Enzymatic Synthesis,Safety Evaluation,and Anti-obesity Effects Of Medium- And Long-chain Triacylglycerols

Obesity is associated with an increased risk of metabolic syndrome and chronic diseases including cardiovascular diseases,type 2 diabetes,hypertension,and other diseases.In general,obesity is closely related to an excess dietary fat intake.Common foods and edible oils are the major sources of oils and fats,each contributing 50%.Edible oils are import for food taste and flavor besides providing basic nutrition.This makes it very difficult to reduce fat intake.In this respect,many studies focus on the modification of the oil structures to develop oil and fat substitutes with lower energy that are safe and less accumulated in vivo.At the same time,the structured lipids should have better safety and nutrition characteristics in addition to their application potentials compared to traditional oils.Among them,medium-and long-chain triacylglycerols(MLCT)have attracted more attention due to their low energy content and little effect on food flavor.Thus,MLCT have been recognized as a perfered oil substitute with good safety performance,physical properties,and cooking performance.In this thesis,novel MLCT with different contents of medium-chain fatty acids(MCFA)were synthesized through enzymatic interesterification,and their safety,obesity prevention potential,effect on lipid metabolism,and gut microbiota composition in mice and rats were systematically evaluated.The results provide further insights into the development of structured lipids.At first,MLCT were prepared via enzymatic interesterification of medium-chain triacylglycerols(MCT)and rapeseed oil(RSO)with Lipozyme TL IM in a packed bed reactor.Single-factor and orthogonal experiments were employed to optimize the substrate ratio,reaction temperature and time,and enzyme loading.As a result,the prefered condition was determined as: the ratio(w/w)of MCT and RSO was 3:2,the reaction temperature was 70 oC,the reaction time was 1 h,and enzyme loading was 12%(w/w).A crude oil containing 72%(w/w)MLCT could be obtained under the reaction condition.After molecular distillation,bleaching,and deodorization,the percentage of MLCT could be improved to 91.5%(w/w),which consisted of 30%(w/w)MCFA.The content of MCFA at the sn-2 position was 48.17%.Furthermore,the acid value,peroxide value,soap value,iodine value,color,smoking point,and freezing time of the obtained MLCT were all in the range of Chinese standards for refined oils.In addition,the obtained MLCT showed good stability under different storage and utilization conditions,indicating its potential as a cooking oil.Second,MLCT with 30%(w/w)MCFA was evaluated for its safety as a dietary fat in mice and rats.The acute oral toxicity study showed that the maximum tolerated dose exceeded 54.33 g/kg body weight(kg bw)/day.Genetic toxicity tests including Ames' test,mouse bone marrow polychromatic erythrocyte micronucleus test,and mouse spermatid malformation test were all negative.In the 90-day feeding study,no dose-related adverse effects were observed in rats.Further safety assessment in pregnant rats did not show any maternal toxicity,embryotoxicity,and teratogenicity.Based on the aforementioned results,three medium-and long-chain triacylglycerols(MLCT)with different contents of medium-chain fatty acids(MCFA)(10%-30%,w/w)were prepared and evaluated for their potential in reducing the body weight gain in C57BL/6J mice.The MLCT with 30%(w/w)MCFA showed the best performance on suppressing fat accumulation of rats.Compared to a high fat diet containing rapeseed oil,the body weight and body fat decreased by 11.0% and 38.4%,respectively.Furthermore,the serum parameters including glucose,total triacylglycerol,total cholesterol,low-density lipoprotein cholesterol,and apolipoprotein B decreased by 46.4%,38.8%,46.0%,and 23.9%.High-density lipoprotein cholesterol increased by 50.1%.Moreover,intake of MLCT with 30%(w/w)MCFA can further reduce the liver weight and liver triacylglycerol content as well as the size of adipocytes.These results suggested the outstanding ability of MLCT with high content of MCFA in reducing high risk of obesity.At last,the effects of MLCT with different contents of MCFA on lipid metabolism and gut microbiota composition were further investigated.MLCT with 30%(w/w)MCFA showed the best performance in decreasing body weight gain as well as optimizing serum parameters and liver triacylglycerol content.The ?-oxidation of triacylglycerols in liver can be enhanced through up-regulating the gene expressions of PPAR? and LCAD,and de novo fatty acid biosynthesis was effectively suppressed by down-regulating the gene expression of FAS,ACC-1,SREBP-1c,and ME.Notably,the dietary intake of MLCT can further improve the ratio of Firmicutes to Bacteroidetes and down-regulate the relative abundance of Proteobacteria.In addition,the intake of MLCT can effectively down-regulate the abundance of Helicobacter,and increase total short-chain fatty acids concentrations.Thus,it is rational to speculate that the regulation of gene expression,the variation of microbiota,and the special metabolic pathway of MCFA in vivo may be synergistically responsible for the anti-obesity of MLCT with relatively high contents of MCFA.