Human milk is recognized as the gold standard of infant nutrition because it provides not only the energy and optimum balanced nutrition for growing infants, but also immunological protection to newborns (Wang et al., 2019Wang X, Jiang C, Xu W, Miu Z. 2019. Enzymatic synthesis of structured triacylglycerols rich in 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol in a solvent-free system. Lwt-Food Sci. Technol. 118(C), 108798-108798. https://doi.org/10.1016/j.lwt.2019.108798 ). A population-based birth cohort study of neonates from Brazil showed that the durations of breastfeeding positively correlated with the intelligence quotient (IQ), educational attainment, and income of adults (Victoria et al., 2015Victoria CG, Horta BL, de Mola CL, Quevedo L, Pinheiro RT, Gigante DP, Goncalves H, Barros FC. 2015. Association between breastfeeding and intelligence, educational attainment, and income at 30 years of age: a prospective birth cohort study from Brazil. Lancet. Glob. Health. 3, E199-E205. https://doi.org/10.1016/S2214-109X(15)70002-1 ). Thus, breastfeeding has not only clear short-term benefits, but also influences long-term consequences on human capital. However, the rate of breastfeeding continues to decline because of medical or personal reasons. In these situations, some infants are partly or even fully fed infant formulas. Due to the demand for high-quality infant formulas in recent years, extensive simulation studies on the composition and distribution of HMF fatty acids have been undertaken.
Human milk contains about 2 to 6% fat, of which more than 98% is in the form of triacylglycerols (TAGs), providing approximately 50-60% of the dietary energy required for infants (Wang et al., 2019Wang X, Jiang C, Xu W, Miu Z. 2019. Enzymatic synthesis of structured triacylglycerols rich in 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol in a solvent-free system. Lwt-Food Sci. Technol. 118(C), 108798-108798. https://doi.org/10.1016/j.lwt.2019.108798 ). Commercial infant formulas are usually manufactured from cow milk and vegetable oils which may have similar fatty acid (FAs) compositions to human milk, but quite different position distribution in TAGs. In human milk, saturated fatty acids (SFAs), predominantly PA (20-30% of total FA), are largely located at the sn-2 position (70% of all PA); whereas the sn-1 and sn-3 positions are occupied by unsaturated fatty acids (USFAs), such as oleic acid (OA) (Ghide and Yan, 2021Ghide MK, Yan Y. 2021. 1,3-Dioleoyl-2-palmitoyl glycerol (OPO)-Enzymatic synthesis and use as an important supplement in infant formulas. J. Food Biochem. 45 (2), 13799. https://doi.org/10.1111/jfbc.13799 ). Thus, the characteristic component of human milk TAGs is OPO. However, cow milk and vegetable oils generally contain SFAs esterified to the sn-1 and sn-3 positions and USFAs at sn-2 position. These differences not only affect the nutrition and digestion of fats, but also cause stool hardness and constipation due to the preferential hydrolysis of PA in the sn-1,3 positions by pancreatic lipase to form calcium soap which is water-insoluble and can hardly be absorbed in the intestine of babies (Lee and Chang, 2021Lee JH, Chang HJ. 2021. Regiospecific Positioning of Palmitic Acid in Triacylglycerol Structure of Enzymatically Modified Lipids Affects Physicochemical and In Vitro Digestion Properties. Molecules 26 (13), 4015-4015. https://doi.org/10.3390/molecules26134015 ). In contrast, when PA is esterified to the sn-2 position, the 2-monoacylglycerol formed is readily absorbed (Ghide and Yan, 2021Ghide MK, Yan Y. 2021. 1,3-Dioleoyl-2-palmitoyl glycerol (OPO)-Enzymatic synthesis and use as an important supplement in infant formulas. J. Food Biochem. 45 (2), 13799. https://doi.org/10.1111/jfbc.13799 ), therefore, the kinetics of hydrolysis of different TAGs may induce a different digestive process.
The synthesis of OPO mainly takes place in two steps: the first step is the synthesis of TAGs enriched in PA at position 2 by chemical or enzymatic methods. Zou et al. (2012b)Zou XQ, Huang JH, Jin QZ, Liu YF, Tao GJ, Cheong LZ, Wang XG. 2012b. Preparation of human milk fat substitutes from palm stearin with arachidonic and docosahexaenoic acid: combination of enzymatic and physical methods. J. Agric. Food Chem. 60, 9415-9423. https://doi.org/10.1021/jf3017354 produced TAGs enriched in PA at position 2 by chemical interesterification of palm stearin and sodium methoxide as catalyst. Jimenez et al. (2010)Jimenez MJ, Esteban L, Robles A, Hita E, Gonzalez PA, Munio MM, Molina E. 2010. Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis. Process Biochem. 45, 407-414. https://doi.org/10.1016/j.procbio.2009.10.018 synthesized TAGs enriched in PA at position 2 by enzymatic interesterification of palm stearin and Novozym 435 as catalyst. In the second step, OPO was obtained by the synthesis of TAGs enriched in PA at position 2 with acyl donors using sn-1,3 regiospecific lipase. Esteban et al. (2011)Esteban L, Jimenez M J, Hita E, Gonzalez PA, Martin L, Robles A. 2011. Production of structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3 positions as human milk fat substitutes by enzymatic acidolysis. Biochem. Eng. J. 54, 62-69. https://doi.org/10.1016/j.bej.2011.01.009 obtained TAGs with 66% PA at sn-2 position and 67.5% OA at sn-1,3 positions, by acidolysis of PA-enriched TAGs and several OA-rich free fatty acid fractions in solvent-free media at 50 °C. Besides, OPO was also obtained by enzymatic interesterification. For instance, Lee et al. (2010)Lee JH, Son JM, Akoh CC, Kim MR, Lee KT. 2010. Optimized synthesis of 1,3-dioleoyl-2-palmitoylglycerol-rich triacylglycerol via interesterification catalyzed by a lipase from thermomyces lanuginosus. New Biotechnol. 27, 38-45. https://doi.org/10.1016/j.nbt.2009.10.006 synthesized a high OPO content (31.43%) from a PPP-rich fraction and ethyl oleate by a lipase-catalyzed interesterification.
