CN117512025A - 一种共轭亚油酸甘油酯的制备方法 - Google Patents
一种共轭亚油酸甘油酯的制备方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
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Abstract
本发明提供了一种共轭亚油酸甘油酯的制备方法,以共轭亚油酸CLA和高油酸花生油为原料,采用脂肪酶催化酯交换制备共轭亚油酸甘油酯;CLA与高油酸花生油的质量比为3‑7:1。本发明拓展了单一的共轭亚油酸甘油酯的制备方法,并且本发明方法获得的产物中CLA含量可以达到67.84%。
Description
技术领域
本发明特别涉及一种共轭亚油酸甘油酯的制备方法。
背景技术
共轭亚油酸(Conjugated linoleic acid,CLA)是由人体必需脂肪酸亚油酸在不同位置和立体构型上衍生的含有共轭双键的十八碳二烯酸,是一类同分异构体的总称。CLA存在16种天然同分异构体,其中9c,11t-CLA和10t,12c-CLA这两种异构体被证实具有较强的生理活性。CLA具有降血脂、改善骨质密度、提高免疫力等功能,但目前CLA制品主要以游离脂肪酸的形态存在,口感不佳、稳定性差、易氧化变质,限制了CLA的应用。共轭亚油酸甘油酯(Co njugated linoleic acid glycerides,CLAG)是一种重组脂质,可通过酯交换制备。与CLA相比,CLAG氧化稳定性好,对防止体内脂肪堆积起到重要作用,可广泛用于食品、医药等领域。
CLAG主要通过化学法或酶法制备而成。Niezgoda等利用硫酸催化CLA与乙醇酯化生成CLAG,但化学反应具有随机性,产物种类难以控制,且产物色泽较深、副产物较多。酶法具有特异选择性,条件温和等优点,但一般通过CLA和甘油直接酯化合成,原料比较单一。
发明内容
本发明的目的是提供一种共轭亚油酸甘油酯的制备方法,所述方法以高油酸花生油为原料,拓展了目前较为单一的共轭亚油酸甘油酯的制备方法。
为解决上述技术问题,本发明采用的技术方案如下:
一种共轭亚油酸甘油酯的制备方法,以共轭亚油酸CLA和高油酸花生油为原料,采用脂肪酶催化酯交换制备共轭亚油酸甘油酯;CLA与高油酸花生油的质量比为3-8:1,优选7:1。
所述脂肪酶选自下面一种或一种以上的混合物:Lipozyme TL TM、Lipozyme RMC、Novozyme 435。
优选的,所述脂肪酶为Lipozyme TL TM、Lipozyme RM C、Novozyme 435的混合物。
更进一步,混合脂肪酶的组成为Lipozyme RM C:Lipozyme TL TM:Novozyme 435的质量比为0.76:0.16:0.08。
脂肪酶的加入量为CLA和高油酸花生油质量和的3-12%,优选为10-11%,最优选为10.2%。
酯交换反应在40-50℃进行20-24h,优选在45℃进行24h。
优选的,采用无溶剂法,将CLA和高油酸花生油充分混匀后加入固定化脂肪酶,充氮密封,45℃反应24h;CLA和高油酸花生油的质量比为7.0:1,脂肪酶的加入量为CLA和高油酸花生油质量和的10.2%。
本发明以CLA和高油酸花生油为原料,以产物中CLA含量为指标,采用脂肪酶催化酯交换制备CLAG。花生油是一种优质食用油,营养价值较高,与普通花生油相比,高油酸花生油中油酸含量较高,在75%以上(即不低于75%)。油酸是一种单不饱和脂肪酸(Monounsaturated fatty acid,MUFA),比多不饱和脂肪酸更加稳定,可有效延缓氧化的发生。有研究表明膳食中的MUFA能够调节血压,促进脂代谢,保护心血管。目前尚未见采用花生油为原料制备CLAG的研究报道,所以本发明拓展了单一的共轭亚油酸甘油酯的制备方法,并且本发明方法获得的产物中CLA含量可以达到67.84%。
附图说明
图1为加酶量对酯交换效果的影响;
图2为温度对酯交换效果的影响;
图3为底物质量比对酯交换效果的影响;
