Study On Enzymatic Synthesis And Characterization Of Low-Calorie Structured Lipids

As one of the most important part of food, oil and fat both bring good sensory properties of food and provide calories and essential fatty acids. With the development of economy, high fat dietary pattern with excessive fat has leaded to several obesity-related disorders such ashyperlipidemia, coronary heart disease, atherosclerosis and other cardiovascular disease. The incidence rate of these diseases with gradually rise, has become a major public health security. Low-calorie structured lipids (LCSLs) refers to the structured lipids with calorie only 40?90% of ordinary oil, mainly including medium-and long-chain triglycerides, short- and long-chain triglycerides, diglyceride, etc. The LCSLs could reduce the storage of fat in the body by changing the oil metabolism path. The commercialization of the short-and long-chain triglycerides were made up of short chain fatty acids and saturated long chain fatty acid, while reducing the oil heat, but the lack of essential fatty acids. Therefore, systematic study of LCSLs which contain unsaturated long-chain fatty acids is valuable in terms of the theoretical contribution and potential economic benefits.The purpose of this study is to obtain the novel LCSLs products which are beneficial to health. With soybean oil and short chain fatty acid acyl as raw material, we not only focus on lipase-catalyzed synthesis and quantitative analysis of LCSLs by gas chromatography, but also pay attention to purification of LCSLs by molecular distillation technology. Physi-chemical properties and oxidation stability of LCSLs was analyzed in order to evaluate their function and safety. The main research contents and results are as follows:1. The chemical compositions of LCSLs were analyzed by gas chromatography-mass spectrometry (GC-MS) through mass spectral library searching and characteristic ions. A gas chromatography with flame ionization detection (GC-FID) method was developed for determining the content of LCSLs in edible oil samples using DB-17 ht fused silica capillary column as the analytical column. The results showed a good linear correlation between peak area and concentration in the range of 0.5-20.0 mg/mL. The relative standard deviations for retention time and peak area of each LCSLs were less than 0.05% and 2.0%, respectively, suggesting good reproducibility of the developed method. The average recoveries from spiked samples of transesterified and original soybean oil were 98.62% and 97.59%, respectively. This method allowed accurate and highly repeatable quantification of LCSLs in edible oils.2. LCSLs with high content of SSL-TAG were synthesized by transerification of butyric anhydride (BA) and glycerol monostearate (GMS) in solvent-free system, catalyzed by Lypozyme TL IM. Based on single factor tests, the optimal synthesis process was obtained by and response surface optimization tests. Under the optimal conditions of substrate molar ratio (BA/GMS) 2.4:1, Lypozyme TL IM dosage 3.23% (mass ratio to substrates),4A molecular sieve added ratio (mass ratio to GMS) 0.5, the reaction temperature of 55?, the rotary speed of 170 r/min, the reaction time of 12 h, the content of the SSL-TAG was 92.75% and SL-DAG was 2.11% in transerification reaction. LCSLs product was obtained by molecular distillation and its combustion heat value was only 75% of the soybean oil. In LCSLs product, the content of SSL-TAG was 96.11%. The content of butyric acid was 62.37 mol%.3. LCSLs containing unsaturated fatty acid were synthesized by interesterification of soybean oil and glyceryl triacetate in solvent-free system, catalyzed by Lypozyme RM IM. The optimal reaction condition was achieved by single factor tests and orthogonal tests. Under the conditions of lipase dosage 10%, water content 1%, substrate molar ratio 2.5:1, the reaction temperature 55?, rotary speed 160 r/min, the reaction time 12 h, the content of SSL-TAG was 53.83% and SLL-TAG was 16.34% in interesterification reaction.4. LCSLs were synthesized by lipase-catalyzed interesterification reaction and purified by secondary molecular distillation. The effect of molecular distillation parameters on acid value and purity of low-calorie structured lipids were investigated. The process parameters of secondary molecular distillation were optimized by the combined use of single factor method and response surface methodology. The results showed the degree of fitting of the regression model was good. The optimum secondary molecular distillation process was achieved at 122? with a scraper speed of 240 r/min and a feeding rate of 2.0 mL/min. The acid value of LCSLs was reduced to 0.95 mg KOH/g with a purity of 60.11%.5. LCSLs products containing high content of SSL-TAG were prepared by secondary molecular distillation technology combined with alkali refining. Physical and chemical properties and oxidation stability of LCSLs products was analyzed. The results showed that the clear, transparent, smell, taste of LCSLs products was same as soybean oil. But saponification value, coagulation temperature and melting temperature of LCSLs products were higher than soybean oil. Tiodine value, smoke point and calorific value were lower than that of soybean oil. Main component of LCSLs products were SSL-TAG, in which the content of AAL-TAG was 71.67% and 2.75 times of ALA-TAG content. LCSLs products were given priority to with 62.67 mol% acetic acid and the long chain fatty acids composition was same as soybean oil. But the saturated long chain fatty acids mainly distributed on sn-1 and long-chain unsaturated fatty acids evenly distributed on the glycerin skeleton. Temperature, illumination and O2 had remarkable effect on the oxidation stability of LCSLs and polyphenols antioxidants were conducive to improve the oxidation stability of LCSLs.6. Acute toxicity test in mice and SD rats of acute gastric test of LCSLs containing high content of SSL-TAG were carried out to preliminary evaluate the safety. The results showed that the LCSLs half lethal dose of KM mice were greater than 21.5 g/kg-bw, belonged to the acute toxic dose of grading standard non-toxic level category. Through continuous high dose LCSLs lavage SD rats for 14 d, death and abnormal behavior had not been found. There had not been significantly effect on the organs. The biochemical indexes of serum were within the normal range. Therefore, these results can be totally concluded that LCSLs products were safe.7. The physiological function was evaluated through the SD rats lavage for 28 d with different doses of LCSLs. Compared with the basis of control group, intakes of LCSLs had no significant effect on the heart, liver, kidney and spleen index, but high doses (5.0 g/kg-bw) intakes of LCSLs decreased food intake, weight increment, body fat ratio, and increased waste lipid. While moderate doses (2.5 g/kg-bw) and low dose (1.0 g/kg·bw) intakes of LCSLs had no significant effect on weight increment, body fat, triglyceride and total cholesterol levels of serum. Compared with high-fat model group, TC and TG concentrations of serum were significantly lowered by intakes of LCSLs. Intakes of LCSLs did not affect the fatty acid composition of the liver, but affected the content of main fatty acid.The results showed the intakes LCSLs could not only control weight gain, but also regulate plasma lipid metabolism.