CN101194014A - 脂肪酶粉末组合物 - Google Patents
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Abstract
本发明提供脂肪酶粉末组合物，其含有被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的平均粒径1μm以上且不足300μm的粉碎品和过滤助剂。该脂肪酶粉末组合物提高了脂肪酶活性和操作性，可适用于油脂的酯交换方法和酯化方法。
Description
技术领域
本发明涉及一种可适用于各种酯化反应、酯交换反应等中的脂肪酶粉末组合物，以及使用该脂肪酶粉末组合物的油脂的酯交换方法等。
背景技术
脂肪酶被广泛用于脂肪酸等各种羧酸与一元醇或多元醇等醇类的酯化反应、多个羧酸酯之间的酯交换反应等中。其中，酯交换反应作为对动植物油脂类的改性以及各种脂肪酸的酯、糖酯和类固醇的制备方法是重要的技术。当使用作为油脂水解酶的脂肪酶作为这些反应的催化剂时，可在室温至约70℃左右的温和条件下进行酯交换反应，与以往的化学反应相比，不仅抑制副反应、降低能耗，而且由于作为催化剂的脂肪酶是天然物质，因此安全性也较高。另外，可以根据其底物特异性或位置特异性高效地生产目标物。然而，即使将脂肪酶粉末照原样用于酯交换反应中也不会充分表现出其活性，而且难以将原本为水溶性的脂肪酶均匀地分散到油性原料中，其回收也困难。因此，以往通常将脂肪酶固定到一些载体上，例如阴离子交换树脂(专利文献1)、酚醛吸附树脂(专利文献2)、疏水性载体(专利文献3)、阳离子交换树脂(专利文献4)、螯合树脂(专利文献5)等上来用于酯化反应或酯交换反应等中。
然而，当使脂肪酶固定化到载体上时，脂肪酶活性降低，因而开发了使用脂肪酶粉末的各种技术。
具体来说，提出了以下方案：在非活性有机溶剂的存在下或不存在下，使脂肪酶粉末分散在含有酯的原料中进行酯交换反应，以使酯交换反应时分散脂肪酶粉末颗粒的90％以上保持在1～100μm范围粒径的方法(专利文献6)。此外，提出了使用将含有磷脂质和脂溶性维他命的酶溶液干燥得到的酶粉末的方案(专利文献7)。
专利文献1：日本特开昭60-98984号公报
专利文献2：日本特开昭61-202688号公报
专利文献3：日本特开平2-138986号公报
专利文献4：日本特开平3-61485号公报
专利文献5：日本特开平1-262795号公报
专利文献6：日本特许第2668187号公报
专利文献7：日本特开2000-106873号公报
发明内容
本发明的目的在于，提供提高了脂肪酶活性和操作性的脂肪酶粉末组合物。
本发明的目的还在于，提供使用肪酶粉末组合物的油脂的酯交换方法。
本发明的目的还在于，提供使用肪酶粉末组合物的酯化方法。
本发明是基于下述见解完成的，即，如果同时使用特定的固定化脂肪酶的粉碎物和过滤助剂，则可解决上述课题。
即，本发明提供脂肪酶粉末组合物，其特征在于，其含有被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的平均粒径1μm以上且不足300μm的粉碎品和过滤助剂。
本发明还提供油脂的酯交换方法，其特征在于，在被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的平均粒径1μm以上且不足300μm的粉碎品和过滤助剂的存在下，进行油脂的酯交换反应，接着，回收该粉碎品和过滤助剂，将它们再循环。
本发明还提供酯化方法，其特征在于，在被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的平均粒径1μm以上且不足300μm的粉碎品和过滤助剂的存在下，进行酯化反应，接着，回收该粉碎品和过滤助剂，将它们再循环。
附图说明
图1表示使用本发明的脂肪酶粉末组合物(实施例4)和粉碎前的固定化脂肪酶(Lipozyme TL-IM)的情况下的酯交换率的变化。
具体实施方式
本发明所使用的脂肪酶是源自嗜热真菌属(Thermomycessp.)的脂肪酶，将被固定化到硅石载体上的该脂肪酶粉碎成平均粒径为1μm以上且不足300μm。这里，被固定化到硅石载体上的该脂肪酶的平均粒径优选为300～1000μm左右。这样的固定化脂肪酶，例如可以从Novozymes A/S公司获取Lipozyme TL-IM。
