对称和非对称棕榈酰油酰结构酯合成及营养学初步评价
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摘要
结构甘三酯(Triacylglycerols, TAGs)一般指天然中不存在的,需要经过化学或者酶法使其脂肪酸在甘油的位置上具有预定的组成和分布的、具有特定物理化学特性或者营养特性的结构酯。功能性结构酯可以降低TAG水平和提高免疫力,含多不饱和脂肪酸(Polyunsaturated fattyacids, PUFAs)的结构酯还具有改善心脏功能、血管内皮功能和抗炎等特性。此外,非对称和对称性TAGs的合成对TAG结构和氧化稳定性关系的研究以及TAG结构和功能的研究十分重要。狭义的结构TAG包括单酸型、二酸型对称结构酯和二酸型非对称结构酯。本研究利用油酸和棕榈酸建立了合成对称和非对称TAGs的一般性方法,并利用花生四烯酸(Arachidonic acid, AA)验证了这些方法在合成富含PUFAs结构酯的可行性,最后,对这些合成的TAGs进行了营养学的初步研究。
     首先,以棕榈酸和油酸为原料,酶法合成单酸型对称结构酯(PPP和OOO)。PPP合成的优化条件为:在无溶剂反应体系中,棕榈酸和甘油的摩尔比为3.3:1(6.6mmol和2mmol),酶添加量为10%,在60°C下,反应12h,产品经纯化由1H NMR检测表明为纯的PPP,纯化得率为89.6%。随后,以上述棕榈酸为代表优化的单酸TAGs合成工艺进行三油酸甘油酯(OOO)的合成,进一步验证该方法的可靠性。纯化商业化的油酸得到高纯度的油酸产品。油酸的纯化采用二步分提法,第一步分提去除亚油酸,第二步分提去除硬脂酸。通过对第一步的分提方法、甲醇添加量、分提时间和第二步分提的溶剂量、分提时间的单因素试验。得到优化的分提条件:第一步分提为湿法分提,分提时间为8h,料液比为1:4。第二步也为湿法分提,分提时间为2h,料液比为1:5。在优化分提条件下得到的油酸的纯度为97.4%,总得率为65.3%。纯化的油酸在上述条件下合成OOO,产品纯化得率为84.6%,OOO经纯化后,TLC分析显示只有TAG。
     其次,采用化学酶法合成二酸型对称结构酯(POP, OPO),POP和OPO的合成采用了1,3-DAGs和相应的脂肪酸的化学酯化来合成的。单因素试验结果显示:1,3-二棕榈酸甘油酯合成的优化条件为:以1mL的二氯甲烷为溶剂溶解底物摩尔比为2:1(4mmol和2mmol)的棕榈酸乙烯酯和甘油,以10%Lipozyme RM IM为催化剂,在室温下反应6h,反应粗产物中1,3-二棕榈酸甘油酯的含量为92.1%。纯化后经TLC、GC和1H NMR检测显示1,3-二棕榈酸甘油酯纯度>98%。由于没有商业化的油酸乙烯酯,先在化学催化乙酸乙烯酯和油酸反应合成油酸乙烯酯。反应后粗产物中含有67.6%的油酸乙烯酯,纯化得到纯的油酸乙烯酯,得率为55.7%。随后使用油酸乙烯酯合成1,3-二油酸甘油酯,单因素试验结果显示:油酸乙烯酯和甘油以摩尔比为2:1(4mmol和2mmol)混合,在无溶剂体系中,以8%Lipzozyme RM IM为催化剂,在30°C条件下反应8h。粗产物混合物中含有87.6%的1,3-二油酸甘油酯。纯化得到>97%的1,3-二油酸甘油酯,得率为74.7%。随后,1mmol的DAGs和1.1mmol游离脂肪酸在常温下化学酯化,纯化得到对称性TAGs POP和OPO,得率分别为88.4%和86.5%。为了验证化学方法对合成含有PUFAs TAGs的可行性,使用相同反应条件合成OAO。得率为82.8%。
     再次,用棕榈酸和纯化的油酸为原料,酶法两步法合成非对称性结构酯(OPP和POO)。即先用酶法或者化学酶法合成相应的油酸或者棕榈酸单甘酯(Monoacylglycerols,MAGs),MAGs进而在脂肪酶的催化下和相应的脂肪酸反应,合成目标TAGs。利用棕榈酸乙烯酯与甘油不可逆反应合成棕榈酸MAG的优化条件:以1mL的甲基叔丁基醚为反应溶剂,以10%的脂肪酶添加量为催化剂,反应6h。反应后,粗产物经过正己烷结晶后棕榈酸MAG的纯度为97-98%,得率为89.5%。利用丙酮缩甘油与油酸可逆反应合成油酸MAG的优化条件:丙酮缩甘油和油酸(1mmol:1.2mmol摩尔比)用0.5mL二氯甲烷溶解后,以8%Novozym435为催化剂,在60°C下反应8h。粗产物中油酸丙酮缩甘油酯的含量为94.6%。油酸丙酮缩甘油进行断裂反应合成油酸MAG。得到的粗断裂产物中含有76.5%油酸MAG,纯化得到纯的油酸MAG,得率为72.8%。最后棕榈酸MAG,油酸MAG分别和油酸,棕榈酸反应合成POO和OPP,两种终产物的sn-2油酸、棕榈酸含量分别为78.2%、82.5%。为了验证酶法方法对合成含有PUFAs TAGs的可行性。利用油酸MAG和AA反应,合成OAA。首先从富含AA的油脂中富集AA,AA的富集采用三步法。先水解富含AA油制备游离脂肪酸(Free fatty acids,FFAs),经尿素络合使AA含量从40.2%提高到78.4%,此步骤的FFAs得率为27.6%。经尿素络合后的AA浓缩物再经AgNO3溶液分提。优化的分提条件是FFAs浓度0.1g/mL,添加量0.1g,甲醇的体积分数30%,分提温度-5°C,分提时间1.5h。在此条件下分提得到纯度90.7%的AA,得率为80.4%。采用与OPP和POO相同合成条件来合成OAA,纯化后TLC显示为单一的TAGs,得率为54.9%。
