Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Food Science & Technology
Non Technical Summary
The amount of fats and oils consumed by Americans is still high. It is recommended that we include 2-3 servings of fish a week in our diet in addition to consuming n-9, n-3, and n-6 fatty acids containing lipids. Infant formula contains short-, medium- and long chain polyunsaturated fatty acids. These highly unsaturated fatty acids are susceptible to oxidation depending on the number of double bonds. For food products containing these types of fatty acids, there is a need to stabilize them with antioxidants to maintain good shelf-life, provide nutrition, and safety. Some of the antioxidants currently in use are chemically synthesized and may be harmful to health. We propose the enzymatic synthesis of novel antioxidants with food grade enzymes (lipases). These novel antioxidants will be characterized, tested in emulsions, and in food products alone or with other natural antioxidants.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Goals / Objectives
1) To synthesis healthful and functional structured lipids using enzymes2) To continue using enzymes to synthesize human milk fat analogues3) To use lipases to synthesis novel antioxidants (hydrophilic and lipophilic) with improved solubility4) To characterize the new enzymatically produced antioxidants and test their ability to also stabilize emulsions5) To characterize the modified lipids and formulated food products
Project Methods
Enzymatic Synthesis of Healthful Lipids and SL as Infant Formula Fat AnalogsPreparation of FFAs from DHASCO® and ARASCO®. DHASCO and ARASCO (~40% DHA, or ARA, DSM Nutritional, Columbia, MD) from algal (Crypthecodinium cohnii) and Mortierella alpina, respectively, will be converted to FFAs according to Vázquez and Akoh (2011).Enzymatic Synthesis. Acidolysis of palm stearin with ARA-FFA and DHA-FFA will be performed in stirred batch bioreactor. Palm stearin 200-400 g will be mixed with ARA-FFA + DHA-FFA at different molar ratiosand 10 wt% immobilized Lipozyme TLIM lipase (sn-1,3 specific) and mixed at 200 rpm for 24 h at 60 ºC.Reactions involving TAG and another TAG (interesterification) will also be explored. The amount of lipase was based on our previous work (Kleiner et al., 2012).Analysis ofProducts. Products will be seprated using silica gel G TLC plates(Nagachinta and Akoh, 2012, 2013a,b).The MAG and TAG bands will be recoveredand converted to fatty acid methyl esters (FAME) following AOAC official method 996.01 (1998) and Pina-Rodriguez and Akoh (2009).Purification by Short-Path Distillation. Short-path distillation (KDL-4 unit, UIC Inc.) will be used to remove FFAs from the SL.After short-path distillation, the FFA content will be determined according to AOCS Official Method Ac 5-41 (2009).sn-2 FA Positional Analysis. Ethanolysis reaction catalyzed by Lipozyme® 435 as reported by Willett et al. (2019) will be used to determine the FAs at the sn-2 position of the TAG because it is betterfor PUFAs than Luddy et al. (1964) and later modified by Pina-Rodriguez and Akoh (2009).Fatty Acid Composition Analysis. The fatty acid composition of substrates and all SLs products will be analyzed in triplicate 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 (Pina-Rodriguez and Akoh (2009).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 as described by Pande and Akoh (2012) and Teichert and Akoh, (2011b, c).Encapsulation of SLs and their Properties. Microencapsulation by spray-drying will follow established methods (Kosaraju et al., 2009; Nagachinta and Akoh, 2013c). Encapsulation efficiencies will be calculated. Other encapsulating materials and technologies will be explored also. Free oil (solvent extractable) as % (w/w) of microcapsules will be estimated.Total oil will be estimated 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) followingAOCS (2009) Method Cd 12b-92. Natural antioxidants will be evaluated for their ability to delay oxidation of the SLs and infant formula emulsions.Lipases-Catalyzed Synthesis of New Antioxidants with Improved SolubilityEnzymatic Esterification of Gallic Acid and Glycerol. Ülger et al. (2017) showed that the transesterification of propyl gallate and methanol by Candida antarctica lipase B (CALB) was possible, and suggests that CALB is capable of overcoming the steric hinderance and electron-donating of gallic acid moiety. Hence, the synthesis