Cocoa butter (CB), an essential fat in chocolate production, is derived from cacao beans. CB is composed mainly of 1,3-dipalmitoyl-2-oleoyl glycerol (POP), 1(3)-palmitoyl-2-oleoyl-3(1)-stearoyl glycerol (POSt), and 1,3-distearoyl-2-oleoyl glycerol (StOSt) (Liu et al., 2007, Simoneau et al., 1999). These molecular structures feature oleic acid at the sn-2 position and saturated fatty acids at the sn-1 and sn-3 positions. This structural characteristic contributes to the pleasing texture of chocolate consumption by promoting quick dissolution and a cooling effect in the mouth (Shukla, 1995).

Due to the increase in chocolate demands and the decrease in cocoa bean production as a result of global warming, demands of substitutes for CB have been increased. Cocoa butter equivalents (CBE) are fats structurally very similar to CB, and thus exhibit comparable physical and chemical characteristics to CB (Smith, 2001) and as a result can be used to partially or fully replace CB (Bahari and Akoh, 2018). In tropical or subtropical climates, chocolate melts easily at room temperature, leading to products with defects. To overcome this problem, a harder fat known as cocoa butter improver (CBI), can be added during the production of chocolate. CBI is a subclass of CBE that contains a higher proportion of high-melting triglyceride (TAG) like StOSt, which contributes to firming soft CB (Lipp and Anklam, 1998). The melting temperature of StOSt (43.7 °C for the β1 polymorph) is significantly higher than those of POP and POSt. StOSt-enriched TAG not only enhances the heat resistance of chocolate but also helps to prevent fat blooming (Ghazani and Marangoni, 2019, Hou et al., 2024).

Solvent fractionation and enzymatic interesterification are the two most effective processes for producing StOSt-enriched TAG. In numerous studies, StOSt-enriched TAG were prepared from various natural fats such as, mango kernel fat (Jin et al., 2019, Jin et al., 2021), illipe butter, sal fat, shea butter, and kokum butter (Jin et al., 2021) using solvent fractionation. However, studies on the preparation of StOSt-enriched TAG using enzymatic methods are negligible, whereas numerous studies have focused on the synthesis of various other symmetrical TAGs (SMTs) using enzymatic methods (Ghide and Yan, 2021, Rosu et al., 1999, Tang et al., 2015). Acyl migration is a major problem in the enzymatic synthesis of the SMTs, such as CBE, human-milk fat substitutes, and 1,3-dibehenoyl-2-oleoyl-glycerol (Mao et al., 2023, Meng et al., 2013, Vereecken et al., 2010). Acyl migration is influenced by several factors, such as water content in the substrate mixture, reaction temperature, the chain length of acyl donor, and reactor type. Moreover, the packed-bed reactor (PBR) has been known to operate with less acyl migration and higher reaction efficiency compared to traditional batch reactors (Choi et al., 2012, Çiftçi et al., 2009, Xu, 2003).

In the present study, StOSt-enriched TAG was prepared in a PBR via a combination of a two-step enzyme-catalyzed interesterification and solvent fractionations. In the first step, enzymatic interesterification of high oleic sunflower oil (HOSO) with ethyl stearate was carried out in a PBR using Lipozyme RM IM (Rhizomucor meihei) as a biocatalyst. The effects of temperature, water content in the substrate mixtures, and molar ratio of the substrates were investigated as a function of reaction time. Repeated lipase-catalyzed interesterification was carried out with ethyl stearate and TAG isolated from the first reaction mixture using the optimum conditions determined from the first reaction. In the second step, two solvent fractionation trials were performed with TAG obtained from a two-step enzymatic interesterification using n-hexane and acetone. At the first fractionation using n-hexane, StOSt was enriched in the liquid fraction, whereas diacylglycerol (DAG) as well as StStSt were removed in the solid fraction. The highest StOSt-enriched TAG was obtained in the solid fraction after the second fractionation using acetone. The fatty acid compositions in TAG and at the sn-2 position of TAG obtained at each step in the overall procedures for producing StOSt-enriched TAG were also investigated.