     最后,以合成的结构酯为原料,分别研究了TAG结构和固脂含量对空腹响应因子的影响。膳食组中以5%(w/w)的大豆油为空白的低脂组,实验组以36%合成脂肪和5%大豆油为测试脂肪。小鼠自由采食,实验组分为四组,其中合成的OPP和商业化的POP用来研究TAG结构影响,而合成的POO与OOO, PPP和POP的混合物用来研究TAG的固脂含量影响。结果表明:小鼠饲喂6周后,膳食脂肪的固脂含量不影响小鼠的自由采食量、体重,空腹血脂和葡萄糖浓度。但是相比POP膳食组,小鼠对OPP组的食物摄取量更少;6周后的体重显著低于其他组。此外,饲喂PPO小鼠的非酯化脂肪酸的浓度显著高于饲喂POP的小鼠组。而饲喂PPO小鼠的血清葡萄糖浓度则显著低于饲喂POP的小鼠组。综合以上得出:膳食TAG的固脂含量或熔点不影响餐后脂质代谢和空腹血脂水平,而TAG结构影响餐后脂质代谢和空腹非酯化脂肪酸和血糖水平。
Generally, structured triacylglcyerols (TAG) are the TAGs with certain physicochemicalproperties and desired structure, which are not found in form of native oils. Functionalstructure TAGs can lower TAG level and enhance immunity. In addition, TAGs rich inpolyunsaturated fatty acids (PUFAs) can improve the cardiac, vascular endothelial functionsand anti-inflammatory activity. Finally, the synthesis of structured TAGs is very important forthe study on the relationship between TAG structure and oxidation stability, and between TAGstructure and function. Structured TAGs include monoacid symmetrica l, symmetrical diacidand unsymmetrical diacid TAGs. In this study, general methods were established bysynthesizing symmetrical and unsymmetrical TAGs using palmitic and oleic acid as startingmaterials. The feasibility of these methods was confirmed by synthesized the structured TAGswith PUFAs. Finally, these TAGs were preliminary evaluation by animal study.
     Firstly, plamitic acid and purified oleic acid were used to synthesize tripalmitin (PPP)and triolein (OOO), respectively. When3.3mmol palmitic acid and1mmol glycerol weremixed under solvent-free system at60°C for12h using10%Novozym435as catalyst, PurePPP was obtained at89.6%yield after purification. The feasibility of this method was testedby synthesizing OOO. Commercial oleic acid was firstly purified by two-step method.Fractionation type, the addition amount of the solvent and time at the first step and additionamount of the solvent and time at the second step were optimized at the second step toremove stearic acid and linoleic acid, repectively. The optimal conditions for wet fractionationwere8h and1:4ratio of oleic acid to methanol for the one-step purification. In the secondstep,2h and1:5ratio of oleic acid to methanol was selected for the optimal conditions. Underthe optimal conditions,97.4%oleic acid was obtained at65.3%yield. OOO wasenzymatically synthesized by the same conditions. The results showed that only OOO spotwas detected by TLC plates and pure OOO obtained at84.6%yield after purification.