使用通常的粉碎机将这样的固定化脂肪酶粉碎成平均粒径为1μm以上且不足300μm，优选平均粒径为1～200μm，更优选平均粒径为1～100μm，特别优选平均粒径为20～100μm。这里，作为粉碎机，可列举出研钵、剪切摩擦式粉碎机、切割式粉碎机、磨石(MYCOLLOIDER、MASSCOLLOIDER)、咖啡磨、power mill、销棒粉碎机(pin mill)、冲击式粉碎机(锤碎机、球磨机)、辊式粉碎机和气流式粉碎机、均化器、超声波粉碎机等。
另一方面，作为本发明所使用的过滤助剂，可列举出氟镁石等无机过滤助剂；和纤维素等纤维或其粉碎物等有机过滤助剂。这些当中，优选有机过滤助剂、特别是有机高分子过滤助剂，其中优选纤维素等，可列举出NIPPON PAPER CHEMICALSCO.，LTD以商品名KC Flock出售的产品等。过滤助剂也优选为粉状，优选平均粒径为10～90μm。
上述脂肪酶粉碎品与过滤助剂的质量比优选为1/10～10/1，特别优选为1/7～2/1。
本发明的脂肪酶粉末组合物必须含有上述脂肪酶粉碎品和过滤助剂。
本发明的脂肪酶粉末组合物可照原样用于油脂的酯交换反应和酯化反应，但通过使其与长链脂肪酸三甘油酯和中链脂肪酸三甘油酯接触后收集，可进行纯化，同时可使脂肪酶活性提高。
作为这里所使用的长链脂肪酸三甘油酯，优选其构成脂肪酸的碳原子数为14～24的三甘油酯，特别优选选自菜籽油、大豆油、葵花籽油、红花油、玉米油所组成的组中的植物油。
作为中链脂肪酸三甘油酯，优选其构成脂肪酸的碳原子数为6～12的三甘油酯。这样的脂肪酸三甘油酯可通过公知的制造方法制造，也可使用市售品。作为市售品，例如由日清奥利友集团株式会社出售的商品名为ODO的商品。
优选以质量比计为95∶5～50∶50的比例使用长链脂肪酸三甘油酯和中链脂肪酸三甘油酯，优选使相对于脂肪酶总质量为2倍～100倍质量的三甘油酯与脂肪酶接触。
作为使用本发明的脂肪酶粉末组合物的酯化反应，期望在该脂肪酶粉末组合物的存在下，进行油脂的酯化反应，接着，回收脂肪酶粉末组合物并再循环。
另外，优选在被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的粒径1μm以上且不足300μm的粉碎品和过滤助剂的存在下，进行油脂的酯化反应，接着，回收该粉碎品和过滤助剂，将它们再循环，从而对油脂进行酯化。
根据本发明，可提高脂肪酶活性，并提高在酯化反应和酯交换反应中的易使用性(操作性)，可再循环而多次用于这些反应，因此，可适用于工业规模下通过油脂的酯交换等进行的油脂改性。另外，根据本发明的方法，可提供提高了风味的油脂。
接着，通过实施例，更详细地说明本发明。
实施例
实施例1
使用特殊机化工业(株)制造的L型MYCOLLOIDER，粉碎5g Novozymes A/S公司的Lipozyme TL-IM。使用堀场制作所公司制造的粒度分布计LA-500，测定粉碎后的脂肪酶的粒径，结果，平均粒径为66.4μm。向该粉末中加入5g作为过滤助剂的纤维素粉末(NIPPON PAPERE CHEMICALS CO.，LTD：平均粒径为30μm)，制成脂肪酶粉末组合物。通过下述方法，测定该脂肪酶组合物的酯交换活性，以相对值示于表1。
脂肪酶活性的测定方法
向以1∶1(w)的比例混合三油酸甘油酯和三辛酸甘油酯得到的油中，添加脂肪酶组合物，使其在60℃下反应。随时间取样10μl，用1.5ml己烷稀释后，过滤脂肪酶组合物，将所得溶液作为气相色谱(GC)用试样。用GC(柱：DB-1ht)分析，由下式求出反应率。GC条件为柱温：初始150℃，升温15℃/分钟，最终370℃。
反应率(％)＝{C34area/(C24area+C34area)}×100
式中，C24代表三辛酸甘油酯，C34代表三辛酸甘油酯的一个脂肪酸被替换为油酸的物质，area是它们的峰面积。根据各时间的反应率，利用分析软件(Origin ver.6.1)求得反应速率常数k值。
脂肪酶活性以将Lipozyme TL-IM的k值设为100时的相对活性来表示。
实施例2
使用研钵，粉碎5g Novozymes A/S公司制造的Lipozyme TL-IM。使用堀场制作所公司制造的粒度分布计LA-500，测定粉碎后的脂肪酶的粒径，结果，平均粒径为52.1μm。向该粉末中加入2.5g作为过滤助剂的纤维素粉末(NIPPON PAPERECHEMICALS CO.，LTD)，制成脂肪酶粉末组合物。通过与实施例1同样的方法，测定该脂肪酶组合物的酯交换活性，以相对值示于表1。
实施例3
向5g Novozymes A/S公司制造的Lipozyme TL-IM中加入50g菜籽油，使用DREMEL公司制造的均化器(Multipro395)，在冰冷却下以12500rpm.粉碎3分钟。使用堀场制作所公司制造的粒度分布计LA-500，测定粉碎后的脂肪酶的粒径，结果，平均粒径为91.5μm。向其中加入5g作为过滤助剂的纤维素粉末(NIPPON PAPERE CHEMICAL S CO.，LTD)，减压过滤菜籽油，得到脂肪酶组合物。通过与实施例1同样的方法，测定该脂肪酶组合物的酯交换活性，以相对值示于表1。