HMF contains long-chain polyunsaturated fatty acids (LC-PUFAs) such as docosahexaenoic acid (DHA, 0.2~0.4 mol%, Table 3), arachidonic acid (ARA, 0.3~0.6 mol%, Table 3), which are crucial for infant growth and development (Qin et al., 2014Qin XL, Zhong JF, Wang YH, Yang B, Lan DM, Wang FH. 2014. 1,3-dioleoyl-2-palmitoylglycerol-rich human milk fat substitutes: production, purification, characterization and modeling of the formulation. Eur. J. Lipid Sci. Technol. 116, 282-290. https://doi.org/10.1002/ejlt.201300343 , Haddad et al., 2012Haddad I, Mozzon M, Frega NG. 2012. Trends in fatty acids positional distribution in human colostrum, transitional, and mature milk. Eur. Food. Res. Technol. 235, 325-332. https://doi.org/10.1007/s00217-012-1759-y ). Specifically, DHA and ARA are present in large amounts in the membranes of the brain and retina (Hoffman et al., 2009Hoffman 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. Plefa Medline 81,151-158. https://doi.org/10.1016/j.plefa.2009.05.003 ). Appropriate amounts of these fatty acids play a complex role in the development and function of the neuronal and retinal tissues after birth (Hoffman et al., 2009Hoffman 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. Plefa Medline 81,151-158. https://doi.org/10.1016/j.plefa.2009.05.003 ). They can be synthesized from linolenic and linoleic acid precursors by adults but infants lack this capacity and for this reason an adequate supply of LC-PUFAs are recommended in infant formulas (Ab et al., 2017Ab Ed, S M, Zou X, Ali AH, Jin Q, Wang X. 2017. Synthesis of 1,3-dioleoyl-2-arachidonoylglycerol-rich structured lipids by lipase-catalyzed acidolysis of microbial oil from Mortierella alpina. Biores. Technol. 243, 448-456. http://dx.doi.org/10.1016/j.biortech.2017.06.090 ).
When preparing HMFS products, the oxidative stability of HMFS must be acceptable during processing and storage. Some unsaturated fatty acids in HMFS, especially DHA and ARA, are easily oxidized to form lipid hydroperoxides and other small oxidation compounds that produce off-flavors via further decomposition. Researchers have reported that the ingestion of a diet high in polyunsaturated fatty acids which are insufficiently protected by antioxidants may increase the risk of atherosclerosis (Sakai et al., 1995Sakai T, Kuwazuru S, Yamauchi, K, Uchida K. 1995. A lipid peroxidation-derived aldehyde, 4-hydroxy-2-nonenal and omega 6 fatty acids contents in meats. Biosci Biotech Bioch. 59, 1379-1380. 10.1271/bbb.59.1379 ). Oxidation reduces the nutritional value and safety of HMFS and in order to prevent the oxidation of oil, different methods have been developed to protect against the lipid oxidation effect, although adding antioxidants remains the most effective one.
The aim of this work was to produce HMFS containing OPO and LC-PUFAs, whose structures and contents are highly similar to HMF’s. The first step involved enzymatic interesterification of the PPP-rich fraction with ethyl oleate to prepare OPO-HMFS and the second step involved the blending of the OPO-HMFS with other oils in order to adjust the contents of LC-PUFAs to simulate the fatty acid profiles in HMF based on some mathematical model. In order to improve the yield of OPO and reduce production costs, the interesterification reaction conditions were optimized and a linear restraint function was established to calculate the physical mixing ratio of oils in the second step. Improvement in the oxidative stability of HMFS-Emulsion by adding antioxidants was also achieved.
2. MATERIALS AND METHODS⌅
2.1. Chemicals and enzymes⌅
Lipozyme RM IM (Rhizomucormiehei immobilized in an ion exchange resin) and Lipozyme TL IM (Thermomyces lanuginosus immobilized on a silica support) were purchased from Novozymes A/S (Bagsvaerd, Denmark). Pancreatic lipase powder was purchased from Sigma-Aldrich (Shanghai, China). Palm stearin (PS), coconut oil (CO) and soybean oil (SO) were donated by Shanghai Kerry Oils & Grains Industries Co., Ltd. (Shanghai, China). Algal Oil (AO) and microbial oil (MO) were purchased from Fuxing Biotechnology Co., Ltd. (Wuhan, Hubei). The fatty acid profiles of CO, SO, MO and AO are shown in Table 1. Analytical or chromatographic-grade reagents were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other reagents were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China).
