Enzymatic Synthesis, Purification And Characterization Of Anovel Structured Lipid

Obesity has become a global problem, threatening to human health. The incidence ofobesity is closely related to the dietary pattern, particularly to the excessive intake ofhigh-calorie fatty foods. Fat is an essential part of diet foods, and it contributes to the taste,flavor and satiety of the food. Nowadays, the fat intake by people is more than30%of thetotal calories from food. Under such circumstance, varieties of low-calorie fats and fatsubstitutes, such as sucrose fatty acid polyesters, medium-chain triglycerides, short-andlong-chain triglycerides and diglycerides, have been designed and developed. The relativelysuccessful products are Caprenin product (P&G), Salatrim product (Nabiso) and diglycerides(Kao, Japan). They can control the accumulation of fat in the body either by providing lowercalorie or altering the metabolism of fats. As an ideal fat substitute, it should have a highdegree of universality with traditional oils and fats, that means it should not change a lot inthe usage method, usage conditions and sensory characteristic. Moreover, the fat substitutesshould have good in nutritional properties and high safety. However, most of the fat substituteproducts have their inherent shortcomings which make them only partially meet the abovecriteria. From the point of view to better improve human health, the market requirement fornew low-calorie fat substitutes will continue growing. Therefore, it has important researchmerit and potential market application to develop low-calorie fat substitutes with highsimilarity to the ordinary oils and fats.We designed a novel structured lipid, which contained a short chain fatty acid at sn-2position. The novel structured lipid can reduce postprandial lipid levels by the differentmetabolism of triglyceride molecules and thus reduce the accumulation of fat in the body.When an edible oil is took into digestive tract, the majority of ingested triacylglycerols (TAGs)are partially hydrolyzed by pancreatic lipase to2-monoacylglycerols (2-MAGs) containinglong-chain fatty acids and fatty acids (FAs), the2-MAGs are immediately resynthesized intoTAGs molecules after absorbed by the small intestinal epithelial cells. The resynthesizedTAGs will be transferred throughout the body by the lymphatic system and thoracic duct. Inthe case of the novel structured lipids, they are partially hydrolyzed by pancreatic lipase to2-MAGs containing short-chain fatty acids (SFAs) and FAs. The SFAs are rapidly absorbedand metabolized to carbon dioxide in liver. Thereby, the resynthesized TAGs by lipases insmall intestinal epithelial cells could be reduced, resulting in reducing postprandial lipidlevels and preventing fat accumulation in body.Firstly, the novel structured lipids were synthesized by transesterification of tributyrin and soybean oil fatty acid methyl esters using Lipozyme RM IM lipase. Central compositerotatable design (CCRD) was used to optimize the reaction variables, i.e, reaction time,reaction temperature and lipase dosage. The incorporation of long chain fatty acids, acylmigration level and content of long-chain triglycerides (LLL-TAG) were set as evaluationcriteria. The optimal reaction conditions were as follows: reaction temperature is51.4℃,reaction time is6.77h and lipase dosage is8.11wt%, and the resulting long chain fatty acidsincorporation rate is43.2%, acyl migration level is5.33%and LLL-TAG content is0.42%under optimized conditions.Secondly, the novel structured lipids were synthesized by two-step method using twoenzymes systems. It was found that a higher content of2-monobutyrin was obtained by usinglower temperature and higher molar ratio of ethanol to tributyrin. The optimal reactionconditions were as follows:65:1of of ethanol to tributyrin,25℃of reaction temperature,5wt%of enzyme amount and2h of reaction time. Under these conditions, the content of2-monobutyrin was21.6%and the yield was62.8%. The incorporated long chain fatty acidscontent reached to52%in the final product, and the acyl migration level was only1.1%.However, the method is complex and large amount of organic solvents is required to purify2-MAGs. Moreover, it’s difficult for scale-up production. Thus, one-step interesterificationmethod was selected to synthesize the novel structured lipids for the following work in thisthesis.Thirdly, a scale-up synthesis of novel structured lipids by transesterification of tributyrinand soybean oil fatty acid methyl esters using Lipozyme RM IM lipase was carried out.Molecular distillation was employed to purify the structured lipids and the effect ofevaporation temperature on the purification was examined. Results showed that betterseparation of transesterification product was achieved under the following conditions:2g/minof the feed rate,1Pa of the distillation system pressure,40℃of the cooling watertemperature at light phase,250r/min of the scraping membrane motor speed and200℃ofthe evaporation temperature. Furthermore, circulating method of scale-up synthesis of novelstructured lipid was studied. The results showed that the total yield was about64%, thecontent of LSL-TAG was80.9%, and most of the butyric acids were distributed in Sn-2position of the TAGs. The physicochemical properties of the novel structured lipids wereevaluated, and the results indicated that the appearance, acid value, saponification value,refractive index and other indicators were very similar to soybean oil which was the acyldonor of long-chain fatty acids. However, the structured lipid had a lower smoking point due to a smaller molecular presented in the product. The results of physicochemical propertiessuggested that the ordinary diet oil could be completely replaced by the structured lipid.TBHQ can effectively improve oxidation stability of the structured lipid.Finally, the lipid lowering property of novel structured lipids were evaluated by rapidfasting experiment and animal growth experiment. It was found that the novel structured lipidcould reduce the postprandial plasma triglyceride levels of mice. After feeding for six weeks,the group received high fat diet containing high dosage of structured lipids had lowerbodyweight, comparing with control group of high fat diet containing high dosage of lard.The plasma cholesterol (TC) content in the group received high fat diet containing mediumand high dosage of structured lipid showed a decreasing trend comparing with the controlgroup. For the tested group, the histological slices of liver and kidney were observed undermicroscope, the number and size of fat droplets in liver were found to be reduced, and theresults of kidney histological slices showed no abnormal symptom. In summary, our resultsshowed that the novel low-calorie structured lipid could control the plasma lipid and preventthe accumulation of body fat.