     Symmetrically structured TAGs were synthesized by chemoenzymatic method byreacting1,3-DAGs with corresponding fatty acids. Optimization results for the synthesis of1,3-dipalmitin showed that when vinyl palmitate and glycerol were mixed with2:1(4and2mmol) molar ratio in1mL dichloromethane at room temperature for6h using10%LipozymeRM IM as catalyst, the maxiumal1,3-dipalmitin content reached to92.1%. The purity of1,3-dipalmitin after purification was confirmed by TLC, GC and NMR.1,3-Diolein wassynthesized by enzymatic esterification of glycerol with vinyl oleate which obtained from thechemical transvinylation between vinyl acetate and oleic acid. Pure vinyl oleate afterpurification was obtained at55.7%yield. Optimization results for the synthesis of1,3-dioleinshowed that when vinyl oleate and glycerol were mixed with2:1(4and2mmol) ratio at30°C for8h using8%Lipozyme RM IM, the maximal1,3-diolein content was87.6%incrude mixture. Pure1,3-diolein was obtained at74.7%yield after purification. Subsequently,TAGs were chemically synthesized by reacting1,3-DAGs with corresponding fatty acids with1:1.1molar ratio. Pure POP and OPO as confirmed by NMR and TLC were obtained at88.4%and86.5%respectively. OAO was also synthesized based on the same reaction conditions.
     Secondly, palmitic acid and purified oleic acid were used to synthesize unsymmetricallystructured TAGs (PPO and POO) by two-step method. Namely, monoacylglycerols (MAGs)were synthesized by chemical or enzymatic methods, which were used further for thesynthesis of unsymmetrical TAGs. The optimized conditions for the synthesis ofmonopalmitin by irreversible reaction were the usage of1mL methyl tert-butyl ether assolvent and10%Novozym435as catalyst. After6h,97-98%monopalmitin was obtained at89.5%yield after recrystallizaiton. In contrast, reversible reaction was used for the synthesisof monoolein. When the reaction was conducted at60°C for8h by reacting1mmol oleicacid with1.2mmol acetonide glycerol in0.5mL dichloromethane with8%Novozym435ascatalyst,94.6%1,2-acetonide-3-oleoylglycerol was produdced in crude reaction mixture andwas used further without purification for the synthesis of monoolein by cleavage. At the endof cleavage reaction, there was76.5%monoolein produced and pure monoolein was obtainedat72.8%yield after crystallization in hexane. Subsequently, OPP and POO wereenzymatically synsthesized by reacting purified monoolein and monopalmitin with palmticacid and oleic acid, respectively. Results showed that there was78.2%oleic acid located atthe sn-2position of POO, whereas palmitic acid content at the sn-2position of OPP was82.5%. In order to confirm the feasibility of enzymatic synthesis of unsymmetrical TAGcontaining PUFAs, AA was selected as model for the synthesis of OAA based on the sameconditions metioned above. Thus, AA was enriched firstly. AA was enriched by three-stepmethod. Free fatty acids (FFAs) were first prepared by hydrolysis and AA content wasincreased from40.2to78.4%at27.6%yield after urea inclusion. The enriched FFAs werepurified further by AgNO3fractionation.. The results showed that when the fractionation wasconducted at-5°C for1.5h by mixing0.1g FFAs with1mL hexane and5mol/L AgNO3solution prepared with30%methanol,90.7%AA was obtained at80.4%yield after AgNO3solution fractionation. When purified AA was reacted with monoolein, pure OAA wasobtained at54.9%yield after purification.
     Finally, synthetic structured TAGs were used to investigate the effects of TAG structureand solid fat content. There was5treatments in this study. The control only contained5%(w/w) soybean oil, while other groups contained the36%synthetic fats and5%soybean oil astest fats. Synthetic OPP and commercial POP were used to investigate the effect of TAGstructure. Synthetic POO and a mixture of POP and OOO were used to study the effect ofsolid fat content of test fats. At the end of six-week feeding, the results showed that the solidfat content of test fats did not change the food intake, body weight, and fasting lipemia andglucose responses. By comparison, food intake in group OPP was less than group POP,resulting in a lower body weight compared to other groups. Non-esterified fatty acids (NEFAs)concentration in mice fed OPP was higher compared to that fed POP, whereas glucoseconcentration in group OPP was lower than that in POP. Thus, we concluded that solid fatcontent of test fats did not affect postprandial lipid metabolism and lipe mia response.However, TAG strucuture changes the fasting NEFA and glucose concentrations.
引文
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