图4为不同脂肪酶酯交换产物中CLA含量变化;
图5为加酶量和底物质量比交互作用3D曲线图;
图6为高油酸花生油和CLAG的气相色谱图;
图7为脂肪酶重复性实验的结果。
具体实施方式
以下以具体实施例来说明本发明的技术方案,但本发明的保护范围不限于此:
以下实施例中所采用的高油酸花生油选自正阳邦农食品有限公司产品,油酸含量在75%以上,但该来源对于结果并无影响。
采用无溶剂法,称取一定量CLA,按一定比例加入高油酸花生油,底物总质量为10.0g,充分混匀后加入一定量固定化脂肪酶(加入比例以底物总质量计),充氮密封,置于恒温振荡器100r/min反应24h。反应完成后低速离心去除酶,收集反应液。用正己烷多次洗涤固定化脂肪酶,收集洗涤液,将洗涤液和反应液合并,40℃旋蒸脱溶,定容到10mL以备测定CLA含量。共轭亚油酸甘油酯是本试验(即酯交换反应)的产物,通过测产物中CLA(共轭亚油酸)的含量来表征本试验的效果。
实施例1-6
底物CLA和高油酸花生油质量比为4:1,反应温度为50℃,脂肪酶采用Lipozyme RMC、Lipozyme TL TM、Novozyme 435复配,质量比为0.76:0.16:0.08的条件下,变化加酶量分别为底物质量的3%、5%、8%、10%、12%、15%,获得的底物中甘油酯脂肪酸含量分别为40.7%、50.79%、54.96%、59.96%、60.64%、60.75%,详见图1。
可见,随着加酶量从3%增加到10%,CLA的含量逐渐增加,在底物足量且3%的低酶负荷下,含量仅为40.7%,此时酶的催化位点不足,酯交换程度较弱。随着加酶量增加到10%,含量达到最大,酶与底物分子充分结合,作用位点达到饱和,反应速度加快,酯交换程度提高。但超过10%后,CLA含量无明显增加,可能是因为过量的酶发生聚集,底物分子扩散较难,不利于酰基供体和甘三酯的结合。
实施例7-11
底物CLA和高油酸花生油质量比为4:1,脂肪酶采用Lipozyme RM C、Lipozyme TLTM、Novozyme 435复配,质量比为0.76:0.16:0.08,加酶量为底物质量的10%的条件下,变化反应温度分别为40℃、45℃、50℃、55℃、60℃,获得的底物中甘油酯脂肪酸含量分别为54.38%、62.38%、59.17%、60.05%、58.96%,详见图2。
CLA含量随着温度的升高呈现先升高后降低的趋势,45℃时达到最大,为62.38%,此温度下脂肪酶的活性最高。温度较低时,酶的催化活性较低,底物间酯交换作用较弱,反应速率低。随着温度的升高,反应体系的黏度下降,底物分子的热运动加快,原料间有效碰撞几率增加。此外酶制剂扩散充分,也有利于反应的进行。但过高的温度使酶的蛋白质变性,空间结构和构象发生改变,导致酶失活,酶解效果下降。
实施例12-17
变化底物CLA和高油酸花生油质量比分别为3:1、4:1、5:1、6:1、7:1、8:1,脂肪酶采用Lipozyme RM C、Lipozyme TL TM、Novozyme 435复配,质量比为0.76:0.16:0.08,加酶量为底物质量10%,反应温度为50℃,获得的底物中甘油酯脂肪酸含量分别为50.37%、58.96%、60.41%、64.03%、64.31%、62.53%,详见图3。
随着底物质量比由3:1升高至6:1,产物中CLA的含量快速增加,由50.37%升至64.03%,但随着底物质量比继续增加,CLA含量增长缓慢趋于平缓,7:1时含量为64.31%,与6:1时无显著性差异,此时反应接近平衡。底物质量比3:1时,酰基供体较少,酯交换不完全,CLA接枝到甘三酯的甘油骨架的量较少。随着底物质量比升高,酰基供体增加,酯交换反应正向进行,但随着底物质量比继续增加达到8:1时,CLA含量反而有所降低,这可能是因为体系中游离脂肪酸含量过高,阻碍脂肪酶螺旋盖的打开,降低部分脂肪酶的活性,影响底物与酶活性位点的结合,导致酯交换反应逐渐停止。另一方面,体系中过多的CLA会增加后期产物分离纯化的难度。
实施例18-31