-: compound not detected because its content was too low. CO: Coconut Oil; SO: Soybean Oil; AO: Algal Oil; MO: Microbial Oil. Results are expressed as mean ± standard deviation (n = 2)
2.2. Preparation of PPP-rich fraction⌅
Palm stearin was mixed with acetone (1:5 w/v) and placed in a vacuum drying oven at 30 °C for 4 h. The PPP-rich fraction was collected after filtration and the removal of acetone was carried out in a vacuum-rotary evaporator at 60 °C and 1.33 × 103 Pa for 1 h (Zou et al., 2012bZou XQ, Huang JH, Jin QZ, Liu YF, Tao GJ, Cheong LZ, Wang XG. 2012b. Preparation of human milk fat substitutes from palm stearin with arachidonic and docosahexaenoic acid: combination of enzymatic and physical methods. J. Agric. Food Chem. 60, 9415-9423. https://doi.org/10.1021/jf3017354 ). The percentage of PPP in the sample increased from 52.8 to 85.4%.
2.3. Lipase-catalyzed synthesis of OPO⌅
The synthesis of OPO was carried out in a solvent-free system. The PPP-rich fraction was mixed acyl (oleic acid or methyl oleate or ethyl oleate) donors at substrate molar ratios of 1:8 and then lipases (10% based on the weight of the reaction mixture) were added to start the reaction in a shaking water bath at 55 °C and stirred at 200 rpm. The synthesis conditions were selected on the basis of previous research presented in this paper.
2.4. Fatty acid composition analysis⌅
The fatty acid composition of the products was analyzed through GC-MS using a Shimadzu GC2010A (Kyoto, Japan) gas chromatography instrument coupled with a GCMS-QP2010 quadrupole mass spectrometer (Shimadzu) and a Rtx®-Wax capillary column, 30 m length, 0.25 mm i.d and 0.25 μm film, consisting of cross-bond polyethylene glycol (Restek). The GC analysis method was reported by Zou et al. (2012a)Zou XQ, Guo Z, Huang JH, Jin QZ, Cheong LZ, Wang XG, Xu XB. 2012a. Human milk fat globules from different stages of lactation: a lipid composition analysis and microstructure characterization. J. Agric. Food Chem. 60, 7158-67. https://doi.org/10.1021/jf3013597 .
2.5. Analysis of C52 content by GC-FID⌅
C52, PPP and diacylglycerols (DAGs) in the reaction products were detected through GC using an Agilent 8890A GC platform equipped with a flame ionization detector and a fused silica capillary column DB-1HT (15 m length × 0.25 mm internal diameter × 0.25 μm film thickness, Agilent, Santa Clara, USA). The GC analysis method was reported by Liu et al. (2020)Liu CS, Tian JJ, Zhang RW, Xu JT, Nie K, Deng L, Wang F. 2020. Solvent-Free Alcoholysis of Tripalmitin to Produce 2-Monoglyceride as Precursor for 1, 3-Oleoyl-2-Palmitoyl-glycerol. Appl. Biochem. Biotechnol. 190, 867-879. https://doi.org/10.1007/s12010-019-03136-5 .
2.6. Sn-2 Positional analysis of TAGs by pancreatic lipase⌅
The hydrolysis of TAGs and determination of FA composition and sn-2 FA composition in TAGs were carried out according to the method described by Lee et al. (2010)Lee JH, Son JM, Akoh CC, Kim MR, Lee KT. 2010. Optimized synthesis of 1,3-dioleoyl-2-palmitoylglycerol-rich triacylglycerol via interesterification catalyzed by a lipase from thermomyces lanuginosus. New Biotechnol. 27, 38-45. https://doi.org/10.1016/j.nbt.2009.10.006 .
2.7. Molecular distillation⌅
KDL1, UIC GmbH (Alzenau, Germany) was used to purify TAGs in the products under the following conditions: the operating pressure was set at 1 × 10−3 mbar, the rotor velocity at 300 min−1, and the feed flow rate at 1.5 mL/min (feed temperature was 60 °C). When the heating oil temperature was increased to 210 °C, ethyl oleate, DAGs and free fatty acids were separated from the product.
2.8. OPO-HMFS blending with selected oils to prepare Final-HMFS⌅
OPO-HMFS TAGs are the main components in the fat in infant formula, and are rich in OA and PA. Nevertheless, the contents in other major fatty acids, such as linoleic acid, lauric acid and some LC-PUFAs should also be within the ranges of their corresponding fatty acids in HMF. According to the fatty acid composition and distribution of typical human milk fat, a linear restraint function for the development of HMFS formula was established as follows:
i is the serial number of the selected oils and x1, x2, x3, x4 and x5 represent the OPO-HMFS, coconut oil, soybean oil, algal oil and microbial oil, respectively; ai1, ai2, a i3, ai4, ai5, ai6 and ai7 are the contents in lauric acid (C12:0)+myristic acid (C14:0), palmitic acid (C16:0), oleic acid (C18:1n-9), linoleic acid (C18:2n-6), Linolenic acid (C18:3n-3), docosahexaenoic acid (DHA) and arachidonic acid (ARA) in the selected oils, respectively. c2, c3, c4 and c5 represent the marketing cost of coconut, soybean, algal and microbial oils, respectively.
2.9. Oil properties analysis⌅
2.9.1 DSC melting profile⌅
The melting profiles of oil samples were determined by differential scanning calorimetry (DSC) using a DSC Q2500 according to the method Cj1-94 (AOCS, 2009AOCS. 2009. Official methods and recommended practices of the American Oil Chemists’ Society. 6th. edition, Cj1-94 Champaign, USA.).
2.9.2 Acid value (AV), peroxide value (PV) and p-anisidine value (p-AV)⌅
The AV, PV and p-AV were determined according to methods 5a-40 Cd, 8b-90 and p 2.4 (AOCS, 1997AOCS. 1997. Official methods and recommended practices of the American Oil Chemists’ Society. 5th. edition, 383 Champaign, USA.), respectively.
