.B. 2019. Source, extraction, characterization and applications of novel antioxidants from seaweed. Annu. Rev. Food Sci. Technol. (invited review)10:541-568.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Hermund, D, Jacobsen, C., Chronakis, I.S., Pelayo, A., Yu, S., Busolo, M., Lagaron, J.M., J�nsd�ttir, R., Kristinsson, H.G., Akoh, C.C., and Garcia-Moreno, P.J. 2019. Stabilization of fish oil-loaded electrosprayed capsules with seaweed and commercial natural antioxidants: effect on the oxidative stability of capsule-enriched mayonnaise. Eur. J. Lipid Sci. Technol. 121:1-10 (1800396).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yesiltas, B., Garcia-Moreno, P.J., S�rensen, A.D.M., Akoh, C.C. and Jacobsen, C. 2019. Physical and oxidative stability of high fat fish oil-in-water emulsions stabilized with sodium caseinate and phosphatidylcholine as emulsifiers. Food Chem. 276:110-118.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Qi, J., Wang, X., Wang, X., Akoh, C.C. and Jin, Q. 2019. Effect of oil type and emulsifier on oil absorption of steam-and-fried instant noodles. J. Oleo Sci. 68:559-566.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Willett, S. A. and Akoh, C.C. and Martini, S. 2019. Enzymatic modification of menhaden oil to incorporate caprylic and/or stearic acid. J. Am. Oil Chem. Chem. 96:761-775.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Willett, S.A. and Akoh, C.C. 2019. Physicochemical characterization of yellow cake prepared with structured lipid oleogels. J. Food Sci. 84:1390-1399.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, S. and Akoh, C.C. 2019. Solvent-free enzymatic synthesis of 1-o-galloylglycerol optimized by the Taguchi method. J. Am. Oil Chem. Soc. 96:877-889.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Willett, S.A. and Akoh, C.C. 2019. Encapsulation of menhaden oil structured lipid oleogels in alginate microparticles. LWT-Food Sci. Technol. 116:1-8 (108566). https://doi.org/10.1016/j.lwt.2019.108566.
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Lai, O.M., Lee, Y.Y., Phuah, E.T. and Akoh, C.C. 2019. Lipases/Esterase: Properties and Industrial Applications. In Encyclopedia of Food Chemistry. (Melton, Shahidi, Varelis, eds.), Elsevier Inc., Waltham, MA, p.158-167.
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Willett, Sarah A. PhD 2019. Dissertation: Production of menhaden oil-based structured lipids: organogelation, microencapsulation, and application in yellow cake.


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project director and the students attended professional meetings such as IFT, AOCS, Euro Fed Lipids and ISBAB to present results of their findings and to enrich their knowledge by listening to other presentations. How have the results been disseminated to communities of interest?Through publications and seminar presentations at industry and professional meetings. What do you plan to do during the next reporting period to accomplish the goals?Will continue our research on enzymatic production of functional and healthful lipids and lipid analysis.

Impacts
What was accomplished under these goals? Cocoa butter equivalents (CBEs) are fats that can mimic the properties of cocoa butter by having similar triacylglycerols profile to cocoa butter (CB). CBE was produced from illipe butter and palm mid-fraction by enzymatic interesterification. The major TAGs of the interesterified product (IP) were 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) at 42.7 ± 1.0%, 1,3-distearoyl-2-oleoylglycerol (SOS) at 29.9 ± 0.3%, and 1,3-dipalmitoyl-2-oleoylglycerol (POP) at 19.1 ± 1.0% which were very similar to CB. Thermal behavior, polymorphism, solid fat content, and crystal microstructure of IP were analyzed and were shown to match CB as well. IP was formulated into dark and white 'chocolates' and their textural, rheological, particle size distribution and fat bloom properties analyzed. The analysis results showed that properties of the 'chocolates' made from IP and CB were similar and therefore suggested that IP is compatible with CB and can be used in chocolate as a CBE. In another experiment, we used mango kernel fat-third stearin (MKF-TS), hard palm-mid fraction (HPMF) and CB to produce hard chocolate fats via physical blending and enzymatic interesterification. The optimal physical blend (PB) produced from 10% HPMF, 55% MKF-TS and 35% CB contained 60.9 g/100g 1,3-distearoyl-2-oleoyl-glycerol, 21.1 g/100g 1-palmitoyl-2-oleoyl-3-stearoylglycerol and 9.5 g/100g 1,3-dipalmitoyl-2-oleoylglycerol. The optimal interesterified blend (IB) enzymatically synthesized from 20% HPMF, 60% MKF-TS and 20% CB shared similar triacylglycerol composition to PB. Both PB and IB showed improved thermostabilities compared to CB in terms of sold fat contents, melting and crystallization behaviors. They also exhibited β polymorphic forms and 50-120 μm featherlike crystals, which were close to those of CB. The melting of IB tended to be sharper than non-interesterified blend (Non-IB) and its crystals were more continuous and uniform, indicating