同时变化底物比、酶加入量、反应温度进行反应,获得的甘油酯脂肪酸含量详见下表。下表中采用的脂肪酶为Lipozyme RM C:Lipozyme TL TM:Novozyme 435的质量比为0.76:0.16:0.08的混合酶。
表1
实施例32-36
底物CLA和高油酸花生油质量比分别为4:1,分别采用以下五种脂肪酶:固定化脂肪酶Lipozyme RM IM、Lipozyme RM C、Lipozyme TL TM、Novozyme 435、Novozyme 40086,加酶量为底物质量10%,反应温度为不同酶的最适温度反应24h(详见表2),获得的底物中甘油酯脂肪酸含量分别为31.83%、35.15%、42.26%、37.04%、31.25%,详见图4。
表2
所述脂肪酶来源如下:固定化脂肪酶Lipozyme RM IM(酶活25BIU/g)、LipozymeRM C(酶活401.39IUN/g)、Lipozyme TL TM(酶活175IUN/g)、Novozyme 435(酶活10000PLU/g)、Novozyme 40086(酶活275IUN/g),皆为诺维信(中国)生物技术有限公司产品,可直接购买获得。
可见,CLA和高油酸花生油的酯交换效果受酶种类的影响,这5种酶催化剂来源于不同的微生物,其固定化材料也不尽相同,因此,在不同酶催化剂的反应体系中,得到的CLA含量不同。在最适作用条件下,经Lipozyme TL TM催化酯交换的产物中,CLA含量显著高于其他酶(P<0.05),这可能与酶的催化能力有关,已有的研究结果证明,反应体系不同,脂肪酶的催化活性有较大差异,Lipozyme TL TM对此反应体系的活力高于其他脂肪酶,因此产物中CLA含量较高。其次是Novozyme 435,5种酶中,Novozyme 435属于广源性酶,专一性不确定,位置随机性较强,在反应过程可作用在甘三酯的3个位置,其他酶为Sn-1,3位特异性脂肪酶,作用位点比Novozyme 435少Sn-2位。综上Lipozyme RM C、Lipozyme TL TM和Novozyme 435三种酶效果较好。
实施例37-48
底物CLA和高油酸花生油质量比分别为4:1,采用不同的脂肪酶,加酶总量为底物质量10%,反应温度为50℃反应24h,对应的脂肪酶选择以及获得的底物中甘油酯脂肪酸含量详见下表3。
其中,按Lipozyme RM C、Lipozyme TL TM和Novozyme 435总量为1,A为LipozymeRM C占比、B为Lipozyme TL TM占比,C为Novozyme 435占比。
表3
可以看出,使用单种酶催化酯交换的产物中CLA含量低于复合酶。即,三种酶单一使用以及三种酶交互使用对CLA和高油酸花生油的酯交换均起到促进作用,贡献大小分别为B>C>A和BC>AB>AC。单酶中Lipozyme TL TM起着重要作用,且Lipozyme RM C和Lipozyme TL TM、Lipozyme TL TM和Novozyme435、Lipozyme RM C和Novozyme 435的共同酯交换及三种酶共同酯交换作用显著提高CLA在高油酸花生油中的插入率。
综上,本发明中,由于采用高油酸花生油为原料,对于其他反应条件进行了研究,确定了底物质量比、酶的选择、加酶量、反应温度等因素来实现本发明对于共轭亚油酸甘油酯的制备。其中,底物质量比影响最为显著,加酶量对结果的影响显著。交互项中加酶量与底物质量比对结果的影响显著。图5是底物质量比、加酶量对CLA含量影响的响应面图。当加酶量固定在10%不变时,底物质量比由4:1升至8:1,CLA的含量呈现先升高后降低的趋势,加酶量和底物质量比交互作用显著(P<0.05)。
脂肪酶的重复利用性
试验方法:将反应后的固定化脂肪酶过滤回收后晾干,用于下一批试验,重复10次,测定每次重复试验产物中CLA的含量。采用的脂肪酶为Lipozyme RM C:Lipozyme TLTM:Novozyme 435的质量比为0.76:0.16:0.08的混合酶。工艺参数为底物CLA和高油酸花生油质量比7.0:1、加酶量10.2%、反应温度45.0℃。
