Structured Lipids for Food and Nutraceutical Applications
Structured lipids (SLs) are lipids that have been modified from their natural form for specific food and nutraceutical applications. This modification involves a change in the chemical structure of the lipid brought about by addition or rearrangement of fatty acids on the glycerol backbone . The starting material can be acylglycerols or phospholipids (PLs) and modifications may be carried out using chemical catalysts or enzymes (biocatalysts). Chemical modification is commonly used in industry, but only randomized products can be made. Enzymatic modification is advantageous because only mild reaction conditions are required, minimal byproduct formation improves product yield, and the specificity of lipases allow for better control over product characteristics and applications . There has been growing interest in the use of enzymes to produce SLs with nutritional and functional benefits and examples are summarized in Table 1. SL products include medium and long chain triacylglycerols (MLCTs), human milk fat (HMF) analogues, cocoa butter substitutes, reduced calorie and trans-free fats, monoacylglycerols (MAGs), diacylglycerols (DAGs), and modified PLs.
SLs may be produced via different reaction types. Acidolysis reactions involve the incorporation of fatty acyl groups from free fatty acids (FFAs) to triacylglycerol (TAG) molecules. Interesterification reactions involve exchange of fatty acyl groups between two or more TAG molecules. Alcoholysis is a substitution reaction in which fatty acyl groups from TAG molecules are transferred to an alcohol such as glycerol (glycerolysis) or ethanol (ethanolysis) . The reaction schematics can be seen in Fig. 1. Both chemical and enzymatic interesterification reactions can take place in a batch reactor where all substrates and catalyst are added and stirred over time, and also in continuous reactors where catalysts are continuously added to substrates to reduce mass transfer limitations [3,4].
Fig. 1 Reaction schematic for structured lipid synthesis showing interesterification, acidolysis, and alcoholysis (glycerolysis) reactions; Other reaction products can be formed due to reactions being random in this example.
Chemical interesterification is used widely in industry because it is relatively inexpensive compared to using enzymes; however, only randomized products can be produced as previously mentioned. The reaction involves hydrolysis of TAG molecules into FFAs followed by reesterification of the FFAs onto the glycerol backbone. This reaction is catalyzed by alkali metals or metal alkylates under anhydrous conditions at high temperatures. Water must be continuously removed to prevent undesired hydrolysis and maximize reaction yield. Commonly, sodium methoxide is used because it is inexpensive and can react at lower temperatures .
For enzymatic modification, lipases and phospholipases are the preferred enzymes to use as biocatalysts to modify fats and oils. They selectively hydrolyze fatty acids on TAG substrate and then re-esterify the glycerol with a new fatty acid while in a hydrophobic environment. Lipases can be used for acidolysis, interesterification, and alcoholysis reactions, and reactions can occur in organic solvent or a solvent-free medium. Different enzymes can be used to achieve certain TAG structures. Non-specific enzymes randomly hydrolyze fatty acids on TAG molecules while sn-1,3-specific enzymes preferentially hydrolyze fatty acids on the sn-1 and 3 positions. Candida rugosa and Candida antarctica (Novozym 435) lipases are examples of non-specific enzymes, while Rhizomucor miehei (Lipozyme RM IM) and Thermomyces lanuginosus (Lipozyme TL IM) lipases are sn-1,3-specific enzymes where the names in parenthesis represents commercially available immobilized versions. Immobilization on supports such as silica gel or acrylic resins allows for recovery and reuse of enzyme over time as well as increased thermal stability .