表1
| 平均粒径(μm) | 相对于脂肪酶制剂质量的相对酯交换活性 | |
| 粉碎前(TL-IM)实施例1实施例2实施例3 | 80066.452.191.5 | 100201304202 |
实施例4
对5g由实施例1得到的脂肪酶组合物添加90g菜籽油和10gODO(日清奥利友集团株式会社)，在25℃下搅拌5小时，然后过滤，回收脂肪酶组合物。
接着，向150gODO(日清奥利友集团株式会社)和850g菜籽油(日清奥利友集团株式会社)中添加通过上述方法进行前处理得到的脂肪酶组合物0.5％，在50℃下搅拌15小时，进行酯交换反应。随时间求出酯交换率，确认反应的进展。另外，使用气相色谱仪分析甘油酯组成，算出测定试样中的酯交换反应产物的比率，从而求出酯交换率。本实施例和LipozymeTL-IM(粉碎前)的酯交换率的变化示于图1。
反应后过滤脂肪酶组合物并回收。通过常用方法对所得到的油进行脱酸、脱色和脱味，纯化酯交换油。由经过训练的专门小组对所得到的酯交换油的风味进行评价。其结果，相对于使用未被菜籽油和ODO前处理的脂肪酶组合物同样进行酯交换而得到酯交换油，风味显著良好。
另外，使用回收的酶，进行酯交换反应，结果，可没有问题地进行10次再循环。
实施例5
使用Hosokawa Micron公司制造的销棒粉碎机(精密冲击式粉碎机100UPZ)，以17600rpm粉碎1Kg Novozymes A/S公司制造的Lipozyme TL-IM。使用堀场制作所公司制造的粒度分布计LA-500，测定粉碎后的脂肪酶粒径，结果，平均粒径为13.8μm。向该粉末中加入1Kg作为过滤助剂的纤维素粉末(NIPPONPAPERE CHEMICALS CO.，LTD：平均粒径30μm)，制成脂肪酶粉末组合物。
实施例6
向5g由实施例5得到的脂肪酶组合物中添加90g菜籽脱色油和10g ODO(日清奥利友集团株式会社制造)，在60℃下搅拌2小时，然后过滤回收脂肪酶组合物。以与实施例1同样的方法测定该脂肪酶组合物的酯交换活性，以相对值示于表2。
实施例7
向5g由实施例5得到的脂肪酶组合物中添加90g菜籽脱色油和10g ODO(日清奥利友集团株式会社制造)，在室温下搅拌24小时，然后过滤回收脂肪酶组合物。以与实施例1同样的方法测定该脂肪酶组合物的酯交换活性，以相对值示于表2。
实施例8
向5g由实施例5得到的脂肪酶组合物中添加50g菜籽脱色油和50g ODO(日清奥利友集团株式会社制造)，在室温下搅拌24小时，然后过滤回收脂肪酶组合物。以与实施例1同样的方法测定该脂肪酶组合物的酯交换活性，以相对值示于表2。
表2
| 相对于脂肪酶制剂质量的相对酯交换活性 | |
| 粉碎前(TL-IM)实施例5实施例6实施例7实施例8 | 100474557714600 |
Claims (10)
1.脂肪酶粉末组合物，其特征在于，其含有被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的平均粒径1μm以上且不足300μm的粉碎品和过滤助剂。
2.根据权利要求1所述的组合物，粉碎品的平均粒径为1～200μm。
3.根据权利要求2所述的组合物，粉碎品的平均粒径为1～100μm。
4.根据权利要求1～3任一项所述的组合物，粉碎品与过滤助剂的质量比为1/10～10/1。
5.根据权利要求1～4任一项所述的组合物，过滤助剂是纤维素。
6.根据权利要求1～5任一项所述的组合物，过滤助剂是粉状的。
7.根据权利要求6所述的组合物，过滤助剂的平均粒径为10～90μm。
8.根据权利要求1～7任一项所述的组合物，其是酯交换用或酯化用的。
9.油脂的酯交换方法，其特征在于，在权利要求1～8任一项所述的脂肪酶粉末组合物的存在下，进行油脂的酯交换反应，接着，回收脂肪酶粉末组合物并再循环。
10.酯化方法，其特征在于，在被固定化到硅石载体上的源自嗜热真菌属的脂肪酶的平均粒径1μm以上且不足300μm的粉碎品和过滤助剂的存在下，进行酯化反应，接着，回收该粉碎品和过滤助剂，将它们再循环。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8911980B2 (en) | 2009-03-27 | 2014-12-16 | The Nisshin Oillio Group, Ltd. | Method for producing lipase powder compositions |
