INTRODUCTION

Fat is a component of human milk that gives half of the energy required for babies (1-3). Human milk contains 3-5% fat comprising 98% triacylglycerols (TAGs) (4-6). The main fatty acids in the human milk TAGs are oleic (30-35%), palmitic (20-30%), linoleic (7-14%) and stearic (5.7-8%) acids (5). About 60-70% of palmitic acid is located at the sn-2 position, whereas unsaturated fatty acids (i.e. oleic, linoleic and ω-3 polyunsaturated fatty acids (PUFAs)) are at the sn-1,3 positions in the TAG structure of human milk fat (HMF) (6). Palmitic acid at the sn-2 position has a significant importance especially in the absorption and metabolism of lipids and nutrition of infants (7, 8).

The dominant TAGs in HMF are 1,3-dioleoyl-2-palmitoyl-glycerol (OPO; 16-29%) and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL; 13-20%) (9). TAGs with palmitic acid at sn-2 position are very helpful in effectively promoting the absorption of calcium ions, leading to the formation of softer stools and hence reducing the possibility of constipation (1, 6, 10, 11). TAGs containing palmitic acid at sn-2 position can also increase early bone mineralization and growth, affect the composition of intestinal microflora, decrease the level and severity of intestinal inflammation and influence neurobiology, which includes modulation of early infant crying (12).

The composition and distribution of fatty acids in HMF are used as a basis to develop an alternative fat as an ingredient for infant formulas. As sources of nutrients, infant formulas are an alternative to human milk when nursing mother does not produce enough breast milk (13, 14). Fats commonly used for infant formulas are vegetable oils or animal fats, especially bovine milk fat (3). However, the composition and distribution of fatty acids in vegetable oils and mammalian milk fats differ from that of HMF (4). In vegetable oils, palmitic acid is mainly (>80%) esterified to sn-1,3 positions (9). Meanwhile, animal fat such as cow’s milk fat has similar palmitic acid content as HMF, but the percentage of palmitic acid esterified at sn-2 position is only about 40% (4, 14). Hereby, vegetable oils, animal fats or blends of oils are modified to mimic the composition and distribution of fatty acids found in HMF (15, 16). This modified fat is so-called human milk fat substitute (HMFS) (14, 17).

HMFS has been successfully developed and commercialized as energy supplement and considered as a source of essential fatty acids, and as nutritional supplement in infant formulas (4). Based on the categories of the LIPID MAPS® structure database (18), HMFSs are classified into four types based on their lipid structure, namely: (i) sn-2 palmitate (palmitic acid at the sn-2 position) such as OPO, (ii) long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6 ω-3), eicosapentaenoic acid (EPA, 22:5 ω-3) and arachidonic acid (ARA, 20:4 ω-6), (iii) medium-chain triglycerides (MCTs) (having medium-chain fatty acids (MCFAs) with 6-12 carbon length), and (iv) milk fat globule membrane supplements (4).

Synthesis of HMFS is conducted by the enzymatic interesterification of oils and fats. The enzymatic interesterification operates at relatively low temperature, and it is considered cost-effective and environmentally friendly method (19). The interesterification utilizes lipase as a biocatalyst, which has specificity and selectivity to produce desired lipids with relatively low amount of by-products (20). Hereby, the changes in the structure of TAGs can be specifically regulated at sn-1,3, sn-2 or an unspecified position (21-25).

The development of structured lipids using enzymatic process technology has several challenges especially for tailoring higher catalytic efficiency and enzyme stability, which are important for overall productivity (26). HMFS contains more than 70% palmitic acid at sn-2 position and it can be produced by acidolysis in solvent system between tripalmitin and a mixture of hazelnut oil fatty acids and stearic acid using Lipozyme RM IM (15), or tripalmitin and fatty acids from hazelnut oil and γ-linolenic acid (GLA) using Lipozyme RM IM and Lipozyme TL IM (16). He et al. (6) reported that acidolysis of TAGs from Nannochloropsis oculata and fatty acids from Isochrysis galbana using Novozyme 435, Lipozyme TL IM, Lipozyme RM IM and recombinant Candida antarctica Lipase B (recombinant CAL-B) in solvent-free system produced HMFS containing 59.38–68.13% palmitic acid at sn-2 position.