Healthful and Structurally Dependent Designer Lipids
Medium and Long Chain TAGs (MLCTs)
Designer lipids that contain essential long chain fatty acids (LCFAs) at the sn-2 position with short chain (C2:0-C6:0, SCFA) and medium chain (C6:0-C12:0, MCFA) fatty acids at the sn-1,3 positions have gained attention for their nutritional applications. It has been suggested that during digestion the SCFAs and MCFAs at the sn-1,3 positions are hydrolyzed by digestive lipases and absorbed more rapidly than LCFAs . This is because SCFAs and MCFAs are transported directly through the portal vein to the liver due to their smaller size and greater solubility. Therefore, they are a quick energy source and not stored in adipose tissue [1, 5]. The essential LCFAs are more efficiently absorbed from the sn-2 position in the form of a 2-monoacylglycerol (2-MAG) after lipolysis. The TAG structure with LCFAs at the sn-2 position and SCFAs and/or MCFAs at the sn-1,3 positions can be formed by acidolysis . The reaction scheme and structure of these TAGs are shown in Fig. 2. A previous study showed that the structured TAGs have different metabolic pathways despite having the same fatty acid composition compared to physical blends of the substrates used . Another study found that a SL containing rapeseed oil with capric acid (C10:0) at the sn-1,3 positions was more rapidly absorbed due to faster hydrolysis compared to a random interesterification product and a rapeseed oil control . TAGs with these structures have been useful in parenteral and enteral feeding, treatment of lipid malabsorption, and treatment of metabolic syndromes [8,9]. This specific TAG configuration is not found readily in nature. Therefore, enzymatic interesterification is a promising method to produce these designer TAGs.
Fig. 2 Reaction schematic for production of MLCTs using an acidolysis reaction; abbreviations are as follows; LCT, long chain triacylglycerol; LCFAs, long chain fatty acids; MCFAs, medium chain fatty acids; MLCT, medium and long chain triacylglycerol.
Human Milk Fat (HMF) Analogues
Another use of enzymatic modification includes the production of HMF analogues. Lipids are one of the most important macronutrients for infants and comprise of 50% of the energy in human breast milk . For proper growth and function, important fatty acids need to be metabolized efficiently, and this is achieved in HMF due to the stereospecific distribution of the fatty acids . One of the most important fatty acids for the infant is palmitic acid because it is an important source of energy, and it is mainly located at the sn-2 position of HMF [10, 11, 12]. This differs from many vegetable oils used in the production of infant formulas, which contain palmitic acid at the sn-1and sn-3 positions. This can cause digestive problems in infants because when lipases cleave the sn-1,3 positions, the palmitic FFAs readily form insoluble soaps with calcium ions resulting in palmitic acid being poorly absorbed . In order to make an efficient HMF analogue, the positional composition of the fatty acids must be similar to HMF. Therefore, the use of enzymes can generate the unique positional composition comprised of mainly sn-OPO TAGs, where O and P represent oleic and palmitic acids, respectively . Specifically, high oleic oils and oils with palmitic acid located at the sn-2 position, such as tripalmitin, can undergo interesterification reaction catalyzed by sn-1,3-specific lipases to generate OPO TAGs . The benefit of using a SL HMF analogue in infant formula includes improved fat absorption, improved calcium absorption, softer stools, and a decrease in constipation . Many studies have been able to utilize specific enzymes to produce HMF analogues with similar TAG structures [11, 13, 14, 15]. Studies have also compared using different enzymes and different enzymatic reaction methods to determine differences in total and positional fatty acid profiles [15, 16]. A commercial example of SL based infant formula includes Betapol from Loders Croklaan (Unilever). This is achieved through acidolysis using an sn-1,3-specific lipase with tripalmitin rich fats from palm stearin fractions and oleic FFAs from high oleic sunflower oil . A reaction schematic is shown in Fig. 3. Recently, HMF analogues have been produced using enzymes with long chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (ARA) because of their role in membrane structure and function in the brain and the retina . MCFAs have also been added to the sn-1,3 positions of TAGs using enzymes to further increase nutrition and provide rapid energy for infants .
Fig. 3 Reaction schematic for production of an sn-OPO human milk fat analogue; P and O represent palmitic and oleic acids, respectively.
Cocoa Butter Alternatives
Cocoa butter alternatives have also gained attention due to the uncertainty in cocoa butter supply and the fluctuation of cocoa butter prices . Cocoa butter is a major component of chocolate formulations, and its positional TAG structure allows for specific structural properties. An effective cocoa butter substitute will be accomplished if it can be blended at some ratio with cocoa butter and retain the same properties of cocoa butter such as melting point (32-35 °C), solid fat index, polymorphic behavior, positional composition, and TAG species composition . The TAGs in cocoa butter are more than 70% symmetrical with oleic acid being the major fatty acid at the sn-2 position. The fatty acids in cocoa butter are palmitic (P), oleic (O), and stearic (St) acids, and the major TAG species include POP (14-19 wt/wt %), POSt (36-41 wt/wt %), and StOSt (25-31 wt/wt %) . Enzyme technology makes it possible to make cocoa butter substitutes from novel sources by conserving the sn-2 oleic acid in order to mimic the functional properties of cocoa butter. Cocoa butter alternatives can be made using an sn-1,3-specific lipase to incorporate palmitic and stearic FFAs into vegetable oils high in oleic acid at the sn-2 position [19, 20].
