Characterization and Oxidative Stability of Human Milk Fat Substitutes Enzymatically Produced from Palm Stearin

Production of human milk fat substitutes (HMFSs) from three types of palm stearin with palmitic acid (PA) of 91.3, 70.3 and 62.6 %, respectively, was scaled up to a kilogram scale. The physiochemical properties of these products including fatty acid profiles, triacylglycerol compositions, tocopherol contents, oxidative stability and melting and crystallization profiles were compared with those of HMFSs from lard, butterfat and tripalmitin and fats from infant formulas. Based on their chemical compositions, HMFSs from palm stearin with PA contents of 70.3 and 62.6 % produced by enzymatic acidolysis were found to have the highest degree of similarity to human milk fat, which indicated that these HMFSs were the most suitable for use in infant formulas. However, HMFSs from palm stearin with PA content of 91.3 % had the highest tocopherol contents. By investigation of the primary and secondary oxidation products during accelerated oxidation, the oxidative stability of HMFSs was found to be positively correlated to the contents of tocopherols, and the volatile oxidation compounds with the highest relative contents in HMFSs were aldehydes analyzed by solid-phase microextraction-GC–MS. All HMFSs had final melting points lower than body temperature.     

Preparation and Characterization of Human Milk Fat Substitutes Based on Triacylglycerol Profiles

Human milk fat substitutes (HMFS) having similarity in (TAG) composition to human milk fat (HMF) were prepared by Lipozyme RM IM‐catalyzed interesterification of lard blending with selected oils in a packed bed reactor. Four oil blends with high similarity in fatty acid profiles to HMF were first obtained based on the blending model and then the blending ratios were screened based on TAG composition similarity by enzymatic interesterification in a batch reactor. The optimal ratio was determined as lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. This blending ratio was used for a packed bed reactor and the conditions were then optimized as residence time, 1.5 h; reaction temperature, 50 °C. Under these conditions, the obtained product showed high degrees of similarity in fatty acid profile with 39.2 % palmitic acid at the sn‐2 position, 0.5 % arachidonic acid (n‐6) and 0.3 % docosahexaenoic acid (n‐3) and the scores for the degree of similarity in TAG composition was increased from 58.4 (the oil blend) to 72.3 (the final product). The packed bed reactor could be operated for 7 days without significant decrease in activity. The final product presented similar melting and crystallization profiles to those of HMF. However, due to the loss of tocopherols during deacidification process, the oxidative stability was lower than that of the oil blend. This process for the preparation of HMFS from lard with high similarity in TAG composition by physical blending and enzymatic interesterification, as optimized by mathematical models in a packed bed reactor, has a great potential for industrialization.     

Preparation of Human Milk Fat Substitutes from Lard by Lipase‐Catalyzed Interesterification Based on Triacylglycerol profiles

Human milk fat substitutes (HMFSs) with triacylglycerol profiles highly similar to those of human milk fat (HMF) were prepared from lard by physical blending followed by enzymatic interesterification. Based on the fatty acid profiles of HMF, different vegetable and single‐cell oils were selected and added to the lard. Blend ratios were calculated based on established physical blending models. The blended oils were then enzymatically interesterified using a 1,3‐regiospecific lipase, Lipozyme RM IM (RML from Rhizomucor miehei immobilized on Duolite ES562; Novozymes A/S, Bagsværd, Denmark), to approximate HMF triacylglycerol (TAG) profiles, particularly with respect to the distribution of palmitic acid in the sn?2 position. The optimized blending ratios were determined to be: lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. The optimized reaction conditions were determined to be: enzyme load of 11 wt%, temperature of 60 °C, water content of 3.5 wt%, and reaction time of 3 hours. The resulting product was evaluated for total and sn?2 fatty acids, polyunsaturated fatty acids, and TAG composition. A high degree of similarity was obtained, indicating the great potential of the product as a fat alternative for use in infant formulas.     

Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases

This study aims to produce human milk fat substitutes by an acidolysis reaction between lard and the free fatty acids (FFA) from a fish oil concentrate rich in docosahexaenoic acid, in solvent-free media. The immobilized commercial lipases from (1) Rhizomucor miehei (Lipozyme RM IM), (2) Thermomyces lanuginosa (Lipozyme TL IM) and (3) Candida antarctica (Novozym 435) were tested as biocatalyst. Also, the heterologous Rhizopus oryzae lipase (rROL), immobilized in Accurel^® MP 1000, was tested as a feasible alternative to the commercial lipases. After 24 h of reaction at 50 °C, similar incorporations of polyunsaturated fatty acids (c.a. 17 mol%) were attained with Novozym 435, Lipozyme RM IM and rROL. The lowest incorporation was achieved with Lipozyme TL IM (7.2 mol%). Modeling acidolysis catalyzed by rROL and optimization of reaction conditions were performed by response surface methodology, as a function of the molar ratio FFA/lard and the temperature. The highest acidolysis activity was achieved at 40 °C at a molar ratio of 3:1, decreasing with both temperature and molar ratio. Operational stability studies for rROL in seven consecutive 24-h batches were carried out. After the fourth batch, the biocatalyst retained about 55 % of the original activity (half-life of 112 h).     

Production and Characterization of DHA and GLA-Enriched Structured Lipid from Palm Olein for Infant Formula Use

Palm olein was modified via lipase-catalyzed acidolysis reaction to obtain fatty acid composition and positional distribution similar to human milk fat. In the reaction, a free fatty acid mix containing 23.23 % docosahexaenoic (DHA), 31.42 % gamma-linolenic (GLA), and 15.12 % palmitic acid was employed. The DHA and GLA were incorporated into the structured lipid (SL) product to improve its nutritional value. Response surface methodology (RSM) was used to investigate the effects of reaction time and substrate mole ratio (palm olein to a free fatty acid mix) on the amount of palmitic acid at the sn-2 position of SL triacyglycerols (TAG), and on the total DHA and GLA incorporation. Gram-scale production of SL was performed using the conditions predicted by RSM to maximize the content of palmitic acid at the sn-2 position. Verification of the predictions from RSM confirmed its practical utility. The resulting SL had 35.11 % palmitic acid at the sn-2 position, with 3.75 % DHA and 5.03 % GLA. Differential scanning calorimetry and HPLC analyses of the TAG revealed changes in their polymorphic profiles and TAG molecular species of SL compared to palm olein. The SL from this study can potentially be used in infant formula formulations.