The reported studies on HMFS production highlighted the exploration of the use of new oils and fats, finding more cost-effective catalysts, synthesis methods, reactor configurations and purification process. Wei et al. (4) reviewed the achievements and trends of development of HMFS, with focus on nutritional bases, preparation methods and applications of HMFS. As an addition to that comprehensive review, this work is more focused on the utilization of lipase as a biocatalyst and factors that affect lipase-catalyzed synthesis of HMFS. It starts with the biocatalysts used for production of HMFS, followed by substrates, methods and reactor configurations, factors influencing the synthesis and purification of HMFS, with a specific objective to increase the efficiency of HMFS synthesis. Additionally, the developments of HMFS production including challenges and opportunities for future research of HMFS are also presented in this work.

LIPASE FOR HMFS SYNTHESIS

Lipase (triacylglycerol acyl-hydrolase, EC 3.1.1.3) is commonly used for oil or fat hydrolysis. In non-aqueous media, lipase can also catalyze the esterification, acidolysis, alcoholysis and interesterification (20, 27-29). The lipase-catalyzed interesterification involves the reversible reaction of simultaneous hydrolysis and esterification reactions (30). A small amount of water is important for non-aqueous enzymatic catalysis for maintaining active conformational structure of the enzymes during non-covalent interactions (31). The excess water has to be removed to shift the progress of the reaction from hydrolysis to esterification, thus enhancing the reaction yield. When the hydrolysis prevails over esterification, by-products such as glycerol, free fatty acids (FFA), monoacylglycerol (MAG) and diacylglycerol (DAG) are obtained, which eventually hampers the separation process.

As part of non-aqueous reaction, the esterification of HMFS can be carried out by lipase as the biocatalyst. The sources of lipase are mostly microorganisms. The commercial lipases available on the market and mostly studied in recent years for the production of HMFS are derived from Rhizomucor miehei, Thermomyces lanuginosa, Candida antarctica, Candida parapsilosis, recombinant lipase B from Candida antarctica, Candida lipolytica, Candida sp. 99-125, Rhizopus oryzae, Alcaligenes sp. and Mucor miehei (9, 24).

Lipases with regiospecificity and regioselectivity are of interest as the reaction yield can be tuned up by these properties. Additionally, the use of an immobilized lipase with a biocatalytic activity maintained at an industrial scale is required for multiple uses, ensuring the economic viability of the process (32, 33), and thus lowering production costs (34, 35). The immobilized lipase sometimes has a higher stability than the native one from freely suspended enzyme (36).

SOURCES AND TYPES OF SUBSTRATES FOR HMFS SYNTHESIS

Oils, fats or single TAG molecules can be used as substrates for HMFS synthesis (Table 2) (6, 40, 45, 48-52, 65, 66, 68, 75-80). Several reported sources of palmitic acid for HMFS synthesis are tripalmitin (77), lard (48), palm stearin (52, 75), basa catfish oil and its fraction (51), oil from Nannochloropsis oculata (6), palm oil (78) and palm olein (68). Oils that can be used as sources of oleic acids are high oleic sunflower oil (79), hazelnut oil (66), tea seed oil (48), rapeseed oil (49) and olive oil (76). Sunflower and soybean oil can be sources of linoleic acid (45). Flaxseed oil (45) and camelina oil (77) can be used as linolenic acid sources. Oils such as coconut (45) and palm kernel oil (49) can be used as MCFAs. Sources of EPA and DHA are fish oil (65, 80), algal oil (49), microalgal oil from Schizochytrium sp. (40) and DHA single cell oil (76). Microbial oil is one of the sources of arachidonic acid (49). In addition, substrates that can be used as a single fatty acid donor for the synthesis of HMFS are palmitic or ethyl palmitic acid (66) as palmitic acyl donors, oleic acid (75) or ethyl oleate (59) as oleic acyl donors, linoleic acid (75), GLA (16), EPA and DHA (46) or ARA (67) as PUFA donors, and myristic (52), caprylic and capric acids (41) as acyl MCFA donors.