Reduced Calorie and Trans-free Fats
Reduced Calorie Fats
Reduced calorie fats can be produced by the incorporation of SCFAs (C2:0-C4:0) into TAG molecules to lower caloric content. These types of fats are designed to have similar physical and functional properties of regular fat but with fewer calories. LCFAs typically have caloric values of 9.0 kcal/g. However, acetic (C2:0), propionic (C3:0), and butyric acids (C4:0) have caloric values of 3.5, 5.0, and 6.0 kcal/g, respectively . Very long chain fatty acids of 22 carbons or greater can also be used because they are poorly absorbed in humans . Examples of reduced calorie fats include Benefat from Cultor Food Science and Caprenin from Procter & Gamble, both of which contain 5 kcal/g. Both products are made using chemical interesterification, but Benefat contains chemically interesterified SCFA TAGs (triacetin, tripropionin, and tributyrin) with LCFAs (stearic acid), while Caprenin contains behenic acid (C22:0) with caprylic (C8:0) and capric (C10:0) acids [3,4].
Low-Trans and Trans-Free Fats
Margarines and shortenings usually contain high amounts of trans fatty acids due to the partial hydrogenation process of plant oils. The consumption of trans fat has been associated with increased risk of coronary heart disease, increased low density lipoprotein (LDL) cholesterol, and reduction in high density lipoprotein (HDL) cholesterol . Recently, the FDA has banned the use of partially hydrogenated oils in processed foods, so the demand for trans-free solutions is increasing. Over the years, there have been efforts to produce trans-free SL alternatives. A successful method involves the use of enzymatic interesterification of saturated fats with unsaturated oils where the product has similar functionality as trans fat. Saturated fats such as coconut oil and palm stearin have been used to produce trans-free margarines with similar properties to traditional margarines . A commercial example includes NovaLipid by ADM, which is a low-trans shortening and is produced by lipase-catalyzed interesterification of soybean oil and fully hydrogenated soybean and cottonseed oils . MCFAs can also be added which provide better textural properties due to the diversity of the TAGs .
Monoacylglycerols (MAGs) and Diacylglycerols (DAGs)
MAGs in the form of 1-MAGs or 2-MAGs are used as food grade emulsifiers. Recent research has included production of 2-MAGs with unsaturated fatty acids to also serve as sources of dietary fatty acids because 2-MAGs are readily absorbed during digestion. Enzymatic production of 2-MAGs can be performed using sn-1,3-specific enzymes for ethanolysis reactions using various TAGs . Examples include 2-MAGs with oleic and arachidonic acids [25, 26]. DAGs can also be used in combination with MAGs as food grade emulsifiers. However, sn-1,3 DAGs have also gained interest due to anti-obesity and hypotriglyceridemic effects. These effects occur because the sn-1,3 DAGs are not utilized efficiently when the fatty acids are resynthesized into TAGs for chylomicron production as compared to TAG molecules . One method for the synthesis of sn-1,3 DAGs involves using sn-1,3-specific lipases to catalyze direct esterification between FFAs and glycerol . Schematics of both reactions can be seen in Fig. 4.
Fig. 4 Ethanolysis and direct esterification reaction schematics for the production of 2-monoacyglycerols and sn-1,3 diacylglycerols, respectively.
