Enzymatic Synthesis And Property Research Of Human Milk Fat Substitutes

Human milk fat(HMF)is the main nutrients of human milk.The infant formula with human milk fat substitutes(HMFS)is the best food to meet the growth and development demands of infants.The fat of infant formula in market is currently blended with milk fat and vegetable oil.Although these fatty acids composition is close to that of HMF,the position distribution of fatty acids is opposite to that of HMF,and infant formula is lack of some long chain polyunsaturated fatty acids such as docosahexaenoic acid(DHA),arachidonic acid(AA)and eicosapentaenoic acid(EPA),which is indispensable in infants' mental development.In our present study,HMFS was synthesized by a two-step enzymatic procedure,its fatty acids composition and position-distribution was close to HMF,and it was also rich in polyunsaturated fatty acids.The optimal synthesis conditions and suitable purification method of HMFS were explored.On the basis of analysis of physical and chemical characters,fatty acid composition and distribution and tocopherol content of HMFS,the effect of different antioxidants on oxidation stability of HMFS was studied by the storage test,which provided reliable reference for application of HMFS.The main results were as follows:(1)The triacylglycerols(TAGs),rich in palmitic acid(PA)at sn-2 position,were synthesized by Novozym 435 lipase-catalyzed acidolysis of camellia seed oil with PA in organic systems.The optimal alcoholysis conditions were determined using one-factorat-a-time experiments and response surface methodology as follows: n-hexane as reaction solvent at a dosage of 10 mL/g(on the basis of substrate mass),lipase dosage10.1%(on the basis of substrate mass),mass ratio of PA to camellia seed oil 1.1:1,reaction temperature 50?,and time 16.50 h.Under these conditions,TAGs with 66.08% PA at Sn-2 position were obtained.The optimal synthesis conditions of the TAGs rich in PA at sn-2 position in solvent-free system were as follows: mass ratio of PA to camellia seed oil 1.5:1,lipase dosage11.40%(on the basis of substrate mass),reaction temperature 56?,and time 21 h.Under these conditions,TAGs with 61.64% PA at Sn-2 position were obtained.The enzyme activity was 89.03% of the initial enzyme activity in solvent system,was 88.98% of the initial enzyme activity in solvent-free system after ten times of repeated use which showed that the stability of the lipase was good in both reaction systems.Although the reaction in solvent system consumed some reagent,the lipase dosage,reaction temperature and time were lower,and the content of PA at Sn-2 position was higher as compared with solvent-free system.(2)With purity and recovery rate of the product as indexes,the extractioncrystallization method for TAGs rich in PA at Sn-2 position separation and purification optimized.The suitable conditions for crystallization were as follows: crystal solvent–absolute alcohol dosage 3 m L/g,crystallization temperature 25?,mixing time 7 min;suitable conditions of recrystallization as follows: volume ratio of recrystallization solvent-absolute alcohol–water 3:1,recrystallization temperature 30?,mixing time 7 min,under this conditions,the purity of TAGs of the purified product reached 95.83%,recovery rate was 91.21%.(3)HMFS were synthesized with purified TAGs,rich in PA at Sn-2 position,and fish oil catalyzed by the Sn-1,3 specific lipase Lipozyme TL IM.The optimized reaction conditions were as follows: n-hexane dosage 4 m L/g(on the basis of substrate mass),lipase dosage 8%(on the basis of substrate mass),reaction temperature 50?,time 4 h.The separation and purification conditions of HMFS were crystal solvent–absolute alcohol dosage 3 m L/g,crystallization temperature 20?,mixing time 7 min,volume ratio of recrystallization solvent-absolute alcohol–water 3:1,recrystallization temperature 15 ?,mixing time 7 min,under this conditions,the purity of HMFS was 94.98%,recovery rate was 92.24%.The test results of physical and chemical properties of HMFS accord with GB 11765-2003 and SC/T 11765-2000.The content of total PA and PA at Sn-2 position was 27.88% and 55.97%,and relative content of PA at Sn-2 position reached 66.92%,which was,similar to HMF,significantly higher than that of infant formula.Besides,the HMFS contain high content of unsaturated fatty acids.It was determined that the content of monounsaturated fatty acid was 38.55%,the content of polyunsaturated fatty acids such as DHA and EPA was 7.38% and 14.50% respectively.Therefore,the nutritional value of HMFS was high.(4)The results of storage experiment showed that oxidation stability of HMFS prepared by a two-step enzymatic procedure was poorer,which could be improved to varying degrees by adding fat-soluble antioxidants such as TBHQ(tertiary-butyl hydroquinone),VE,L–ascorbyl palmitate(L-AP),?-carotene.Among of these,composite antioxidants(0.01% VE + L-AP + 0.01% citric acid)showed a better ability in slowing down the increas speed of peroxide value(PV),P-anisidine value(P-AV)and total oxidation value(TOTOX),and enhanced the shelf life.