The type of substrate is one of the important factors in the synthesis of HMFS. The composition of the raw material of the substrate that undergoes the interesterification process in the synthesis of HMFS has a significant influence on the final product. In the synthesis of HMFS with high content of palmitic acid at sn-2 position, it is better to use a substrate containing high content of palmitic acid at that position. Zhang et al. (32) produced HMFS containing 79.51% palmitic acid at the sn-2 position by acidolysis between lard (81.92% palmitic acid at the sn-2 position) and oleic acid (r=1:2) at 45 °C for 10 h using 10% additives β-cyclodextrin and Candida sp. 99-125. Yang et al. (39) also reported the production of HMFS with 71.1% palmitic acid at the sn-2 position obtained from the acidolysis between lard (67.3% palmitic acid at the sn-2 position) and fatty acids from soybean oil (r=1:2.4) at 61 °C for 1 h using 13.7% Lipozyme RM IM. In another study, Zou et al. (50) produced HMFS containing 58.43% palmitic acid at the sn-2 position by acidolysis between the solid fraction of basa catfish oil (60.42% palmitic acid at the sn-2 position) and fatty acids from high oleic acid sunflower oil (r=1:6) using 12% Lipozyme RM IM at 50 °C for 2 h. The use of basa catfish oil (49.3% palmitic acid at the sn-2 position), which was acidolyzed with fatty acids from sesame oil (r=1:3), using 8% Lipozyme RM IM at 40 °C for 2 h, yielded HMFS containing 48.3% palmitic acid at the sn-2 position (51).

The use of palm stearin containing different amounts of palmitic acid also affects the final quality of the product. Zou et al. (52) produced HMFS containing 62.8% palmitic acid at the sn-2 position from acidolysis between palm stearin (56.8% palmitic acid at the sn-2 position) and a mixture of fatty acids from rapeseed oil, sunflower oil, palm kernel oil, stearic acid and myristic acid (r=1:14.6) at 57 °C for 3.4 h using 10.7% Lipozyme RM IM. Meanwhile, Wang et al. (75) reported the use of fractionated palm stearin containing >90% tripalmitin (91.96% palmitic acid at the sn-2 position), which was acidolyzed with a combination of oleic acid and linoleic acid at r=1:8:4 and 60 °C for 4 h using 8% lipase NS 40086 yielding HMFS with 87.75% palmitic acid at the sn-2 position. In another study, Faustino et al. (77) acidolyzed tripalmitin (purity >85%) with fatty acids from camelina oil (r=1:2) at 60 °C for 24 h using R. oryzae lipase immobilized on 5% Lewatit VP OC 1600 yielding HMFS with 67.7% palmitic acid at the sn-2 position.

Melting points of some substrates for HMFS synthesis are 66-68 °C for tripalmitin (77), 30.3-48 °C for lard (45, 81), 58-61 °C for palm stearin (52, 56, 79), 64.5 °C for fractionated palm stearin (59) and 63 °C for palmitic acid (37). The substrate melting point influences the enzymatic interesterification to obtain the optimal target product. Lee et al. (81) reported that transesterification between lard (27.1% palmitic acid) and olive oil (73.3% oleic acid) or camellia oil (81.6% oleic acid) at 40 °C for 12 h using 8.33% Lipozyme IM-20 in isooctane solvent yielded HMFS with 12.9 or 15.4% OPO, respectively. Transesterification of palm oil (44.3% palmitic acid) with olive oil or camellia oil resulted in HMFS containing 21.8 or 25.2% OPO. Despite having a high palmitic acid content at the sn-2 position, interesterification of lard produced lower OPO content than palm oil. This is related to the used low reaction temperature (40 °C), lower than the melting point of lard, which is 48 °C, therefore the solubility of lard is low in isooctane at 40 °C (81).

METHODS AND REACTORS FOR LIPASE-CATALYZED HMFS SYNTHESIS

Synthesis of HMFS can be carried out either through one-step reaction (i.e. acidolysis or transesterification) or two-step reaction (i.e. alcoholysis and esterification, acidolysis and acidolysis, or transesterification and acidolysis). The lipase-catalyzed reaction schemes for the synthesis of HMFS are shown in Fig. 1.