2.9.3 Induction period (IP)⌅
The oxidative stability was determined in terms of induction period (IP) using the Rancimat method (Olajide et al., 2020Olajide TM, Liu T, Liu HA, Weng XC. 2020. Antioxidant properties of two novel lipophilic derivatives of hydroxytyrosol. Food Chem. 315, 126-197. https://doi.org/10.1016/j.foodchem.2020.126197 ). 3 Grams of sample with and without added antioxidants were carefully weighed into Rancimat tubes and subjected to accelerated oxidation at 100 °C under an air flow of 20 L/h.
2.9.4 Emulsion preparation and oven test⌅
For convenience, infant formula liquid milk is also popular in the market. Most papers only tested the oxidative stability of HMFS itself, but did not evaluate the oxidation stability of HMFS in emulsion. HMFS-in-water emulsions (500 g) were prepared as mixtures of purified HMFS, Tween 80 and a phosphate buffer solution (pH 7.0) according to methods described by Chen et al. (2020)Chen QQ, Pasdar H, Weng XC. 2020. Butylated methyl caffeate: a novel antioxidant. Grasas Aceites, 71, 352. https://doi.org/10.3989/gya.0226191 . These samples were divided into experimental groups (0.02% antioxidants added) and a control group (no antioxidant added). Particle size distributions of the emulsions were measured by Mastersizer 2000 (triplicate, at room temperature) according to the method by Zou et al., (2012a)Zou XQ, Guo Z, Huang JH, Jin QZ, Cheong LZ, Wang XG, Xu XB. 2012a. Human milk fat globules from different stages of lactation: a lipid composition analysis and microstructure characterization. J. Agric. Food Chem. 60, 7158-67. https://doi.org/10.1021/jf3013597 . The average emulsion droplet size 14.90±0.76 nm was similar to HMF, and there was no visible significant change in the state of the emulsions over the course of the oven test. The emulsions were held in an oven at 63 ± 1 °C and the induction period of the samples was dependent on the oil reaching a peroxide value of 80 meq O2/kg oil (Shi et al., 2017Shi G, Liao X, Olajide TM, Liu J, Jiang X, Weng XC. 2017. Butylated caffeic acid: An efficient novel antioxidant. Grasas Aceites, 68, e201. https://doi.org/10.3989/gya.1278162 ; Olajide et al., 2020Olajide TM, Liu T, Liu HA, Weng XC. 2020. Antioxidant properties of two novel lipophilic derivatives of hydroxytyrosol. Food Chem. 315, 126-197. https://doi.org/10.1016/j.foodchem.2020.126197 ).
2.10. Statistical analysis⌅
All assays were carried out in duplicate or triplicate and the data are presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) was carried out using IBM SPSS 22.0, followed by Duncan’s multiple range test (P < 0.05).
3. RESULTS AND DISCUSSION⌅
3.1. Influence of reaction time and acyl donors on the contents in C52 and sn-2 PA⌅
The whole process for obtaining OPO consisted of two reactions: first, obtaining TAGs rich in tripalmitin (PPP) from palm stearin and second, obtaining OPO by interesterification or acidolysis of TAGs enriched in PA at position 2 and acyl (oleic acid or methyl oleate or ethyl oleate) donors, catalyzed by sn-1, 3 specific lipases.
Acyl donors are important raw materials for the preparation of OPO. Srivastava, et al., (2006)Srivastava A, Akoh CC, Chang SW, Lee GC, Shaw JF. 2006. Candida rugosa lipase lip1-catalyzed interesterification to produce human milk fat substitute. J. Agric. Food Chem. 54, 5175. https://doi.org/10.1021/jf060623h reported that methyl ester is a better acyl donor than free acid for producing HMFS. Three kinds of oleic acyl donors (oleic acid, methyl and ethyl oleate) were compared to investigate their influence on the contents in C52 and sn-2 PA in our study. The initial rate of C52 synthesis using methyl or ethyl oleate with TAGs rich in PPP was higher than the oleic acid tested (Figure 1A). This was mainly due to better solubility of methyl or ethyl oleates in solvent-free systems than oleic acid. It should be noted that PPP with a high melting point (about 60 °C) hardly melt into liquid at the optimal reaction temperature of lipase, so organic solvent must always be added to the reaction system to reduce the PPP dissolution temperature and the viscosity of the reaction system so as to ensure the high activity of the lipase. However, large scale use of solvents may bring potential safety hazards. Methyl or ethyl oleates can act as both substrate and solvent to accelerate the dissolution of PPP at appropriate temperatures. There was no significant difference in the initial rate of C52 synthesis between methyl or ethyl oleate use. Ethyl oleate was eventually selected because ethanol hydrolyzed by ethyl oleate in the interesterification reaction is much safer compared to methanol. Moreover, the boiling point of ethyl oleate (220 °C) is lower than that of oleic acid (241 °C) at the same pressure, hence easier to remove during the purification of OPO. When the heating oil temperature of molecular distillation was increased to 210 °C, the by-products of interesterification reaction including DAGs, MAGs and ethyl oleate were almost completely removed. The purified product did not contain ethyl oleate and only a small amount of DAGs (1.6 ± 0.2%, Table 2). The C52 content increased with time, but was practically constant from 6 h and its maximum content of over 40% was similar to all the acyl donors tested.