Structured phospholipids are produced in order to improve the nutritional properties of PLs by adding important fatty acids to the glycerophospholipid backbone by using phospholipase A1 (PLA1), phospholipase A2 (PLA2), or lipases. This is done because phospholipids are more bioavailable and may serve as more efficient carriers of fatty acids for incorporation into membranes and tissues compared to TAGs . Previous studies have modified phosophtidylcholine with n-3 and MCFAs using phospholipases [29, 30]. Lysophosphatidylcholine, a food emulsifier, can also be produced by enzymatic interesterification in which 1 out of the 2 acyl groups are removed from phosphatidylcholine. Lysophosphatidylcholine is synthesized by using PLA2, which cleaves the acyl group at the sn-2 position of the phospholipid. The reaction schematic is shown in Fig. 5. This emulsifier has good applications in water in oil emulsions and has good stabilizing effects . Phospholipases can also be used for the degumming process during edible oil refining, and the benefits of enzymatic degumming include increases in production yields and decreases in operation costs and wastewater production .
Fig. 5 Reaction schematic for lysophophatidylcholine; X represents the choline head group and R1 and R2 represent fatty acids in the phospholipid.
SLs have many applications in the food and nutraceutical industries. While chemical interesterification is still widely used by industry, enzymatic interesterification has gained attention due to the specific structures of products. Some examples of SLs used today include those that contain MLCTs for rapid energy and essential fatty acids, HMF analogues for proper lipid digestion in infants, cocoa butter alternatives that have similar properties as cocoa butter, reduced calorie fats and oils, low-trans and trans-free margarines to reduce the health implications of trans-fatty acids, MAGs and DAGs as emulsifiers and nutraceuticals, and modified phospholipids as bioavailable sources of physiologically important fatty acids. Therefore, chemical and enzymatic interesterification are promising techniques to provide beneficial lipids for various applications.
- Akoh CC, Kim BH (2015) Recent research trends on the enzymatic synthesis of structured lipids. J Food Sci 80:C1713-C1724
- Weete JD, Lai O, Akoh CC (2008) Microbial lipases. In: Akoh CC, Min DB (eds) Food lipids, chemistry, nutrition, and biotechnology, 3rd ed, CRC Press, Boca Raton, pp 767-794
- Rousseau D, Marangoni A (2008) Chemical interesterification of food lipids: theory and practice. In: Akoh CC, Min DB (eds) Food lipids, chemistry, nutrition, and biotechnology, 3rd ed, CRC Press, Boca Raton, pp 268-292
- Willis WM (2008) Enzymatic interesterification. In: Akoh CC, Min DB (eds) Food lipids, chemistry, nutrition, and biotechnology, 3rd ed, CRC Press, Boca Raton, pp 808-834.
- Jandacek RJ, Whiteside JA, Holcombe BN, Volpenhein RA, Taulbee JD (1987) The rapid hydrolysis and efficient absorption of triglycerides with octanoic acid in the 1 and 3 positions and long-chain fatty acid in the 2 position. Am J Clin Nutr 45:940-945
- Lee K-T, Akoh CC, Flatt WP, Lee J-H (2000) Nutritional effects of enzymatically modified soybean oil with caprylic acid versus physical mixture analogue in obese zucker rats. J Agric Food Chem 48:5696-5701
- Høy C-E, Straarup E (2000) Structured lipids improve fat absorption in normal and malabsorbing rats. J Nutr 130:2802-2808
- Nagao K, Yanagita T (2010) Medium-chain fatty acids: Functional lipids for the prevention and treatment of the metabolic syndrome. Pharmacol Res 61:208-212
- DeMichele SJ, Karlstad MD, Bistrian BR, Istfan N, Babayan VK, Blackburn GL (1989) Enteral nutrition with structured lipid: effect on protein metabolism in thermal injury. Am J Clin Nutr 50:1295-1302
- Bar-Yoseph F, Lifshitz Y, Cohen T (2013) Review of sn-2 palmitate oil implications for infant health. Prostaglandins Leukot Essent Fatty Acids 89:139-143
- Zou L, Pande G, Akoh CC (2016) Infant formula fat analogs and human milk fat: new focus on infant developmental needs. Annu Rev Food Sci Technol 7:139-165
- Innis SM, Dyer R, Quinlan P, Diersen-Schade D (1995) Palmitic acid is absorbed as sn-2 monopalmitin from milk and formula with rearanged triacylglycerols and results in increased plasma triglyceride sn-2 and cholesteryl ester palmitate in piglets. J Nutr 125:73-81
- Álvarez CA, Akoh CC (2015) Enzymatic synthesis of infant formula fat analog enriched with capric acid. J Am Oil Chem Soc 92:1003-1014
- Rønne TH, Yang T, Mu H, Jacobsen C, Xu X (2005) Enzymatic interesterification of butterfat with rapeseed oil in a continuous packed bed reactor. J Agric Food Chem 53:5617-5624
- Sproston MJ, Akoh CC (2015) Enzymatic modification of anhydrous milkfat with n-3 and n-6 fatty acids for potential use in infant formula: comparison of methods. J Am Oil Chem Soc 93: 251-265
- Teichert SA, Akoh CC (2011) Characterization of stearidonic acid soybean oil enriched with palmitic acid produced by solvent-free enzymatic interesterification. J Agric Food Chem 59:9588-9595
- Hoffman DR, Boettcher JA, Diersen-Schade DA (2009) Toward optimizing vision and cognition in term infants by dietary docosahexaenoic and arachidonic acid supplementation: a review of randomized controlled trials. Prostaglandins Leukot Essent Fatty Acids 81:151-158
- Yamada K, Ibuki M, McBrayer T (2005) Cocoa butter, cocoa butter equivalents, and cocoa butter replacers. In: Akoh CC, Lai OM (eds) Healthful lipids. AOCS Press, Champaign, pp 642-664.