Fig. 1

Schemes of lipase-catalyzed reactions for human milk fat substitute synthesis

Acidolysis is a reaction between TAG and FFA (4). Enzymatic acidolysis is usually considered as a reversible two-step reaction, i.e. hydrolysis of the ester bonds with the released fatty acid followed by esterification of FFA to the backbone of glycerol (5). Table 3 (6, 15, 16, 32, 39, 40, 44-53, 60, 62, 67-69, 75, 76, 81) shows the reacting conditions for the HMFS production, where the resulting HMFS generally contains more than 60% palmitic acid at the sn-2 position. The main by-product of the acidolysis reaction are FFAs, which can be removed through neutralization (65, 82), liquid-liquid extraction (83) or molecular distillation (84, 85). In acidolysis, there is usually acyl migration in which fatty acids at the sn-2 position migrate to either sn-1 or sn-3 position (9). This entails the formation of various mixed TAGs as by-products in the reacting mixture. In general, acyl migration increases the separation complexity of the target HMFS from other by-products (24). Hence, the recovery of structured TAGs (i.e. target HMFS) is relatively lower than of those obtained by the two-stage process (9).

Transesterification is a reaction between TAG and TAG or ester (4). HMFS-based transesterification reaction is carried out with TAGs that are rich in palmitic acid at the sn-2 position, using sn-1,3 regioselective lipase. In the transesterification reaction, the final product is a mixture of different TAGs with similar physical properties. To some extent, it is difficult to purify and obtain the target HMFS. Nevertheless, this issue is specific, depending on the type and proportion of oil (5, 9). Table 4 (43, 55-59, 66, 69, 86-92) summarizes the reaction conditions for HMFS synthesis by transesterification.

The one-step enzymatic process has been used in many studies due to its simplicity, but its drawbacks are: (i) difficulties in converting intermediate DAGs into desired HMFS resembling HMF, and (ii) complexity of purification due to the presence of by-products (24). To overcome these drawbacks, a multi-step enzymatic process such as alcoholysis followed by esterification has been proposed (9, 24). The synthesis of HMFS via multi-step enzymatic process results in a higher OPO purity (74-95%) than in one-step enzymatic process (about 43%). However, this approach also has bottlenecks, especially the reaction complexity and high solvent consumption (45).

The two-step process for the HMFS synthesis can be alcoholysis route followed by the esterification reaction (42, 93, 94) (Table 5). The two-step process is proposed in the synthesis of HMFS to overcome the drawbacks of acidolysis and transesterification. This method exploits the regioselectivity of lipase at sn-1,3 (9, 24). Two-step synthesis consists of alcoholysis of TAG using sn-1,3 specific lipase to produce sn-2 MAG rich in palmitic acid and followed by esterification of sn-2 MAG rich in palmitic acid with FFAs (4, 95) or esterified fatty acids (93). Generally, the final product of interesterification between sn-2 MAG rich in palmitic acid and oleic acid contains 92-94% palmitic acid at sn-2 position and 83-89% oleic acid at sn-1,3 position, while the yield of OPO reaches 70-72% (9). The alcoholysis followed by esterification process avoids the acyl migration and obtains a purely structured TAG (HMFS) (5, 9, 24). However, this process is not commonly used in industrial production due to the complexity of the steps, which leads to an increased overall cost (9).

Two-step synthesis of HMFS can also be carried out through two-step acidolysis (61, 65) (Table 4). Esteban et al. (61) conducted acidolysis of palm stearin and palmitic acid at r=1:3, temperature 37 °C in solvent system using Novozyme 435, which produced TAGs containing a high palmitic acid content at the sn-2 position (74.5%). After the first acidolysis, the obtained TAGs were used as intermediates for the second acidolysis with oleic acid at r=1:6 using R. oryzae, Mucor miehei, RM IM, TL IM and Alcaligenes sp. lipases. The final product contained 67.8% palmitic acid at sn-2 position and 67.2% oleic acid at sn-1,3 positions (61). In addition, Pina-Rodriguez and Akoh (96) carried out a two-step interesterification (transesterification followed by acidolysis) for the synthesis of the DHA-containing amaranth oil structured lipid. First, a customized amaranth oil was produced by transesterification of amaranth oil and ethyl palmitate using Novozyme 435. The second step was acidolysis of the obtained oil with DHA using Lipozyme RM IM. The final product contained 28% palmitic acid and 33% palmitic acid at sn-2 position.

The interesterification for HMFS synthesis can be carried out in batch and continuous reactors (5). The batch reactor is easy to operate and suitable for small scale production. However, at the industrial scale, for an economical production process, continuous operation is preferred rather than batch-wise operation (53, 97). In continuous reactor system, such as continuous stirred tank reactor (CSTR), plug flow reactor (PFR) or packed bed reactor (PBR), substrate is continuously introduced into the reactor and the product is subsequently withdrawn (98). PBR is more suitable for industrial-scale production than CSTR (24).