|Fatty acid (area%)||Total||Sn-2 position||Sn-1,3 position|
Results are expressed as mean ± standard deviation (n = 3). Operational conditions: temperature, 55 °C; substrate molar ratio, 1:10 (PPP-rich fraction/ ethyl oleate); enzyme load, 10 wt %; water content, none; reaction time, 3 h. C52: primarily OPO and its isomeric compounds; PPP: tripalmitin; DAG: diacylglycerols. Fatty acid content at sn-1,3 positions of TAGs is determined by equation:
The content in PA at the sn-2 position decreased with the extension of reaction time (Figure 1B). Although Lipozyme RM IM exhibited the sn-1, 3-specificity, TAG with OA at the sn-2 position was possible to produce due to acyl-migration in the form of DAGs. DAGs are inevitable intermediates in lipase-catalyzed interesterification and they could be converted from 1,2-DAGs and 1,3-DAGs (Xu et al., 1998Xu X, Skands ARH, Hoy CE, Mu H, Balchen S, Adler-Nissen, J. 1998. Production of specific-structured lipids by enzymatic interesterification: elucidation of acyl migration by response surface design. J. Am. Oil. Chem. Soc. 75, 1179-1186. https://doi.org/10.1007/s11746-998-0309-z ). The thermal instability of these DAGs may lead to acyl transfer as reaction time increases, reducing the yield of OPO (Xu et al., 1998Xu X, Skands ARH, Hoy CE, Mu H, Balchen S, Adler-Nissen, J. 1998. Production of specific-structured lipids by enzymatic interesterification: elucidation of acyl migration by response surface design. J. Am. Oil. Chem. Soc. 75, 1179-1186. https://doi.org/10.1007/s11746-998-0309-z ). The percentage content of PA in the sn-2 position, determined by GC analyses, represents acyl migration. The acyl migration rates of oleic acid, methyl oleate and ethyl oleate in 3 h were 7.8, 5.7 and 5.1%, respectively. Compared to oleic acid, the PA content at the sn-2 position decreased more slowly in a short reaction time (3 h) with ethyl oleate tested, thus showing a lower acyl migration rate, which may be related to the polarity difference between oleic acid and ethyl oleate (Figure 1B). Li et al. (2010)Li W, Du W, Li Q, Li RW, Liu DH. 2010. Dependence on the properties of organic solvent: study on acyl migration kinetics of partial glycerides. Bioresource Technol. 101, 5737-5742. https://doi.org/10.1016/j.biortech.2010.03.018 have reported that organic solvents with low polarity could inhibit the acyl-migration of sn-2 fatty acids in TAGs. To obtain a higher C52 and sn-2 PA contents in short reaction times, ethyl oleate was selected to be the most ideal acyl donor.
3.2. Optimization of interesterification conditions⌅
3.2.1. Effect of temperature on the contents of C52 and sn-2 PA⌅
Higher temperature decreases the viscosity of substrates and increase mass transfer, which in turn reduce reaction time to equilibrium, but also enhance the rate of occurrence of acyl-migration and other negative impacts, such as fat oxidation. Previous studies show that substrate could be dissolved at low temperature in solvent system, which partitions in a more stable environment (Esteban et al., 2011Esteban L, Jimenez M J, Hita E, Gonzalez PA, Martin L, Robles A. 2011. Production of structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3 positions as human milk fat substitutes by enzymatic acidolysis. Biochem. Eng. J. 54, 62-69. https://doi.org/10.1016/j.bej.2011.01.009 ; Wei et al., 2015Wei W, Feng YF, Zhang X, Ca X, Feng FQ. 2015. Synthesis of structured lipid 1,3-dioleoyl-2-palmitoylglycerol in both solvent and solvent-free system. Lwt-Food Sci. Technol. 60, 1187-1194. https://doi.org/10.1016/j.lwt.2014.09.013 ). However, the addition of solvents is harmful to the environment and/or economy. Solvent-free is a much safer, much more environmentally-friendly and economical industrial application. Thus, all the experiments in this study were based on a solvent-free system.
The reaction accelerated as temperature increased from 50 to 55 °C and the C52 content increased with it (Figure 2A). However, the C52 content, when above 55 °C, changed only slightly. At 55 °C, the C52 content was 45.4% and PA content at the sn-2 position was 75.2% at the end of the 3-h reaction. Compared to other temperatures, the products at 50 °C were characterized by higher PA content (i.e. 77.0%) at the sn-2 position; while the C52 content was only 39.8%, which may be due to lower catalytic efficiency. Moreover, higher temperature could decrease the sn-2 PA content significantly and might cause adverse consequences on enzyme properties and so, 55 °C was selected as the most ideal reaction temperature.
3.2.2 Effect of substrate ratio on the contents of C52 and sn-2 PA⌅
According to the reaction formula, OPO can be synthesized under ideal conditions by maintaining the ratio of the PPP-rich fraction to ethyl oleate at 1:2. However, the interesteri- fication reaction is reversible, and the feedback regulation of the product will inhibit the reaction. To obtain the highest OPO yield, the effect of substrate ratio was compared in this experiment. From Figure 2B, the C52 content in the enzymatic products at the substrate ratios of 1:2, 1:4, 1:6, 1:8, 1:10 and 1:12 were 14.7, 25.7, 35.1, 41.7, 44.1 and 44.4%, respectively. The C52 content between the molar ratios of 1:10 and 1:12 were nearly identical, indicating that increasing the amount of ethyl oleate continuously would not necessarily improve the OPO yield. At the same time, using high amounts of ethyl oleate would mean a limitation for industrial production of OPO-rich HMFS. In addition, no significant difference in the sn-2 PA content with all the molar ratios tested indicated that the amount of ethyl oleate had very little influence on the acyl migration reaction. Thus, to improve catalytic and economic efficiency, an optimum molar ratio of 1:10 between the PPP-rich fraction and ethyl oleate is required.
