Synthesis of lipids with sn-2 palmitic acid and sn-1,3 oleic acid from hard palm stearin and oleic acid

Human milk fat substitute (HMFS) is a fat that contains 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) as the main triacylglycerol. Hard palm stearin (HPS) is a palm oil fraction containing high palmitic acid (PA), which can be used as a raw material in OPO synthesis. The challenge in OPO synthesis using HPS is to maintain sn-2 PA and increase sn-1,3 oleic acid (OA). This research was conducted to synthesize OPO through enzymatic acidolysis of the HPS-solid fraction and OA (1:6, mole ratio) using Lipozyme TL IM 10% of the substrate weight at 60 °C for 2 – 24 h. The results showed that acidolysis caused increased partial acylglycerol, decreased TAG, and decreased sn-2 PA while increased sn-2 OA and sn-1,3 OA. The presence of acyl migration caused the incorporation of OA using Lipozyme TL IM at the sn-1,3 positions after 2 h was not significantly different. The product obtained at 2 h contained TAG with 74% sn-2 PA and 17% sn-2 OA, 78% sn-1,3 PA, and 11% sn-1,3 OA.


Introduction
One of the specialty fat products from fat or oil is the human milk fat substitute (HMFS), commonly used in infant formula.The production of HMFS is generally carried out through an approach to the characteristics of HMF fatty acids (FAs) to obtain HMFS resembling HMF [1,2].Generally, triacylglycerol (TAG) in HMF contains most palmitic acid (PA) occupying the sn-2 position and the majority of unsaturated FAs occupy the sn-1,3 positions [2,3].The main FA content in the TAG structure of HMF is 25-35% oleic acid (OA) and 20-30% PA, where about 60-70% sn-2 PA and sn-1,3 OA [1,4,5,6] HMFS is an analog HMF made with oils and fats modification, which is generally carried out through enzymatic interesterification [7].The most commonly prepared HMFS is sn-2 palmitate, with most TAGs containing 1,3-dioleoyl-2-palmitoyl-glycerol (OPO).HMFS synthesis (especially sn-2 palmitate) can be carried out using two processes: a one-stage and two-stage synthesis.One-stage sn-2 palmitate synthesis is a lipase-catalyzed reaction of TAGs with TAGs or FA esters or a reaction of TAGs with FAs.The two-step synthesis starts with an alcoholysis reaction of TAG to generate sn-2 monoacylglycerol (MAG) and then esterification between sn-2 MAG and FAs is carried out [1].Synthesis through the two-step reaction requires high costs compared to a one-step reaction.
OPO synthesis generally uses tripalmitin (PPP) with OA donors or ethyl oleate (EO) using sn-1,3 lipase [1,8].However, in large-scale production, the problem with PPP is the high cost [9].Palm oil (PO) and its fractions can be used as raw materials in the manufacture of HMFS because they contain high PA [10], besides that, they are easy to obtain, very cheap, and food-grade materials.However, the challenge in OPO synthesis using PO fractions is to maintain sn-2 PA and increase sn-1,3 OA [11,12], it is caused by PO generally containing high sn-1,3 PA [13].Exploration of the high PA-rich PO fraction at the sn-2 position is an opportunity in downstream PO to fill the demand for raw materials in the synthesis of HMFS.In addition, PO also has the opportunity to produce PPP and PA as PA donors in the synthesis of HMFS [12,14].One of the PO fractions that can be used as a raw material for HMFS is hard palm stearin (HPS) because it contains high PPP and PA [12,15].
Hasibuan (2021) [11] reported OPO content was higher, sn-2 PA was lower, and diacylglycerol (DAG) was higher compared to Lee et al (2010) [17].The high contents of MAG and DAG in transesterification products are thought to be caused by high levels of DAG in raw materials, where DAG can trigger acyl migration and cause increased DAG levels in the product Hasibuan (2021) [11].In this research, it is necessary to study OPO synthesis by acidolysis of PA-rich PS and OA using Lipozyme TL IM.This work evaluated the acylglycerol composition and disturbance of the FA composition.

Materials
HPS was taken from PT. Asianagro Agung Jaya (Indonesia).OA was procured from Tokyo Chemical Industry (Japan).Lipozyme TL IM was procured from Novozyme A/S (Denmark).FA ester was procured from Supelco (USA).2-palmitoyl glycerol, TAGs standards, sodium cholate, and pancreatic lipase were procured from Sigma Chemical Co.(USA).Thin layer chromatography (TLC) and other chemicals were procured from Merck (Germany).

Production of High PA fraction from HPS
HPS was fractionated using Hasibuan et al (2021b) procedure [15].HPS and acetone (1:5 g/mL) was heated (50 °C), then cooled (30°C) for 3 h.The solid fraction was separated and acetone is removed by evaporation.The solid fraction was characterized by its total and distribution of FA composition.

Acidolysis of HPS-Solid Fraction and OA
Acidolysis between the HPS-solid fraction and OA was performed using the procedure from Tecelão et al (2019) [6].A 5 g of the substrate from a mixture of HPS-solid fraction and OA (1:6 mole ratio) was reacted using Lipozyme TL IM 10% (w/w substrate) at an orbital shaker (200 rpm, 60 ºC) for 2, 4, 6, 8, 16, and 24 h.The interesterification product is separated from the enzyme by filtration.Acylglycerol composition and distribution of FAs in acidolysis products were evaluated.Each experiment was carried out in 2 repetitions.

