Breast milk is the most ideal food source for infants, containing only 3% to 5% lipids yet providing 50% to 60% of infants’ energy requirements, as well as essential fatty acids [1,2]. Approximately 98% of the fat in breast milk exists in the form of triacylglycerols (TAGs), and their structural distribution determines the physiological functions of breast milk fat. The saturated fatty acids in TAGs are primarily located at the sn-2 position of the glycerol backbone (especially palmitic acid (PA), which accounts for about 70% of the saturated fatty acids at the sn-2 position), while the sn-1 and sn-3 positions of TAGs are mainly occupied by unsaturated fatty acids (such as oleic acid, linoleic acid, linolenic acid, etc.). This structure facilitates the digestion and absorption of breast milk fat by infants [3].

1,3-dioleoyl-2-palmitoyltriglyceride (OPO structured lipids) is the most abundant triglyceride in breast milk, playing a crucial role in the digestion and absorption process of infants and young children, and a high-level sn-2 palmitate diet increased calcium absorption from 42% to 57% [4]. As shown in Figure 1, OPO structured lipids with palmitic acid at the sn-2 position and oleic acid at the sn-1 and sn-3 positions, upon hydrolysis by pancreatic lipase in the gastrointestinal tract, release a large amount of free unsaturated fatty acid oleic acid (OA) and sn-2 monopalmitin (sn-2 MAG). Regular milk powder will form calcium soap, which can cause babies to cry. However, milk powder rich in OPO structured lipids will not form calcium soap, and babies can better absorb the nutrients in the milk powder. The free unsaturated fatty acids do not bind with calcium ions in the intestine to form fatty acid calcium salts, thereby reducing the risk of constipation. Meanwhile, sn-2 monopalmitin serves as an easily absorbed energy source for intestinal cells, promoting rapid growth in infants. Clinical studies on OPO structured lipids have demonstrated its additional functions such as enhancing bone strength, reducing inflammation, and improving memory. Consequently, research on the preparation of OPO structured lipids is receiving increasing attention.

Currently, the main global producers of OPO are Bunge Loders from the United States, Wilmar International from Singapore, Aarhus Karlshamn from Sweden, and so on; China still relies on imports of OPO structured lipids from brands such as Betapol^TM from the Netherlands and InFat^TM from Israel [5].Therefore, the research and development of OPO structured lipids can promote the upgrading and sustainable development of China’s infant formula industry. At present, the preparation methods of OPO structured lipids are mainly divided into chemical synthesis and enzymatic synthesis. Chemical synthesis utilizes chemical reagents to catalyze the synthesis of OPO structured lipids, but this method involves vigorous reactions and poses risks of chemical solvent residues and environmental pollution [6]; On the other hand, enzymatic synthesis employs lipases as catalysts, offering advantages such as mild reactions, high catalytic specificity, and environmental friendliness, thus exhibiting broader application prospects.

The enzymatic synthesis of OPO structured lipids mainly includes alcoholysis, transesterification, and acidolysis. In enzymatic alcoholysis, oils rich in palmitic acid (such as tripalmitin (PPP) or lard) are first subjected to alcoholysis with ethanol under the catalysis of specific lipases to generate sn-2 monopalmitin (2-MAG). Subsequently, 2-MAG is esterified with oleic acid to produce OPO structured lipids (Figure 2). Liu et al. [7] used PPP as the raw material to produce 2-MAG through solvent-free enzymatic alcoholysis. They then esterified 2-MAG with ethyl oleate under the catalysis of Candida sp. 99-125 lipase, resulting in an OPO yield as high as 85.06%. This method is confronted with challenges in terms of solvent usage, cost, reaction efficiency, the treatment of by-products, and environmental impact. Optimizing the solvent usage, reducing the cost of catalysts and alcohols, enhancing reaction efficiency, minimizing waste generation, and effectively treating these wastes are the key issues that need to be addressed.

Enzymatic transesterification involves the ester–ester interchange reaction between two different triglycerides or between a triglyceride and an acyl ester, catalyzed by sn-1,3-specific lipases, to generate a new triglyceride, namely OPO structured lipids (Figure 3) [4]. Lee et al. [8] used PPP and ethyl oleate as substrates and catalyzed the ester–ester interchange with lipase from Thermomyces lanuginosus, obtaining an OPO content of 31.43%. Sarah A et al. [9] utilized soybean oil and PPP as raw materials, catalyzed by the commercial enzyme Novozym 435, ultimately achieving a sn-2 palmitic acid content of 60.84%. Enzymatic transesterification does not generate by-products, but both the substrates and products are triglycerides, which are similar in nature. As a result, product separation is difficult, and the yield is not high. To improve the separation efficiency and purity of the product, strategies such as using excess reactants, removing the product, selecting appropriate solvents and catalysts, and employing methods like distillation, extraction, and crystallization can be applied.

Compared to the multi-step reactions of alcoholysis and the challenging separation of transesterification, acidolysis is more favored by scientists. Enzymatic acidolysis involves the generation of OPO structured lipids through the catalysis of lipases using triglycerides rich in sn-2 palmitic acid and raw materials rich in oleic acid (Figure 4). The substrate triglycerides mainly include tripalmitin, basa catfish oil, lard, palm stearin, etc., while the raw materials rich in oleic acid primarily comprise oleic acid or oleic acid-rich vegetable oils (such as sunflower oil, rapeseed oil, camellia oil, etc.). Zheng et al. [10] used PPP and OA as substrates and performed acidolysis under the catalysis of Candida lipolytica lipase, achieving an OPO yield of 46.5%. Zhang et al. [11] catalyzed the reaction between lard and oleic acid at a mass ratio of 1:2 using Candida lipolytica lipase and simultaneously added β-cyclodextrin to enhance the yield of enzymatically catalyzed synthesis of OPO structured lipids. After 10 h of catalysis, the final OPO yield was 55.3%. This method facilitates easier product separation, and compared to the separation costs of transesterification and the multi-step reactions of alcoholysis, it is more conducive to large-scale industrial production.