3.2.3 Effect of enzyme load on the contents in C52 and sn-2 PA⌅
Enzyme load is related to reaction rate. Higher enzyme load will accelerate the reaction rate and improve the incorporation rate of the acyl donor in the interesterification reaction. When the enzyme load was less than 10%, the content in C52 increased as enzyme load increased and reached its highest point (43.8%) at 10%, but later decreased as enzyme load increased (Figure 2C). Meanwhile, the content in sn-2 PA decreased slowly and continuously as the enzyme load increased. The sn-2 PA content was only 71.8% when the enzyme load reached 12%. These findings are understandable because the water content in the Lipozyme RM IM may have promoted the amount of DAG produced from TAGs, thereby enlarging the possibility of acyl-migration. An enzyme load of 10% (w/w) generated the C52 content (43.8%) and sn-2 PA content (73.9%) after 3 h, showing preferable yield and catalytic efficiency.
3.2.4 Effect of water content on the contents in C52 and sn-2 PA⌅
Researchers have found that the water content in the synthetic reaction of TAG favors the content in DAG, which is due to hydrolytic side reactions (Zou et al., 2012bZou XQ, Huang JH, Jin QZ, Liu YF, Tao GJ, Cheong LZ, Wang XG. 2012b. Preparation of human milk fat substitutes from palm stearin with arachidonic and docosahexaenoic acid: combination of enzymatic and physical methods. J. Agric. Food Chem. 60, 9415-9423. https://doi.org/10.1021/jf3017354 ). Moreover, the presence of water on the surface of lipase is important for maintaining its activity and the flexibility of its protein structure. With increased water content in the system, the yields of C52 and sn-2 PA decreased consistently (Figure 2D). The content in C52 was 44.9% in the system without water, which was 3.6% higher than that in the system with 10% water content. Similarly, the content in sn-2 PA was 77.1% in the system without water, which was 7.8% higher than that in the system with 10% water content. A similar study was also reported in a study by Liu et al., (2020)Liu CS, Tian JJ, Zhang RW, Xu JT, Nie K, Deng L, Wang F. 2020. Solvent-Free Alcoholysis of Tripalmitin to Produce 2-Monoglyceride as Precursor for 1, 3-Oleoyl-2-Palmitoyl-glycerol. Appl. Biochem. Biotechnol. 190, 867-879. https://doi.org/10.1007/s12010-019-03136-5 . The results indicated that high moisture in the reaction system can accelerate the hydrolysis of TAGs to DAGs and cause more acyl-transfer, reducing the content in sn-2 PA. It has been speculated that there are some hydrophilic groups on the surface of Lipozyme RM IM itself, which maintains its activity. As a result, the reaction was carried out in the absence of water.
Overall, the optimal OPO synthetic conditions were as follows: temperature, 55 °C; substrate molar ratio, 1:10 (PPP-rich fraction/ethyl oleate); enzyme load, 10 wt %; water content, none; reaction time, 3 h. Under these conditions, using molecular distillation, the composition of fatty acid and acyl glycerol of the OPO-HMFS are shown in Table 2. The OA and PPP contents in the OPO-HMFS were 53.3 and 2.9%, respectively, showing that OA was effectively incorporated into the substrate. A relatively small amount of PA on the TAGs migrated from the sn-2 to sn-1, 3 positions, since 75.3% of PA can be observed at the sn-2 position of OPO-HMFS.
3.3. OPO-HMFS blending with selected oils to prepare Final-HMFS.⌅
Compared to the fatty acid composition and location distribution of HMF (PA, 19.6% - 29.0%; OA, 26.0% - 40.6%; linoleic acid, 7.1% - 20.1%; myristic acid+ lauric acid < 20.0%; linoleic acid/linolenic acid, 5-15; DHA, 0.2%-0.4% and ARA, 0.3%-0.6%), the synthesized OPO-HMFS contained a smaller amount of linoleic acid and no medium-chain fatty acids or LC-PUFAs, but was characterized by much higher PA content (33.6%). In order to add LC-PUFAs into infant formula, many studies used the acidolysis or interesterification method to prepare structural lipids containing LC-PUFAs, which was not efficient and thus increased production costs (Hoffman et al., 2009Hoffman 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. Plefa Medline 81,151-158. https://doi.org/10.1016/j.plefa.2009.05.003 ). Consequently, in our study, OPO-HMFS was physically blended with CO, SO, AO, and MO to adjust the contents in major and important fatty acids (ARA and DHA) to the proper ranges and maintain regional specificity of fatty acids like HMF.
