Establishment Of An Evaluation Model For Human Milk Fat Substitutes And Their Enzymatic Preparation

The physical existing state of human milk fat and its chemical composition were analyzed in thisstudy. On the basis of chemical composition, an evaluation model was established and then thedegrees of similarity of different mammalian milk fat were assessed by this model. Meanwhile,different types of palm stearin were selected as raw materials for preparation of HMFS by differentmethods, and their properties were compared with the reported HMFS and the fat from thecommercial infant formulas.The study of the physical properties of human milk fat globule (HMFG) revealed that thedescending order of average diameters of HMFG from different lactation stages was colostrum>mature milk> transitional milk; the zeta potential of HMFG increased with the progress of lactationand the content of human milk fat showed the similar tendency. The observation of microstructureof human milk fat globule membrane (HMFGM) labeled by Rh-DOPE fluorescent probe underconfocal laser scanning microscopy (CLSM) at storage (4°C), room (20°C) and physiologicaltemperature (37°C) indicated that a phospholipid rearrangement occurred on the HMFGM duringthe variation of temperature. However, the physiological significance of the microstructure changesstill remained unknown.The investigation of chemical composition of colostrum, transitional and mature milk fat byvarious analytic techniques demonstrated that the contents of chemical constituents in human milkfat at different stages of lactation were different, but had some regularity. The SFA contents of milkfat from different lactation stages were not significantly different. However, in terms of palmiticacid, the content in colostrum fat was significantly higher than that in mature milk fat. Meanwhile,the content of PUFA in colostrum fat was significantly higher than those in transitional and maturemilk fat, whereas it contained significantly less content of SFA at sn-2position. The major fattyacids in human milk fat with contents more than1%were C10:0, C12:0, C14:0, C16:0, C16:1,C18:0, C18:1, C18:2, the sn-2fatty acids with contents more than1%were C12:0, C14:0, C16:0,C16:1, C18:0, C18:1, C18:2and the PUFA with contents more than0.1%were C18:3, C20:2,C20:3, C20:4, C20:5, C22:2, C22:4, C22:5, C22:5, C22:6. The TAG species in human milk fatdidn’t change, but their contents were different. The TAG with contents more than1%were OLaLa,MMLa, LLaO, MOLa, PMLa, LaOO, POLa, MPL, SMM, POL, PPL, PPM, OOO, POO, PPO, POS.Based on the chemical composition of human milk fat, an evaluation model for assessment of thedegrees of similarity of human milk fat substitutes (HMFS) from the aspects of fatty acidcomposition and distribution, PUFA and TAG composition was established and the validity of thesystem was verified by the selected oils and fats with representative chemical composition. Theverification results showed that the results of the evaluation system could reflect the compositiondifference between HMFS and human milk fat, which indicated the validity of the model. Thedegrees of the similarity of milk fats from cow, buffalo, sheep, donkey and camel obtained from themarket were then evaluated by the newly established model and the results showed that the milkfats had low degrees of similarity to human milk fat, which were not suitable to be used aslong-term fat substitutes for infant. Based on the chemical composition of human milk fat, three types of palm stearin were selectedas the starting materials, the sn-1,3-specific lipase, Lipozyme RM IM, was used as the catalyst anddifferent methods in accordance with the characteristics of palm stearin were developed to prepareHMFS.(1) Two-step method including enzymatic acidolysis and physical blending was developedfor preparation of HMFS by use of high melting point palm stearin prepared by fractionation (PS1).Enzymatic acidolysis was used to decrease the content of total PA and increase the relativepercentage of sn-2PA among total PA (%sn-2PA). After optimization, the optimized acidolysisconditions were obtained as substrate molar ratio10:1, temperature60°C, enzyme load8wt%,reaction time4h, water content3.5wt%and the total PA, sn-2PA and%sn-2PA of the enzymaticproduct under these conditions were39.6%,83.7%and70.5%, respectively. At the physicalblending stage, to guarantee the quality of the final product and the maximum yield, a model wasestablished to calculate the fatty acid profiles of the blended oils and the Matlab R2010a was usedto optimize the entire blending process. Based on the model prediction, a desirable formulaconstituted enzymatic product/rapeseed oil/sunflower oil/palm kernel oil/algal oil/microbial oil at amole ratio of1:0.28:0.40:0.36:0.015:0.017was obtained; and the final product had PA, sn-2PAcontent and%sn-2PA at23.5%,43.1%and61.1%and the contents of AA and DHA at0.4and0.3%, respectively, with the contents of other fatty acids within the ranges of corresponding fattyacids in human milk fat.