The advantages of PBR over the batch reactor for the production of structured lipids can be seen in the following aspects (53): (i) the slow substrate flow through the enzyme column to avoid the damage of the enzyme structure and increase the stability of the enzyme, (ii) the production can be carried out continuously, and (iii) it reduces the occurrence of acyl migration due to the excessive use of the enzyme. To some extent, the continuous process at a high volumetric flow rate is more advantageous than operation at a slow volumetric flow rate. At a high flow rate, possibility of acyl migration reduces, thus increasing the productivity (23). The acyl migration in a PBR is lower than in a stirred batch reactor (24). Nielsen et al. (97) reported that the reaction equilibrium in acidolysis of lard and soybean oil fatty acids in a PBR was reached in <1.5 h residence time. Zou et al. (53) reported that Lipozyme RM IM could be used for 10 days in a PBR without a significant loss of activity in interesterification between palm stearin and mixed of stearic acid, myristic acid and fatty acids from rapeseed oil, sunflower oil and palm kernel oil. Wang et al. (40) also reported that the number of reuses of lipase in a packed reactor increased 2.25-fold compared to that of batch reactor.

FACTORS INFLUENCING HMFS SYNTHESIS

Some aspects considered for HMFS synthesis are biocatalyst concentration, reaction type, substrate composition and mode of operation (5). Table 3, Table 4 and Table 5 show selected works on HMFS synthesis using various substrates, enzymes and other relevant parameters for optimizing the process in order to obtain products that resemble HMF.

PURIFICATION OF HMFS

The synthesis of structured lipids by the enzymatic interesterification produces TAGs, partial glycerides (DAG and MAG) and FFAs. The acidolysis between TAG and fatty acids gives products with a high FFA content. Products of acidolysis between palm stearin and palmitic acid at an amount of substance ratio 1:3 contain 50% FFAs (37). The transesterification between TAG molecules gives products with low content of FFAs (0.5-7%) (56, 58, 69). Thus, each type of enzymatic interesterification or utilization of different substrates can result in different complexity in the purification of HMFS. This complexity, as indicated earlier, depends on the number of by-products contained in the reaction mixture. Purification after HMFS synthesis is intended to increase TAG fraction by removing FFAs and partial glycerides. The removal of FFAs can be carried out by neutralization (57, 61, 62, 65, 82), liquid-liquid extraction (55, 83) and evaporation using molecular distillation (45, 48, 50, 51, 53, 84, 85). Molecular distillation is also applied to remove both FFAs and partial glycerides simultaneously (48).

Neutralization is carried out through saponification of FFAs using an alkaline solution such as KOH. The acylglycerol fraction is then extracted using hexane (57, 61, 62, 65, 82). Ilyasoglu (57) reported that the neutralization of the transesterification product of tripalmitin and a mixture of olive oil and flaxseed oil (1:1) (r=1:2.67) using 0.8 M KOH enhanced TAG content up to 78%. Robles et al. (65) also reported the neutralization of the acidolysis product of palm stearin rich in palmitic acid at the sn-2 position and oleic acid (r=1:6) using 0.5 M KOH at 37 °C. The TAG yield was up to 80%. Esteban et al. (61) confirmed the neutralization of acidolysis product of palm stearin rich in palmitic acid at the sn-2 position and oleic acid using 0.5 M KOH in the presence or absence of hexane. With (at room temperature) and without solvent at 50 °C, the neutralization can increase TAG purity to 99% with the yield of 96%. Yuan et al. (55) reported the removal of FFAs from the interesterification product using liquid-liquid extraction with 85% ethanol at a volume ratio of 1:1.