Based on the fatty acid profiles of HMF, OPO-HMFS contained a smaller amount of linoleic acid (6.2±1.2%, Table 2) and no medium chain fatty acids or LC-PUFAs; therefore, CO, SO, AO, and MO, which are rich in lauric acid (48.9%), linoleic acid (54.8%), DHA (48.3%), and ARA (48.1%), respectively, were selected to blend with the OPO-HMFS to supplement the lack in fatty acids in OPO-HMFS (Tables 1 & 2). These selected oils are common in the market with low cost and convenient for industrialized production. To guarantee good quality and lower production costs of the Final-HMFS, Matlab R2018b was used to optimize the blending process. The final desirable formula constituted OPO-HMFS/CO/SO/AO/MO at a weight ratio of 0.70:0.18:0.11:0.004:0.007. The determined and theoretically evaluated fatty acid compositions and positional distributions of the final product under this blending ratio are shown in Table 3. The contents in PA and sn-2 PA were 26.2 and 47.9%, and the contents in LC-PUFA including ARA and DHA were 0.4 and 0.3%, respectively. The main and important fatty acids were almost within the ranges of these in HMF (Table 3), indicating that the fatty acid composition of the final product was highly similar to HMF and may be used as a fat substitute in infant formula. Besides, researches have reported differences between the fatty acid compositions of HMF, especially LC-PUFAs, at different stages of lactation (Sala-Vila et al., 2005Sala-Vila A, Castellote AI, Rodriguez-Palmero M, Campoy C, Lopez-Sabater MC. 2005. Lipid composition in human breast milk from Granada (Spain): changes during lactation. Nutrition. 21, 467-473. https://doi.org/10.1016/j.nut.2004.08.020 ; Haddad et al., 2012Haddad I, Mozzon M, Frega NG. 2012. Trends in fatty acids positional distribution in human colostrum, transitional, and mature milk. Eur. Food. Res. Technol. 235, 325-332. https://doi.org/10.1007/s00217-012-1759-y ) and different proportions of fatty acids can also be formulated through the established model to meet the nutritional needs of infants in different stages of lactation.
|Fatty acid||Theoretical values||Determined Values||HMF|
Results are expressed as mean ± standard deviation (n = 3). Theoretical values: Calculated from Matlab R2018b software. Determined values: OPO-HMFS was blended with coconut, soybean, algal and microbial oils at a weight ratio of 0.70:0.18:0.11:0.004:0.007. HMF (human milk fat): Typical fatty acid composition (mol %) of human milk fat was adapted from previous studies (Qin et al., 2014Qin XL, Zhong JF, Wang YH, Yang B, Lan DM, Wang FH. 2014. 1,3-dioleoyl-2-palmitoylglycerol-rich human milk fat substitutes: production, purification, characterization and modeling of the formulation. Eur. J. Lipid Sci. Technol. 116, 282-290. https://doi.org/10.1002/ejlt.201300343 , Haddad et al., 2012Haddad I, Mozzon M, Frega NG. 2012. Trends in fatty acids positional distribution in human colostrum, transitional, and mature milk. Eur. Food. Res. Technol. 235, 325-332. https://doi.org/10.1007/s00217-012-1759-y ).
3.4. Oil properties analysis⌅
3.4.1. DSC melting curve⌅
The melting curves of OPO-HMFS, Final-HMFS and palm stearin were determined by DSC. A lower melting temperature was observed for the OPO-HMFS product (18.1 °C) compared to palm stearin (57.6 °C), which contributed to the high content in OA and low content in PA in the product. The melting temperatures of both OPO-HMFS and Final- HMFS (14.3 °C) were lower than the normal human body temperature (about 37.5 °C) depicting that they are suitable to be used as fat base materials in infant formulas. The melting range of HMFS is important for its digestion and absorption. The melting range of Final-HMFS was relatively narrower than that of OPO-HMFS (Figure 3), showing that the content in the high melting point component in OPO-HMFS was higher than that of Final-HMFS.
Table 4 summarizes the basic properties of palm stearin, OPO-HMFS and Final-HMFS. TAGs hydrolyze to release free fatty acids during heating or the action of lipase, which influences the stability of oil. AV can be used to indicate the degree of oil hydrolytic rancidity and refining. The undetectable AV of OPO-HMFS showed that molecular distillation was an effective method to remove free fatty acid or fatty acid ethyl ester. The AV of the Final-HMFS was only 0.3 mg KOH/g, indicating that the selected oil blended with OPO-HMFS was of good product quality.
|IP||16.8±0.7a||4.1±0.9 c||6.3±0.6 b|
-: compound not detected because its content was too low. Results are expressed as mean ± standard deviation (n = 3). Means in the same row with different letters are significantly different according to Duncan’s multiple range test (P < 0.05). OPO-HMFS was synthesized using palm stearin enriched in tripalmitin and ethyl oleate by enzymatic interesterification under optimum conditions. Final-HMFS was obtained by blending OPO-HMFS with coconut, soybean, algal and microbial oils at a weight ratio of 0.70:0.18:0.11:0.004:0.007.
3.4.3. PV and p-AV⌅
PV and p-AV were used to evaluate the degree of oil oxidation and deterioration. PV represents the main products of oil oxidation, like hydroperoxides, while p-AV represents the small molecular compounds such as aldehydes and ketones produced by further decomposition of peroxides. High PV and p-AV indicate high oxidation of oil samples. From the Table 4, the PV and p-AV of OPO-HMFS (1.1 meq O2/kg oil and 0.9, respectively) were quite low compared with palm stearin (3.8 meq O2/kg oil and 3.2, respectively), thus implying the purification process had also removed the majority of peroxides. The PV (3.4 meq O2/kg oil) and p-AV (3.0) in Final-HMFS was higher than that in OPO-HMFS but it was still within an acceptable range.