结果详见图7,10次重复试验产物中CLA含量分别为67.84%、67.56%、66.38%、66.07%、64.52%、63.71%、63.04%、62.40%、60.72%、58.39%。这表明,脂肪酶在优化的工艺参数下至少可以重复利用十余次,在第9次反应时固定化脂肪酶的催化活力仍可保持90%左右。该复合脂肪酶良好的重复利用性表明,本工艺可以在很大程度上降低生产成本,具有较好的工业利用前景。
本发明中,甘油酯的脂肪酸测定方法如下:
取100μL待测样品,采用薄层层析法(TLC)将样品中的甘油酯分离,展开剂为正己烷-乙酸乙酯-乙酸(体积比80:20:1)。将硅胶板放在紫外光下显色,得到四个条带,从下到上分别为甘一酯、甘二酯、游离脂肪酸和甘三酯,刮下甘一酯、甘二酯和甘三酯对应的目标条带,加入乙醚提取甘油酯,并定容至10mL。采用三氟化硼-乙醚法(GB/T 17376-2008《动植物油脂脂肪酸甲酯制备》)进行脂肪酸甲酯化制备,采用气相色谱测定,根据面积归一化法对脂肪酸组成进行定量分析,得到产物中CLA的相对百分含量。
气相条件:色谱柱为HP-88(60m×250μm×0.25μm);进样口温度250℃,检测器温度270℃;不分流模式;升温程序为120℃保持1min,以10℃/min升至175℃保持10min,以5℃/min升至210℃保持5min,以10℃/min升至230℃保持10min;进样量1μL。
图6显示了高油酸花生油(上图)和CLAG(下图)气相色谱图,图中可见,高油酸花生的脂肪酸主要为棕榈酸(C16:0)、油酸(C18:1)、亚油酸(C18:2)、硬脂酸(C18:0)和山嵛酸(C22:0)等,主要以不饱和脂肪酸为主,这与前人的研究结果一致。普通花生油脂中亚油酸含量最高,而高油酸花生油脂中油酸含量远高于亚油酸,降低了油脂体系的不饱和度,有利于花生油脂的储存。产物CLAG的脂肪酸在种类和含量上与原料高油酸花生油的差别较大,研究表明高油酸花生油的脂肪酸在Sn-1,3和Sn-2位均有分布,所用的脂肪酶主要作用于Sn-1和Sn-3位,由产物峰图可以看出,花生油脂的脂肪酸峰高均降低,在21min时出现新峰,该化合物为CLA,表明原料间发生了酯交换且CLA接枝到了高油酸花生油脂的甘油骨架上,该方法制备CLAG效果较好。
Claims (8)
1.一种共轭亚油酸甘油酯的制备方法,其特征在于,以共轭亚油酸CLA和高油酸花生油为原料,采用脂肪酶催化酯交换制备共轭亚油酸甘油酯;CLA与高油酸花生油的质量比为3-8:1。
2.如权利要求1所述的共轭亚油酸甘油酯的制备方法,其特征在于,所述脂肪酶选自下面一种或一种以上的混合物:Lipozyme TL TM、Lipozyme RM C、Novozyme 435。
3.如权利要求2所述的共轭亚油酸甘油酯的制备方法,其特征在于,所述脂肪酶为Lipozyme TL TM、Lipozyme RM C、Novozyme 435的混合物。
4.如权利要求3所述的共轭亚油酸甘油酯的制备方法,其特征在于,Lipozyme RM C:Lipozyme TL TM : Novozyme 435的质量比为0.76: 0.16: 0.08。
5.如权利要求2所述的共轭亚油酸甘油酯的制备方法,其特征在于,脂肪酶的加入量为CLA和高油酸花生油质量和的3-12%。
6.如权利要求5所述的共轭亚油酸甘油酯的制备方法,其特征在于,脂肪酶的加入量为CLA和高油酸花生油质量和的10-11%。
7.如权利要求1所述的共轭亚油酸甘油酯的制备方法,其特征在于,所述酯交换反应在40-50℃进行20-24h。
8.如权利要求1-7任一所述的共轭亚油酸甘油酯的制备方法,其特征在于,采用无溶剂法,将CLA和高油酸花生油充分混匀后加入固定化脂肪酶,充氮密封,45℃反应24h;CLA和高油酸花生油的质量比为7.0:1,脂肪酶的加入量为CLA和高油酸花生油质量和的10.2%。
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