Separation using molecular distillation is carried out based on the difference in vaporization temperatures of FFAs, partial glycerides and TAGs. Using molecular distillation, Qin et al. (45) purified the acidolysis product of 34L-leaf lard and camellia fatty acids (r=1:4). At the evaporation temperature of 180 °C and pressure of 6.7-7.5 Pa, the TAGs were rich in OPO with the purity of 91.39% and the yield of 40.75%. Zou et al. (50) also reported the purification of the product of acidolysis between the solid fraction of basa catfish oil and high oleic sunflower oil fatty acids (r=1:6). At the evaporation temperature of 185 °C and the pressure of 2 Pa, the TAG fraction with the yield of 95.7% was obtained. A stepwise evaporation using molecular distillation is also possible for purification of interesterification product. Sørensen et al. (84) produced TAG fraction of 31.3% from the acidolysis between butterfat and a mixture of fatty acids from rapeseed oil and soybean oil (r=1:2). The conditions were pressure of 0.1 Pa and the evaporation temperatures in stages 1 and 2 of 90 and 185 °C, respectively. The ranges of evaporation temperatures and pressures of molecular distillation to remove FFAs during HMFS purification are 180-185 °C and 0.1-7.5 Pa. In addition, the separation of TAGs from partial glycerides is carried out at the evaporation temperature of 230 °C and a pressure of 10⁷ Pa (48).

In the two-step acidolysis (i.e. a multi-stage process), purification starts with the first acidolysis to remove FFAs from the reaction mixture. In the second acidolysis, FFAs and DAGs are also removed from the product mixture. A single-step enzymatic process can also produce nearly pure HMFS. However, it is challenging to convert all of the intermediate DAGs formed during the reaction. In addition, multiple purification steps are required to remove the by-products (24). The concentration of target TAGs containing palmitic acid at the sn-2 position in the final product can be increased by separating the other TAGs through fractionated crystallization (58, 81, 84). Lee et al. (81) reported an increased OPO content from 25.2 to 53.3% in the enzymatic transesterification product of palm oil and camellia oil by fractionation at 22 °C for 16 h. Sørensen et al. (84) reported that HMFS with 56% palmitic acid at the sn-2 position was produced from fractionation of the acidolysis product of butterfat and a mixture of fatty acids from rapeseed and soybean oil. Also, the acidolysis product of solid fractions from fractionation of butterfat and a mixture of fatty acids from rapeseed and soybean oil produced HMFS with 47% palmitic acid at the sn-2 position.

CURRENT DEVELOPMENT OF HMFS PRODUCTION

In the last two decades, HMFS has been developed from a wide variety of substrates and enzymes and under various reaction conditions. In general, the most studied type of HMFS is sn-2 palmitate (OPO) because this TAG is the major component of HMF. Thus, the main consideration in HMFS production is to have palmitic acid at the sn-2 position (107). OPO-enriched HMFS is produced from interesterification between palmitic acid-containing source (i.e. lard, tripalmitin, palm oil and its derivatives: palm stearin or palm olein, catfish oil, palmitic acid or ethyl palmitate) and oleic acid-containing sources (i.e. olive oil, high oleic sunflower oil, oleic acid or ethyl oleate).

A better understanding of the composition and structure of HMF leads to better HMFS investigations (9). Recently, Wang et al. (75) synthesized both OPL and OPO from palm stearin fractions. OPL synthesis has not received much attention. The OPO to OPL ratios in HMF range from 0.5 to 2.0 (108, 109). Apart from sn-2 palmitate, HMF also contains PUFAs and MCFAs, which play an important role during the early human development (4, 110).

Synthesis of HMFS enriched with long-chain polyunsaturated fatty acids can be obtained from fish oil, algal oil, fungal oil, microbial oil, silkworm pupae oil, hazelnut oil, soybean oil, sunflower oil, ALA, GLA, DHA and ARA. Ghosh et al. (56) synthesized HMFS from palm stearin fractions and fish oil (r=2:1) via transesterification using Lipozyme TL IM. The final product contained 75.98% palmitic acid at the sn-2 position, 0.27% ARA, 3.43% EPA and 4.25% DHA. Interesterification between tripalmitin and ARA ethyl ester from ARA-rich single-cell oil from Mortierella alpina (r=1:2) using Lipozyme RM IM resulted in 69.57% ARA incorporation at sn-1,3 position and 62.31% palmitic acid at sn-2 position (43).