3.4.4. Oxidative stability of HMFS⌅
The rancimat assay characterizes the oxidative stability of oils. The oxidative stability indexes of palm stearin, OPO-HMFS and Final-HMFS were measured by the Rancimat test and expressed as IP (Table 4). Higher IP reflects a longer shelf-life of products. The IP value of the OPO-HMFS (4.1 h) and Final-HMFS (6.3 h) were lower compared to palm stearin (16.8 h). For one thing, USFAs in HMFS were more easily oxidized than SFAs in palm stearin; for another thing, the reason may be due to the loss in tocopherols and β-carotene in the process of refining OPO-HMFS by molecular distillation, thus reducing antioxidant capacity. The decrease in oxidative stability after molecular distillation is in agreement with several similar studies (Nielsen et al., 2006Nielsen NS, Yang TK, Xu XB, Jacobsen C. 2006. Production and oxidative stability of a human milk fat substitute produced from lard by enzyme technology in a pilot packed-bed reactor. Food. Chem. 94, 53-60. https://doi.org/10.1016/j.foodchem.2004.10.049 ; Qin et al., 2014Qin XL, Zhong JF, Wang YH, Yang B, Lan DM, Wang FH. 2014. 1,3-dioleoyl-2-palmitoylglycerol-rich human milk fat substitutes: production, purification, characterization and modeling of the formulation. Eur. J. Lipid Sci. Technol. 116, 282-290. https://doi.org/10.1002/ejlt.201300343 ). Fat oxidative rancidity is due to the long-term storage of HMFS under unfavorable conditions, which usually requires some protection from antioxidants.
The efficacy of β-carotene, L-AP, VE and TBHQ (tert-butylhydroquinone) as antioxidants was evaluated using the Rancimat test at 100 °C, and concentrations at 0.02% (w/w) under air saturation conditions. The results were expressed as induction period (IP) corresponding to the oxidative stability of OPO-HMFS (Figure 4). The IPs of the blank, β-carotene, L-AP, VE and TBHQ were 3.8, 5.6, 7.8, 8.1, and 12.7 h, respectively. This indicates that the antioxidant activities of the antioxidants decreased as follows: TBHQ > VE ≈ L-AP > β-carotene. Although TBHQ has strong antioxidant activity compared to other antioxidants, it is a synthetic antioxidant. The existence of a double bond in β-carotene can prevent oil from being oxidized, but the effect is not profound. VE, which contains hydroxyl groups, can inhibit lipid oxidation by capturing free radicals, which is a natural antioxidant and a nutritional component contained in vegetable oil. L-AP is the only unnatural antioxidant allowed to be added to baby food in China. In some instances, combined antioxidants have greater antioxidant activities than their individual effects. The IP of the mixture of VE and L-AP (at a weight ratio of 1:1) is 9.8 h, which is higher than that of VE or L-AP alone, indicating that L-AP has a synergistic effect with VE. When VE and L-AP are used together, L-AP could regenerate the phenoxyl radical of VE back to active VE. The synergistic effect of VE and L-AP cannot only enhance the oxidative stability of OPO-HMFS, but also reduce cost because L-AP is low-priced.
3.4.5. Oxidative stability of OPO-HMFS-Emulsion in oven test⌅
HMFS are widely used in emulsions because of low water solubility. Emulsion can be used as an excellent carrier for wrapping, protecting and transporting HMFS, so that it can reach the designated position in the gastrointestinal tract digestion process (Hu, et al., 2003Hu M, Mcclements DJ, Decker EA. 2003. Lipid Oxidation in Corn Oil-in-Water Emulsions Stabilized by Casein, Whey Protein Isolate, and Soy Protein Isolate. J. Agric. Food Chem. 51, 1696-1700. https://doi.org/10.1021/jf020952j ). Nevertheless, the oxidation of HMFS in emulsion is inevitable. In this study, PV was used to evaluate the oxidative stability of OPO-HMFS-Emulsion during the oven test at 63 ± 1, for 21 days. As shown in Figure 5, the PV of the blank group was 2.7±0.5 meq/kg on the first day, which then began to accelerate until it reached a maximum of 118.6±1.9 meq/kg after 21days of storage. In contrast, the PV of HMFS containing the TBHQ, VE, L-AP, mixture of VE and L-AP increased slightly until day 9, and then gradually increase dynamically with storage time. The changes in PV in the samples spiked with TBHQ, VE and L-AP showed that the addition of antioxidants was better at preventing oxidation of the emulsions and they were 20.4, 70.2, and 80.5 meq/kg on the last day, respectively. This indicates that the antioxidant activities of the antioxidants in emulsions decreased in the following order: TBHQ > VE > L-AP. Similarly, the results from Figure 5 showed that the mixture of VE and L-AP resulted in a synergistic antioxidant effect. Besides, the OPO-HMFS-Emulsion oxidative stability of L-AP and VE at a ratio of 2:3 was greater than that of ratio 1:3. Although increasing the proportion of L-AP in mixed antioxidants can improve the oxidative stability of OPO-HMFS emulsion, the proportion of VE in it should be kept at a higher level because VE also plays an important role in improving immunity and promoting the growth of infants.
This study produced HMFS which contained OPO and LC-PUFAs, whose content and structure of fatty acids are highly similar to HMF. The enzymatic interesterification conditions for OPO-HMFS (enzyme load, substrate molar ratio, temperature, water content) were optimized. A high content in C52 production (46.7%) and a sn-2 PA content of 75.3% were achieved. Moreover, OPO-HMFS was blended with coconut oil, soybean oil, algal oil and microbial oil at weight ratios of 0.70:0.18:0.11:0.004:0.007 to simulate the fatty acid profiles of HMF according to some mathematical models. The main and important fatty acids were closely within the ranges naturally found in HMF. The mixture of L-AP and VE resulted in a synergistic antioxidant effect both in OPO-HMFS and OPO-HMFS-Emulsion.