that enzymatic interesterification is a potential technique for manufacturing hard chocolate fats. We also used a rice bran oil solid fat fraction (RBOSF) to produce cocoa butter alternatives via interesterification reaction catalyzed by immobilized lipase (Lipozyme® RM IM) in hexane. Effects of reaction time (6, 12, and 18 h), temperature (55, 60, and 65ºC), mole ratios of 3 substrates [RBOSF:palm olein:C18:0 donors (1:1:2, 1:2:3, and 1:2:6)] were determined. The substrate system put in 3 ml of hexane and 10% of lipase added. Two sources of C18:0 donors, stearic acid (SAd) and ethyl stearate (ESd) were used. Optimal reaction was 1:2:6 mole ratio of the substrate (RBOSF:PO:SAd), at 65ºC for 12 h. DSC curves showed the melting point of CB at 20.94ºC, while those of the SL were 14.15 and 40.35ºC, respectively. The melting completion temperature (Tmc) of CB was 25.5°C while that of SL was 43.9°C, respectively. Healthful structured lipids (SLs) were produced using menhaden oil and capric acid or ethyl caprate as substrate. Enzymatic reactions conditions were optimized using the Taguchi method L9 orthogonal array with three substrate molar ratio levels of capric acid or ethyl caprate to menhaden oil (1:1, 2:1, and 3:1), three enzyme load levels (5, 10, and 15% [w/w]), three temperature levels (40, 50, and 60?C), and three reaction times (12, 24, 36 h). Reactions with ethyl caprate incorporated significantly more C10:0, at 30.76±1.15 and 28.63±2.37 mol% versus 19.50±1.06 and 9.81±1.51 mol%, respectively, for both Lipozyme® 435 and Lipozyme® RM IM, respectively. Reactions with ethyl caprate as substrate and Lipozyme® 435 as biocatalyst produced more of the desired medium-long-medium (MLM)-type TAGs with polyunsaturated fatty acids (PUFAs) at sn-2 and C10:0 at sn-1,3 positions. One review article on mango kernel fat fractions as potential healthy food ingredient and one essay based on the IFT Babcock-Hart Award received in 2018 for contributions that resulted in improved public health through nutrition or more nutritious food were published.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Pastrana-Bonilla, E., Akoh, C.C. and Cerquera, N.E. 2017. Identification and quantification of anthocyanins in muscadine grapes by HPLC and HPLC-MS. ARPN J. Eng. Appl. Sci. 12:626-631.
  • Type: Other Status: Published Year Published: 2017 Citation: Nielsen, P.M. and Akoh, C.C. 2017. Biodiesel from low-quality feed stocks. Inform. 28: 23-27.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Liang, P., Li, R., Sun, H., Zhang, M., Cheng, W., Chen, L., Cheng, X. and Akoh, C.C. 2018. Phospholipids composition and molecular species of large yellow croaker (Pseudosciaena crocea) roe. Food Chem. 245:806-811.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Willett, S.A. and Akoh, C.C. 2018. Application of Taguchi method in the enzymatic modification of menhaden oil to incorporated capric acid. J. Am. Oil Chem. Soc. 95:299-311.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kadamne, J.T., Moore, M., Akoh, C.C. and Martini, S. 2018. Sonocrystallization of a tristearin-free fat. J. Am. Oil Chem. Soc. 95:699-707.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Jin, J., Jin, Q., Akoh, C.C. and Wang, X. 2018. Mango kernel fat fractions as potential healthy food ingredients: A review. Crit. Rev. Food Sci. Nutr. doi: 10.1080/10408398.2018.1428527 (in press).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jie, L., Qi, C., Sun, J., Yu, R., Wang, X., Korma, S.A. Xiang, J., Jin, Q., Akoh, C.C. and Wang X. 2018. The impact of lactation and gestational age on the composition of branched-chain fatty acids in human breast milk. Food Funct. 9:1747-1754.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Bahari, A. and Akoh, C.C. 2018. Synthesis of cocoa butter equivalent by enzymatic interesterification of illipe butter and palm mid-fraction. J. Am. Oil Chem. Soc. 95:547-555.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jin, J., Akoh, C.C., Jin, Q. and Wang, X. 2018. Preparation of mango kernel fat stearin-based hard chocolate fats via physical blending and enzymatic interesterification. LWT-Food Sci. Technol. 97:308-316.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Bahari, A. and Akoh, C.C. 2018. Texture, rheology and fat bloom study of chocolates made from cocoa butter equivalent synthesized from illipe butter and palm mid-fraction. LWT-Food Sci. Technol. 97:349-354.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hermund, D.B., Heung, S.Y., Thomsen, B.R., Akoh, C.C. and Jacobsen, C. 2018. Improving oxidative stability of skin-care emulsions with antioxidant extracts from brown alga Fucus vesiculosus. J. Am. Oil Chem. Soc. 95:1509-1520.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kosiyanant, P., Pande, G., Tungjaroenchai, W. and Akoh, C.C. 2018. Lipase-catalyzed modification of rice bran oil solid fat fraction. J. Oleo Sci. 67(10):1299-1306.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Akoh, C.C. 2018. Conducting research at the interface of food science and nutrition. J. Food Sci. (invited) 83:2692-2696.