Naturally, MCFAs are present in HMF in the form of medium- and long-chain triacylglycerols (MLCTs). The main composition of TAGs is one MCFA and two long-chain fatty acids (MLL type) (4, 111). MCFAs in HMF mostly consist of lauric acid (C12:0), a small amount of capric acid (C10:0) and caprylic acid (C8:0) (112). MCT-enriched HMFS can also be synthesized from coconut oil, palm kernel oil and MCFAs containing caprylic acid and capric acid. Recently, Yuan et al. (55) synthesized HMFS containing TAG with MLL type from catfish oil and coconut oil (r=1.5:1) via interesterification using Lipozyme RM IM, Lipozyme TL IM, NS 40086 and DF Amano 15 (DF 15) with an enzyme load of 8%, temperature of 60 °C and reaction time of 3 h. The final product contained MLCT and MLL reaching 62.14 and 39.85%, respectively, with the main TAGs in HMFS being C12:0/C16:0/C18:1, C12:0/C18:1/C18:1, C12:0/C14:0/C18:1 and C12:0/C12:0/C18:1. Korma et al. (87) also produced HMFS containing 6.12% medium-medium-medium chain TAGs, 53.75% MLCTs and 40.13% long-long-long chain TAGs from interesterification of fungal oil containing high ARA content from Mortierella alpina and MCT (r=1:1) catalyzed by 8% recombinant CAL-B, temperature of 90 °C and reaction time of 3 h.

A single-step enzymatic transesterification can produce HMFS similar to HMF using the suited amount of substrate ratio. For example, Zou et al. (91, 92) reported a mixture of lard, sunflower oil, canola oil, palm kernel oil, palm oil, algal oil and microbial oil at a mass ratio 1.00:0.10:0.50:0.13:0.12:0.02:0.02 for HMFS synthesis. This substrate in the mixture was transesterified at a temperature of 60 °C, moisture content of 3.5% (on the lipase mass basis), reaction time of 3 h and Lipozyme RM IM 11% (on the total substrate mass basis). The product of HMFS had palmitic acid content of 20.1% with 38.2% palmitic acid at sn-2 position. The resulting HMFS had a high degree of similarity with HMF in the composition of total and sn-2 position fatty acids, PUFA and TAG with the values ​​of 92.5, 90.3, 61.5 and 71.9, respectively (91). Zou et al. (92) also used the substrate at that mixture ratio, which was transesterified using Lipozyme RM IM in PBR at 50 °C and a residence time of 1.5 h. The obtained HMFS had 39.2% palmitic acid at sn-2 position, 0.5% ARA and 0.3% DHA. Based on TAG content and purity, the degree of similarity of HMFS to HMF was 72.3.

At present, the commercial HMFS for inclusion in infant formulas has been successfully produced from various sources of oils and fats (4, 5, 9). The sn-2 palmitate is one of the structured TAGs that is generally supplemented into infant formulas (5, 113). HMFS products that have been commercialized according to the main fatty acid composition are Betapol 60 (C16:0, C18:1, C18:2), InFat™ (C16:0, C18:1), Bonamil (C16:0, C18:1, C18:2, C18:3), Similac Advance (C16:0, C18:1, DHA, ARA), Alsoy (C16:0, C18:1, DHA, ARA), Aptamil 1 (C16:0, C18:1, EPA, DHA), Milu-Milk (C16:0, C18:1, DHA), Bledina Alma (C16:0, C18:1, C18:2), Gallia HA (C16:0, C18:1, DHA, ARA), Cow & Gate Premium (C16:0, C18:1, DHA, GLA, ARA) and Baby Semp 1 (C16:0, C18:1, DHA, ARA) (24).

OUTLOOK: CHALLENGES AND OPPORTUNITIES IN HMFS SYNTHESIS

Structured lipids are designed through the modification of oils and fats to have desired nutritional or physicochemical properties suitable for food industry (9, 114, 115). HMFS is one of the ingredients in infant formula that is potentially and continuously developed to support infant growth according to the needs of each stage of baby’s age (i.e. infant and advanced formulas) and baby conditions (normal or premature and low birth mass babies).

The challenge for developing HMFS is the relatively high production cost. To enhance productivity (thus, reducing overall production cost), the synthesis of HMFS is carried out through a careful selection of the substrate, enzyme, reactor configuration and reaction conditions. Generally, the optimum reaction conditions for HMFS synthesis are at amount of substrate ratios between TAGs and FFAs of 1:2-1:14, temperatures of 40-60 °C, enzyme loads of 8-10% and reaction times of 2-24 h in batch process or 1-3 h in continuous process. Large-scale production of HMFS through a one-stage process using tripalmitin is not attractive because of its high cost and difficulty in obtaining products resembling HMF (24). On the other hand, the multistep reaction can produce higher yield of HMFS that has properties resembling HMF. However, the increase in reaction system complexity will also tend to increase downstreaming costs. It is worth mentioning that the production of HMFS in a solvent-free system is preferred in terms of food safety and costs (5).