- Abigor RD, Marmer WN, Foglia TA, Jones KC, DiCiccio RJ, Ashby R, Uadia PO (2003) Production of cocoa butter-like fats by the lipase-catalyzed interesterification of palm oil and hydrogenated soybean oil. J Am Oil Chem Soc 80:1193-1196
- Çiftçi ON, Fadıloğlu S, Göğüş F (2009) Conversion of olive pomace oil to cocoa butter-like fat in a packed-bed enzyme reactor. Bioresource Technol 100:324-329
- Akoh CC (2008) Lipid-based synthetic fat substitutes. In: Akoh CC, Min DB (eds) Food lipids, chemistry, nutrition, and biotechnology, CRC Press, Boca Raton, pp 654-678.
- Remig V, Franklin B, Margolis S, Kostas G, Nece T, Street JC (2010) Trans fats in America: a review of their use, consumption, health implications, and regulation. J Am Diet Assoc 110:585-592
- Adhikari P, Shin JA, Lee JH, Hu JN, Zhu XM, Akoh CC, Lee KT (2010) Production of trans‐free margarine stock by enzymatic interesterification of rice bran oil, palm stearin and coconut oil. J Sci Food Agric 90:703-711
- Compton DL, Laszlo JA, Appell M, Vermillion KE, Evans KO (2014) Synthesis, purification, and acyl migration kinetics of 2-Monoricinoleoylglycerol. J Am Oil Chem Soc 91:271-279
- Wang X, Li M, Wang T, Jin Q, Wang X (2014) An improved method for the synthesis of 2-arachidonoylglycerol. Process Biochem 49:1415-1421
- Wang X, Liang L, Yu Z, Rui L, Jin Q, Wang X (2014) Scalable synthesis of highly pure 2‐monoolein by enzymatic ethanolysis. Eur J Lipid Sci Technol 116:627-634
- Rosu R, Yasui M, Iwasaki Y, Yamane T (1999) Enzymatic synthesis of symmetrical 1, 3-diacylglycerols by direct esterification of glycerol in solvent-free system. J Am Oil Chem 76:839-843
- Lemaitre-Delaunay D, Pachiaudi C, Laville M, Pousin J, Armstrong M, Lagarde M (1999) Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C] DHA in phosphatidylcholine. J Lipid Res 40:1867-1874
- Li X, Chen JF, Yang B, Li DM, Wang YH, Wang W-F (2014) Production of structured phosphatidylcholine with high content of DHA/EPA by immobilized phospholipase A1-catalyzed transesterification. Int J Mol Sci 15:15244-15258
- Ochoa AA, Hernández‐Becerra JA, Cavazos‐Garduño A, García HS, Vernon‐Carter EJ (2013) Phosphatidylcholine enrichment with medium chain fatty acids by immobilized phospholipase A1‐catalyzed acidolysis. Biotechnol Prog 29:230-236
- Yang B, Zhou R, Yang JG, Wang YH, Wang WF (2008) Insight into the enzymatic degumming process of soybean oil. J Am Oil Chem 85:421-425