  • Type: Book Chapters Status: Published Year Published: 2018 Citation: 49. Kleiner, L. and Akoh, C.C. 2018. Structured Lipids in Commercial Applications. In Lipid Modification by Enzymes and Engineered Microbes. (Bornscheuer, ed.), Elsevier (Academic Press) and AOCS Press (American oil Chemists Society), p. 179-202.
  • Type: Books Status: Published Year Published: 2017 Citation: Akoh, C.C. 2017. Food Lipids: Chemistry, Nutrition, and Biotechnology. Fourth Edition, Revised and Expanded. CRC Press/Taylor & Francis Group, Boca Raton, FL, 1047 pages.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Sahin-Yesilcubuk, N. and Akoh, C.C. 2017. Encapsulation Technologies for Lipids. In Food Lipids: Chemistry, Nutrition, and Biotechnology. (Akoh, ed.), Fourth Edition, Revised and Expanded. CRC Press/Taylor & Francis Group, Boca Raton, FL, p. 453-476.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Lai, O.M., Phuah, E.T., Lee, Y.Y., Akoh, C.C., and Weete, J.D. 2017. Microbial Lipases. In Food Lipids: Chemistry, Nutrition, and Biotechnology. (Akoh, ed.), Fourth Edition, Revised and Expanded. CRC Press/Taylor & Francis Group, Boca Raton, FL, p. 853-898.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Akoh, C.C. and Kim, B.H. 2017. Structured Lipids. In Food Lipids: Chemistry, Nutrition, and Biotechnology. (Akoh, ed.), Fourth Edition, Revised and Expanded. CRC Press/Taylor & Francis Group, Boca Raton, FL, p. 941-972.


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project director and the students attended professional meetings such as IFT, AOCS, EuroFed LIpids and ISBAB to present results of their findings and to enrich their knowledge by listening to other presentations. How have the results been disseminated to communities of interest?Through publications and seminar presentations at industry and professional meetings. What do you plan to do during the next reporting period to accomplish the goals?Will continue our research on enzymatic production of functional and healthful lipids.