One of the potential sources of substrates for HMFS synthesis is palm stearin because of its high palmitic acid content and relatively low price. However, the content of palmitic acid-rich TAGs at the sn-2 position of palm stearin needs to be increased through chemical interesterification (52, 53), enzymatic interesterification (37, 38) or fractionation (47, 56, 59, 116), which is due to the nature of palm stearin that is abundant in oleic acid at the sn-2 position. The acidolysis between palm stearin and oleic acid using an sn-1,3-specific lipase will result in triolein, which is not preferred (75). The HMFS synthetic route using palm stearin has to be started with enhancement of palmitic acid-rich TAGs at the sn-2 position (116). Then, the fatty acids at the sn-1,3 positions from the palmitic acid-rich TAGs are replaced with acyl donors through acidolysis or transesterification. The common acyl donors are single fatty acids (oleic acid, ALA, GLA, EPA, DHA and ARA), FFA mixtures of vegetable oils (such as olive, camelina, rapeseed, sunflower or hazelnut oil), sources of ω-3 PUFAs (such as fish or microalgal oil) (5, 9, 24), or sources of MCFA (such as coconut or palm kernel oil). HMFS that is similar to HMF and has C8:0, C10:0, C12:0, C16:0, C18:1, C18:2, EPA, DHA, GLA and ARA can potentially be commercialized in the future.

In HMFS synthesis, the high ratio of acyl donors is not attractive due to the difficulties in the separation process (such as deacidification) (51). This entails high costs of post-process separation (32). The possibility of producing HMFS with a low ratio of acyl donors is very interesting. However, the main limitation in the reaction process is low mass transfer, thus, a lower reaction rate. To overcome this problem, an enzyme that has a higher specificity and stability is needed. Faustino et al. (77) reported that tripalmitin consumption of 62.7% was achieved at r=1:1.2 at 65 °C using R. oryzae lipase immobilized on Lewatit VPOC 1600 during acidolysis between tripalmitin and FFAs from camelina oil. The isolation and genetic engineering of new lipases with better stability during operation at high temperatures are also of interest for future research (9, 19). The mutagenesis techniques are also promising for creating novel lipases such as an sn-2-specific lipase (22), which would facilitate the production of OPO. In addition, the use of continuous systems other than PBR, such as enzymatic membrane reactor, is also interesting to be developed (117). In enzymatic membrane reactor system, a continuous reaction can be facilitated by having immobilized enzyme retained inside the reactor (117).

CONCLUSIONS

Human milk fat substitute (HMFS) is synthesized by the enzymatic interesterification of vegetable oils, animal fats or blends of oils. The main characteristic of HMFS is having triacylglycerols (TAGs) with palmitic acid located at the sn-2 position and unsaturated fatty acids at the sn-1,3 positions. Selection of substrates, enzymes, batch or continuous reactor configuration and reaction conditions needs to be considered to increase the overall production of HMFS. Lipozyme RM IM, Lipozyme TL IM and Novozyme 435 are widely used for the synthesis of HMFS. Lipozyme RM IM and Lipozyme TL IM are used as biocatalysts due to their regiospecificity towards sn-1,3 positions. Generally, Lipozyme RM IM is used in acidolysis, whereas Lipozyme TL IM is used in transesterification. Novozyme 435 is used due to its regiospecificity towards sn-2 position, which is beneficial for incorporating palmitic acid at sn-2 position of the oils and fats, both in acidolysis and transesterification. Generally, the optimum reaction conditions for HMFS synthesis are amount of substrate ratios of TAGs and fatty acids between 1:2 and 1:14, temperatures of 40-60 °C, enzyme loads of 8-10%, moisture contents of 0.2-2% and reaction times of 2-24 h in batch process or 1-3 h in continuous process. The separation of interesterification product from FFAs in HMFS synthesis is carried out by neutralization using 0.5 M KOH (1.5 times the quantity of KOH required to neutralize the FFAs) or molecular distillation at the evaporation temperatures of 180-185 °C and pressures of 0.1-7.5 Pa.

117SitanggangABDrewsAKraumeM. Development of a continuous membrane reactor process for enzyme-catalyzed lactulose synthesis. Biochem Eng J. 2016;109:65–80. 10.1016/j.bej.2016.01.006