(2) In the process of preparation of HMFS by use of palm stearin withmelting point of58°C (PS2), batch reactor was used and the fatty acid composition and distributionwere primarily adjusted by acidolysis reaction. Through factorial experiments, the major factorswere determined as reaction time, temperature, substrate molar ratio and enzyme load, and themajor indices for optimization experiments were total PA and sn-2PA. The acidolysis reactionswere optimized by response surface methodology (RSM) and the optimum conditions weredetermined as reaction time,3.4h; temperature,57°C; substrate molar ratio,14.6mol/mol; enzymeload,10.7wt%. The PUFA contents of the enzymatic product were adjusted by the addition of AOand MO, whose amounts were calculated by the physical blending model and the ratio wasdetermined as enzymatic product:AO:MO=1:0.008:0.009. The contents of PA, sn-2PA and%sn-2PA in the final product were29.2%,61.3%and67.0%and the contents of AA and DHA were0.5%and0.3%, respectively.(3) In the process of preparation of HMFS by use of palm stearin withmelting point of52°C (PS3), the continuous packed bed reactor was used and acidolysis reactionwas primary used to adjust the fatty acid composition and distribution of the palm stearin. Athree-factor, rotatable five-level central composite design (CCD) combined with RSM was used tooptimize the acidolysis reactions. The optimum conditions were determined as residence time,2.7h;reaction temperature,58°C; substrate molar ratio,9.5mol/mol. The amounts of AO and MO werecalculated by the blending model and the ratio was determined as the enzymaticproduct:AO:MO=1:0.008:0.009. The contents of PA, sn-2PA and%sn-2PA in the final productwere28.3%,52.1%and61.4%and the contents of AA and DHA were0.5%and0.3%, respectively.The degrees of similarity of three types of HMFS from palm stearin (PS1_HMFS, PS2_HMFS,PS3_HMFS), the reported HMFS from lard (lard_HMFS), butterfat (butterfat_HMFS) andtripalmintin (tripalmintin_HMFS) and the fats from six types of infant formulas were assessed bythe evaluation model, the oxidative stability of the synthesized HMFS was analyzed by acceleratedoxidation experiments and then the secondary volatile oxidation products in the HMFS after accelerated oxidation were qualitatively and quantitatively analyzed. The evaluation resultsdemonstrated that HMFS from palm stearin had high degrees of similarity in fatty acid compositionand distribution and TAG composition. However, due to lack of some types of PUFA, theirsimilarity in PUFA composition was relatively low. Lard_HMFS had high degree of similarity infatty acid profile to human milk fat, especially the%sn-2PA, but it had low degrees of similarity inPUFA and TAG composition. Butterfat_and tripalmitin_HMFS had high degrees of similarity infatty acid composition, but their degrees of similarity in PUFA and TAG composition were low.The fats from infant formulas only had similarity in fatty acid composition yet not in other aspects,which indicated they didn’t meet the requirements of HMFS. The results from accelerated oxidationexperiments showed that the descending order of the oxidative stability of HMFS wasPS1_HMFS> tripalmitin_HMFS> PS2_HMFS> PS3_HMFS> butterfat_HMFS> lard_HMFS.Different amounts of antioxidant (TBHQ) were added to HMFS from palm stearin and the resultsshowed that higher contents of antioxidant in the HMFS led to lower PV, p-AV and TOTOX.Finally, the secondary volatile oxidation products in the HMFS were analyzed byHS-SPME-GC-MS and the results revealed that the content of hexanal was the highest amongvarious volatile oxidation products and in various types of oxidation products, aldehydes had thehighest content. Meanwhile, the contents of most of secondary volatile oxidation products werereversely correlated to the contents of antioxidant in the HMFS. Therefore, the addition of a certainamount of antioxidant to HMFS has an important role to enhance their oxidative stability.