Impacts
What was accomplished under these goals? A glucose-cysteine Maillard reaction product (MRP) was produced and its antioxidant effects on lipid oxidation were determined for a structured-lipid enriched with polyunsaturated fatty acids in a complex emulsion. Trolox equivalent antioxidant capacities (TEAC) were determined for MRP heating intervals of 2, 4, and 6 h and were compared to α-tocopherol (TOC), MRP with TOC (TOC-MRP), and TOC with ascorbyl palmitate (TOC-AP). Emulsions were produced with total antioxidant additions of 0.02% of the oil, and lipid oxidation was monitored by peroxide and p-anisidine values over 56 d. Positive correlations between browning and heating time as well as TEAC were observed. Total TEAC values for the MRP at 6 h, TOC, TOC with the MRP at 6 h, and TOC-AP were 2.51, 3.87, 2.68, and 2.76 mg trolox eq/g, respectively. Oxidation results indicated a possible antioxidant effect for the MRP at 6 h on secondary oxidation for days 14 to 28. These results suggest that the MRP at 6 h could be useful in inhibiting secondary oxidation in complex emulsions. In another study we demonstrated that ultrasound along with supercooling can induce a change in the physical properties of interesterified (IE) fat samples including microstructure, isothermal solid fat content (SFC), and rheological properties. Although the application of high intensity ultrasound (HIU) did change the physical properties of both IE with 20 and 30% palmitic acid, the effect was more pronounced on the IE with 30% palmitic acid. This could be due to differences in TAG composition between physical blend (PB) and IE samples. Palmitic acid was mostly present in the OOP + OPP TAG in IE samples while PB samples were composed mainly of PPP. These TAG species are responsible for driving the crystallization of the samples. The higher concentration of these TAG in the IE 30% palmitic acid sample may have corresponded to more nuclei and thus higher degree of crystallization, which may have affected the physical properties of these samples upon sonication. Results from this research suggest that HIU effectiveness is driven by two main factors: (a) the generation of supercooling, and (b) the presence of sufficient saturated fats. That is, HIU is not effective at inducing crystallization in samples with low content of palmitic acid. It is possible that the lack of effect under these conditions is due to physical properties of the material such as its viscosity, which will affect the formation of cavities during sonication. However, with slightly higher content of palmitic acid (30%), sonication and processing conditions can be tailored to obtain various physical properties. By changing the HIU conditions, the extent of change may be modified and this processing technique can be extended to the healthier IE samples for use as trans-free fat alternatives. We performed similar experiment using stearic acid (S) in place of palmitic acid.Sonication promoted crystallization of low melting TAGs and the incorporation of SSS into the crystalline network. The IE samples with stearic acid at the sn-2 position have superior crystallization properties including SFC and rheology than the IE with palmitic acid at the sn-2 position. Although HIU was not as effective at inducing crystallization in the IE C16:0 20% samples due to the lower amount of saturated fats in the system, HIU induced crystallization in both the IE C18:0 20 and 30% samples. This could have been due to the higher melting point of the stearic containing samples compared to the palmitic ones. The induction of superior crystallization properties in these samples upon sonication can make them great candidates as ingredients for trans-fat free applications. Structured lipids were designed for use in an edible film application and their properties compared. Blends [60:40, 70:30, and 80:20 (w/w)] of coconut oil (CO) and high oleic sunflower oil (HOSO) were interesterified using immobilized enzyme, Lipozyme® TL IM. The enzyme was successful in increasing oleic acid at the sn-2 position of the TAGs. The structured lipids were helpful in reducing the opacity of CO in a film use and this is advantageous. The interesterified product (IP) 60:40 provided the strongest tensile strength (TS) although it had the weakest elongation break (EB). The IP 60:40 contained the most oleic acid (33.86 ± 1.55%) at the sn-2 position and also resulted in a fairly translucent product. The IP 60:40 was used to continue further research on the use of SLs in edible films to prepare sports nutrition products in our laboratory. Two review articles were published on the structured lipids for food and nutraceutical application and on biotechnological and novel approaches for designing structured lipids for infant nutrition.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: ?ahin Ye?il�ubuk, N. and Akoh, C.C. 2017. Biotechnological and novel approaches for designing structured lipids intended for infant nutrition. J. Am. Oil Chem. Soc. (Invited review). J. Am. Oil Chem. Soc. 94:1005-1034.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kadamne, J.T., Ifeduba, E.A., Akoh, C.C. and Martini, S. 2017. Sonocrystallization of interesterified fats with 20 and 30% of stearic acid at sn-2 position and their physical blends. J. Am. Oil Chem. Soc. 94:1045-1062.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Sproston, M.J., Ifeduba, E.A. and Akoh, C.C. 2016. Structured lipids for food and nutraceutical applications. AOCS Lipid Library (invited review). August 30, pages 1-7. http://dx.doi.org/10.21748/lipidlibrary.41522
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Sproston, M.J. and Akoh, C.C. 2016. Antioxidative effects of a glucose-cysteine Maillard reaction product on the oxidative stability of a structured lipid in a complex food emulsion. J. Food Sci. 81:C2923-C2931.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jiang, X., Wu, S., Zhou, Z. and Akoh, C.C. 2016. Physicochemical properties and volatile profiles of cold-pressed Trichosanthes kirilowii Maxim seed oils. Int. J. Food Prop. 19:1765-1775.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kadamne, J.T., Ifeduba, E.A., Akoh, C.C. and Martini, S. 2017. Sonocrystallization of interesterified fats with 20 and 30% C16:0 at sn-2 position. J. Am. Oil Chem. Soc. 94:3-18.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Jin, G., Kim, J., Lee, Y., Akoh, C.C., Chun, H.S., Ahn, S. and Kim, B.H. 2017. A nuclear magnetic resonance spectroscopy approach to discriminate the geographic origin of roasted Asian sesame oils. J. Oleo Sci. 66:337-344.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Moore, M.A. and Akoh, C.C. 2017. Enzymatic interesterification of coconut and high oleic sunflower oils for edible film application. J. Am. Oil Chem. Soc. 94:567-576.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang, Y., Zhai, X., Gao, L., Jin, J., Zhong, Q., Sun, C., Yan, L., Liu, R., Akoh, C.C., Jin, Q. and Wang, X. 2017. Quality of wood-pressed rapeseed oil. J. Am. Oil Chem. Soc. 94:767-777.