of l-o-galloylglycerol (IUPAC: 2,3-dihydroxypropyl 3,4,5-trihydroxybenzoate) could be achieved by transesterification of propyl gallate and glycerol catalyzed by CALB. The transesterification reaction by-product is n-propyl alcohol, which is much easier to remove than water. Lipozym® 435 is CALB expressed in Aspergillus niger and immobilized onto acrylic resin. The advantage of Lipozym® 435 is that it is a food-grade enzyme, compared to Novozyme® 435 and products synthesized with it can be used as antioxidants if food formulations.Synthesis of l-o-Galloylglycerol in Solvent-free Media by a Food-grade Enzyme Optimized by Taguchi Method.Typical reaction will be carried out in dry-base water bath under various conditions. According to the literature and preliminary work, reaction time, temperature, substrate molar ratio of glycerol and propyl gallate, enzyme load will be used as four factors to be considered. The results will be analyzed by statistical analysis, such as signal to noise ratio (S/N) optimization and linear regression. Compounds will be analyzed by HPLC-Diode-array detector (DAD)-ELSD), ESI (electrospray ionization)-MS-MS, and Fourier-transform infrared spectroscopy (FT-IR). For compounds containing gallic acid moiety, a strong UV absorption will be shown andDAD used to quantify it. For glycerol, ELSD,a universal detector, will be used. The yield and conversion rate will be quantified by HPLC-DAD. The structure of the reaction product(s) will be analyzed by ESI-MS-MS and FT-IR. Other hydroxybenzoic acids, FAs, and alcohol donors will be explored to produce both water-soluble and oil-soluble antioxidants that can serve as emulsifiers also. Based on the preliminary data, the incorporation of gallic acid onto glycerol is possible by enzymatic esterification. If the incorporation is not achievable, it will confirm the assumption of Otto et al. (2000) that a spacer of at least one methylene bridge between the carboxylate group and the aromatic ring is necessary in order to induce a lipase-catalyzed conversion for CALB.Ability of Enzymatically Synthesized Antioxidants to Stabilize Emulsions Antioxidant Ability Test. One hundred milligrams of each compound and the corresponding unesterified phenolic acid will be added into 100 g of refined soybean oil. The samples will be placed in OSIinstrumentand the test will be performed according to AOCS Official Method Cd 12b-92 (2011). Refined soybean oil without added antioxidant will be used as control. PVand p-AVwillbe measured to evaluate the antioxidant activities of each synthesized compound in bulk oil and in an oil-in-water (o/w) emulsion (refined soybean oil in water). Emulsion will be prepared according to Zou and Akoh (2013) with modification. Briefly, lecithin will be used as emulsifier and dispersed in refined soybean oil. Polytron high-speed batch disperser (Kinematica, Inc., Bohemia, NY) and EmulsiFlex-C5 high-pressure homogenizer (Avestin, Inc., Ottawa, Canada) will be used to prepare the emulsion. Synthesized compounds will be added to emulsionsand stored for 21 days at 45 °C in the dark in a shaking water bath at 200 rpm. Samples and control will be prepared for 7, 14, and 21-day analyses. PV and p-AV will be determined according to AOCS Official Method Cd 8b-90 and AOCS Official Method Cd18-90, respectively (2011). Total oxidation (TOTOX) value will be calculated (Pande and Akoh, 2016). Free radical DPPH test will be analyzed(Sabally et al. 2007) at 517 nm.Antioxidant Ability to Stabilize Emulsion. The preparation of emulsion will be performed as described above. Instead of lecithin, synthesized compounds will be used as emulsifier. As described by Zou and Akoh (2013), the particle size distribution of emulsion will be used as indicator of the ability to stabilize the emulsion. The particle size distribution in each sample will be analyzed by a LS 13 320 MW laser diffraction particle size analyzer (Beckman Coulter, Inc., Miami). Control without synthesized compound analyzed. Other ways of improving the oxidative stability of infant formula fat analogs containingPUFAs and the SL products will be explored and may includeoleogels as described by Willett and Akoh (2019).Data Analysis: The Statistical Analysis System (SAS, 1996) will be used. Duncan's test will be used to determine differences between means. The tested significance level will be 5%. All reactions and analysis will be performed in triplicate.