Analysis of Acylglycerol Composition
The acylglycerol composition was determined according to AOCS (2005) [18], with modifications.A 0.025 g of sample was added with 10 μL tetrahydrofuran and 50 μL N-methyl-N-trimethylsilyltrifluoroacetamide, and vortexed.To the mixture, 2 mL of heptane was added and vortexed.The sample was injected into gas chromatography (DB5-HT capillary column, injector at 390 °C, FID at 390 °C).

Isolation of TAG
Isolation of TAG using a procedure from Faustino et al., (2016).Interesterified product (0.5 g in 2.5 mL chloroform) was developed to the TLC plate using a solvent of hexane/diethyl ether/acetic acid (80:20:1, v/v/v), then air-dried.The band TAG was scraped off.TAG was extracted using 2 mL of diethyl ether (2 times) and centrifuged.The solvent was removed with nitrogen flushing.Total FA composition and those at the sn-2 position in TAG were analyzed (AOCS, 2005).

Statistical Analysis
Determination of differences in samples was performed using ANOVA, Duncan multiple range test (p < 0.05), and independent sample t-test (p < 0.05) using SPSS 22 software.

Results and Discussion
The solid fraction contains PA, stearic acid, OA at sn-1,3 and sn-2 positions (Table 1).The solid fraction contained 88.13 ± 1.83 % sn-2 PA and 92.92 ± 0.43 % sn-1,3 PA.From the FA data in Table 1, it is indicated that TAG in the solid fraction is thought to consist mostly of PPP, PPS, and their isomers PSP.
During the acidolysis reaction between the HPS-solid fraction and OA, there is a change in the acylglycerol composition (MAG, DAG, TAG), as presented in Table 2.The change in acylglycerol composition is due to the acidolysis reaction where two reactions occur simultaneously, namely hydrolysis and further esterification to form TAG, MAG, and DAG [20].The content of MAG and DAG increases along with the decrease in TAG levels during the acidolysis reaction.This shows that enzymatic reactions are reversible [21].Increased levels of MAG and DAG can also be caused by the unavoidable acyl migration during enzymatic reactions to produce structured TAGs.The increase in DAG and decrease in TAG are significant during acidolysis, which indicates that there is a possibility that DAG is not converted to TAG.This is evidenced by the higher DAG content in the interesterification product during 2 to 24 h of reaction compared to the initial DAG content.DAG increase and TAG decrease significantly (p < 0.05) at increasing reaction time.This is caused by the long reaction time which will favor unwanted hydrolysis reactions, accumulating by-products (such as DAG) [21].The expected product is TAG containing sn-2 PA and sn-1,3 OA.In OPO synthesis, OA incorporation is expected at the sn-1,3 positions [22] and sn-2 PA is maintained.Thus, the levels of OA and PA were evaluated at various reaction times.For this reason, acidolysis products are characterized by the levels of PA and OA at sn-2 and sn-1,3 positions.

Figure 1.
PA and OA total of acidolysis product of HPS-solid fraction and OA at mole ratio 1:6, 60 ℃, and 10% Lipozyme TL IM Figure 1 shows that as the reaction time increases, there is an increase in OA contents.At 2 h, total OA content increased sharply, then after 2 h, OA contents increased at a small rate (p>0.05).Meanwhile, Wei et al (2015) stated OA contents increased over time, but remained constant after 6 h [22].In this study, the incorporation of OA at reaction times of 2 -24 h ranged from 13 -19%, relatively lower than in previous studies.Tecelao et al (2019) reported an OA incorporation of around 30% obtained at 4 h using Candida parapsilosis lipase [6].Qin et al (2011) also reported that at 6 h, the OA incorporation was 51.59% [9].An increase in OA levels in TAG tends to decrease PA contents (Figure 1).During 2 -24 h, PA contents can be maintained at > 70%.The sn-2 OA content tends to increase with increasing reaction time (Figure 2).It shows that OA is rapidly incorporated at the sn-2 position, it is caused by acyl migration [3,22].The incorporation of OA at 2 -24 h tended to increase by 17 -30%, whereas at 8 h OA content decreased.This value is relatively greater than in previous studies.Wang et al (2020) infromed that the amount of sn-2 OA was around 15% at 12-14 h [3].Hasibuan (2021), also reported that the sn-2 OA content was 15% at 2 h [11].OA incorporation causes a decrease in PA contents with increasing reaction time, which occurs due to an increase in sn-2 OA, which is caused by acyl migration.Previous research also reported that sn-2 PA decrease over time [3].The total sn-2 PA decrease affects the relative contents of sn-2 PA. Figure 3 also shows that the sn-1,3 OA has increased while PA has tended to decrease.The sn-1,3 OA level during 2 -24 h was not significantly different.interesterification product of PPP-rich TAGs from PS and EO using lipozyme TL IM produced a product with 80.6% sn-2 PA [10].In addition, Hasibuan (2021) also reported that transesterification between PPP-rich TAGs from PS and EO using Lipozyme TL IM produced products with 75.8% sn-2 PA and 58.7% sn-1,3 AO [11].It showed that Lipozyme TL IM was more effectively in the transesterification reaction compared to acidolysis [11].

Conclusions
HMFS containing sn-2 PA and sn-1,3 OA could be produced by acidolysis between the solid fractions of HPS and OA using 10% Lipozyme TL IM at 1:6 (mole ratio of substrate), 60 ℃, and 2 h.Under these conditions, a product obtained containing TAG with > 74% sn-2 PA and > 11% sn-1,3 OA.High DAG content in raw materials can cause acyl migration so that DAG content in enzymatic interesterification products increases.