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students and the project director attended professional meetings such as AOCs and IFt to present the results and enrich their knowledge by listening to other presentations. How have the results been disseminated to communities of interest?Through publications, seminars, industrial presentations, and professional meeting presentations What do you plan to do during the next reporting period to accomplish the goals?To continue our research on production of healthful and functional lipids using enzymes and use them in food applications.

Impacts
What was accomplished under these goals? We compared encapsulation of stearidonic acid soybean oil (SDASO) by complex coacervation in the classical gelatin (GE)-gum arabic (GA) system with that of a Maillard reaction product (MRP). A portion of the control (microcapsules based on the GE-GA system) was cross-linked with transglutaminase (TG) after encapsulation.Based on the amount of oil released from microcapsules during heat treatment (85 C for 30 min) in yogurt milk base, MR-modified microcapsules displayed the highest thermal stability. Furthermore, yogurts formulated with MR-modified microcapsules had the best oxidative stability during 14 days of storage at 4 C demonstrating that the antioxidant components of MR-modified microcapsules had good carry-through properties. High sn-2 docosahexaenoic and arachidonic acid oils (DHAOm and ARAOm, respectively) were produced independently via enzymatic interesterification of DHA-rich and ARA-rich single cell oils (DHASCO and ARASCO, respectively) using a mix of immobilized lipases, Lipozyme® TL IM and Novozym® 435 (weight ratio 1:1) as the biocatalyst system. Final results showed that DHAOm contained 46.53 mol% of total DHA (49.70 % at the sn-2 position), while ARAOm contained 47.25 mol% of total ARA (36.08 % at the sn-2 position). This represents a significant increment in the amount of DHA and ARA at the sn-2 position when compared to DHASCO (47.8 mol%; 30.30 % at the sn-2) and ARASCO (47.79 mol%; 28.50 % at the sn-2), respectively. These products have potential as additions to infant formulas where DHA and ARA supplementation is required. A structured lipid (SL) with a high amount of sn-2 palmitic acid was synthesized from anhydrous milkfat and was then enriched with docosahexaenoic (DHA) and arachidonic (ARA) acids using an immobilized lipase. This SL might be totally or partially used in commercial fat blends for infant formula.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ifeduba, E.A. and Akoh, C.C. 2015. Microencapsulation of stearidonic acid soybean oil in complex coacervates modified for enhanced stability. Food Hydrocolloids 51:136-145.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: �lvarez, C.A. and Akoh, C.C. 2015. Enzymatic synthesis of infant formula fat analog enriched with capric acid. J. Am. Oil Chem. Soc. 92:1003-1014.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ifeduba, E.A. and Akoh, C.C. 2016. Microencapsulation of stearidonic acid soybean oil in Maillard reaction-modified complex coacervates. Food Chem. 199:524-532.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zou, L., Pande, G. and Akoh, C.C. 2016. Infant formula fat analogs and human milk fat: new focus on infant developmental needs. Ann. Rev. Food Sci. Technol.(invited review) 7:139-165.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: �lvarez, C.A. and Akoh, C.C. 2016. Enzymatic synthesis of high sn-2 DHA and ARA modified oils for the formulation of infant formula fat analogues. J. Am. Oil Chem. Soc. 93:383-395.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: �lvarez, C.A. and Akoh, C.C. 2016. Preparation of infant formula fat analog containing capric acid and enriched with DHA and ARA at the sn-2 position. J. Am. Oil Chem. Soc. 93:531-542.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Sproston, M.J. and Akoh, C.C. 2016. Enzymatic modification of anhydrous milkfat with n-3 and 6 fatty acids for potential use in infant formula: comparison of methods. J. Am. Oil Chem. Soc. 93:251-265.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ifeduba, E.A., Martini, S. and Akoh, C.C. 2016. Enzymatic interesterification of high oleic sunflower oil and tripalmitin or tristearin. J. Am. Oil Chem. Soc. 93:61-67.


Progress 07/01/15 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nothing to report. Project is new.

Publications