The Formation Of High-melting-temperature Solid Fat In Milk And Its Digestion,Absorption And Metabolism Mechanism

Cooling and storage of milk fat result in the crystallization of triacylglycerols（TAG）in the fat globules,and the crystalline TAG is mainly composed of long-chain saturated fatty acids,which tend to form a stableβcrystal form at low temperatures.The melting temperature of the solid TAG fraction（>37℃）is higher than the human body temperature.The dissolution,emulsification,lipolysis,digestion and absorption of high-melting-temperature solid TAG in milk fat are scientific issues to be explored.In this thesis,the formation and formation mechanism of high-melting-temperature solid TAG,in vitro and in vivo digestion and absorption characteristics,as well as the metabolism mechanism of high-melting-temperature solid TAG and its postprandial effects were studied.Firstly,the morphology of native milk fat globules and the thermal properties and polymorphism of TAG in the fat globules were analyzed.The effects of different processing techniques（cooling rate,homogenization pressure and storage time）on the crystallization and melting properties of TAG were studied.The average particle size of native milk fat globules was 3.11μm in diameter,and the initial crystallization temperature(T_onset)and final melting temperature(T_endset)of TAG in the fat globules were 19.0℃and 37.4℃,respectively.Fast cooling（5℃/min）forced the TAG molecules to arrange into a fine metastableαcrystal form,while slow cooling（1℃/min）was conducive for the formation of thermodynamically stableβ-type spherulites in the fat globules,with a higher crystallization T_onset（20.8℃）.High-pressure homogenization destroyed milk fat globule membrane and significantly reduced the size of the fat globules,and inhibited the formation of high-melting-temperature solid fat.When the homogenization pressure was 20 MPa,30 MPa and 40 MPa,the melting T_endset of the TAG in the fat globules was 37.7℃,36.7℃and 36.4℃,respectively.During storage at 4℃for 72 h,The TAG in the fat globules underwent a polymorphic transformation ofα→β’→β,and the percentage ofβ-form increased from 45.2%to 77.0%,accompanied by an increase in melting T_endset from 37.8℃to 41.6℃.Temperature fluctuations during processing and refrigeration induced crystallization and recrystallization of milk fat.TAG molecules in the fat globules underwent polymorphic transformation and structural rearrangement to form a thermodynamically stable high-melting-temperature solid fat.Six solid/liquid fractions of 30S/30L,20S/20L and 15S/15L were obtained from the repeatedly freeze-melted milk fat at three temperature nodes of 30℃,20℃and 15℃by a multi-step dry fractionation.Among them,30S and 20S were the high-melting-temperature fractions.The melting T_endset and the content of long-chain saturated TAG were 42.1℃/38.9℃and 19.3%/3.2%,respectively;20L and 15L were the low-melting-temperature fractions.The corresponding melting T_endse and long-chain saturated TAG content were 22.0℃/17.1℃and1.8%/0.1%,respectively.Reconstitution of milk fat globules was carried out using whole milk fat（WF）,20L,20S and 30S as the TAG core.The volume-surface average diameter D^[3,2]（0.5μm）and the specific surface area S（12.3 m²/g）of four reconstituted milk fat globules were similar to those of the native homogenized milk.A three-stage semi-dynamic in vitro digestion model was used to evaluate the digestion characteristics of WF-,20L-,20S-,and 30S-reconstituted milk fat globules.TAG,as well as diacylglycerol（DAG）,monoacylglycerol（MAG）and free fatty acid（FFA）released by hydrolysis during digestion were monitored using HPLC-ESI-Q-TOF-MS/MS and GC-FID.In the mouth,the reconstituted milk fat globules were slightly flocculated due to the bridging by amylase.In the stomach,a large amount of hydrolysis（57.3%）occurred in 20L,accompanied by an apparent hydrolysis rate constant k of 6.1×10^-3 min^-1 and a large number of liquid-ordered domains appearing at the interface of fat globules;while the high-melting-temperature 30S and 20S tended to aggregate and settle,accompanied by a hydrolysis degree（29.8%and 28.0%）and hydrolysis rate(2.4×10^-3 min^-1and2.1×10^-3 min^-1)being 1/3～1/2 of 20L.In the small intestine,20L was almost completely hydrolyzed,and the aggregates formed by 30S and 20S in the stomach were also deeply dissociated under the emulsification and hydrolysis of bile salts and pancreatic lipase,but there was a small amount of residual lipids（29.9μmol/g and 19.9μmol/g）.These undigested lipids existed in the form of mixed micelles or vesicles,however,there was still 5.8%TAG residue in 30S even after 120 min of digestion.The differences of in vivo gastric emptying,lipolysis,absorption,satiety signal release and postprandial lipemia response were studied in adult healthy rats fed with WF-,30L-and 30S-reconstituted milk fat globules.In the stomach,30L and 30S presented a soft watery curd and a solid clot,respectively.The digestibility of TAG in 30S（39.2%）was significantly less than that of 30L（60.1%）,accompanied by lower DAG release（208.2 and 315.5μmol/g,respectively）.In the small intestine,DAG was further hydrolyzed into sn-2 MAG and FFA which were absorbed by small intestinal epithelial cells,and the absorption rate of 30S hydrolysate（68.2%）was lower than that of 30L（75.7%）.In addition,the postprandial lipemia response（total triglycerides,TG;total cholesterol,TC）caused by 30S administration was weaker than that of 30L,but the release level of satiety signals（cholecystokinin,CCK;glucagon-like peptide1,GLP-1;peptide YY,PYY）in the 30S group was 5.9%～11.4%higher than that of the 30L group.The slower hydrolysis rate of 30S in the gastrointestinal tract of rats produced a stable postprandial lipemia response and a strong sense of satiety.The effects of high-melting-temperature solid fat on the growth of SD rats,physiological and biochemical indicators,tissue morphology and the expression of lipid-metabolism-associated enzymes were studied using WF,30L and 30S as the only lipid source.The daily food intake of rats in the 30S group（18.70 g/d）was less than that in the 30L group（19.65 g/d）,and corresponded to a lighter body weight（370.18 g vs.390.23 g）.The difference in organ indexes and serum biochemical indexes was not significant between the two groups,but the level of apolipoprotein B（Apo B）in the liver of the 30S group was higher,and the size of perirenal adipocytes（7.73 mm²）was significantly smaller than that in the 30L group（10.62 mm²）.The amount of total lipids lost in the feces of the 30S group was 29.09%higher than that of the 30L group,which mainly composed of calcium soap（41.21μmol/g）and esterified fatty acids（67.62μmol/g）.Compared to 30L,30S increased the expression of lipolysis-associated enzymes（adipose triglyceride lipase,ATGL;hormone-sensitive lipase,HSL;protein kinase A,PKA;cyclic adenosine monophosphate,cAMP）in the liver.At the same time,it reduced the expression level of lipid-synthesis-associated enzymes（acetyl-Co A carboxylase,ACC）in adipose tissue.The high-melting-temperature solid fat,30S,had a low bioavailability,effectively reducing body fat accumulation and improving lipid metabolism in rats.