| CN107805647A (zh) * | 2017-12-14 | 2018-03-16 | 广州白云山汉方现代药业有限公司 | 一种酶法合成中长链结构酯的方法 |
| CN111630161A (zh) * | 2017-11-24 | 2020-09-04 | 诺维信公司 | 用于酯交换的小的酶颗粒 |
| CN113939589A (zh) * | 2019-05-29 | 2022-01-14 | 诺维信公司 | 用于酯化和酯交换的脂肪分解聚合物颗粒 |
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| DK402583D0 (da) | 1983-09-05 | 1983-09-05 | Novo Industri As | Fremgangsmade til fremstilling af et immobiliseret lipasepraeparat og anvendelse deraf |
| DK153762C (da) | 1985-02-27 | 1989-01-09 | Novo Industri As | Fremgangsmaade til fremstilling af et immobiliseret lipasepraeparat |
| GB8729890D0 (en) * | 1987-12-22 | 1988-02-03 | Unilever Plc | Improvements in & relating to fat processes |
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| JP2794201B2 (ja) | 1989-07-31 | 1998-09-03 | 味の素株式会社 | 固定化リパーゼ酵素剤 |
| JP2668187B2 (ja) * | 1993-09-17 | 1997-10-27 | 日清製油株式会社 | リパーゼ粉末を用いたエステル交換法 |
| WO1995022606A1 (en) | 1994-02-21 | 1995-08-24 | Novo Nordisk A/S | Method for production of an immobilized enzyme preparation and use of the immobilized enzyme preparation |
| US5989599A (en) * | 1995-04-24 | 1999-11-23 | Nestec S.A. | Process for the interesterification of phospholipids |
| US5902738A (en) * | 1996-04-18 | 1999-05-11 | Roche Vitamins Inc. | Enzymatic acylation |
| AU1556699A (en) | 1997-12-23 | 1999-07-19 | Novo Nordisk A/S | A process for immobilisation of enzymes |
| US6156548A (en) * | 1997-12-23 | 2000-12-05 | Novo Nordisk A/S | Immobilization of enzymes with a fluidized bed for use in an organic medium |
| JP2000106873A (ja) | 1998-10-06 | 2000-04-18 | Nisshin Oil Mills Ltd:The | 熱安定性酵素およびその製造法 |
| US6372472B1 (en) * | 1999-09-24 | 2002-04-16 | Swim Pure Corporation | Filter media containing powered cellulose and immobilized lipase for swimming pool and spa water filteration |
| JP3690951B2 (ja) * | 1999-12-22 | 2005-08-31 | 日清オイリオグループ株式会社 | 油脂のエステル交換反応方法 |
| PL1709143T3 (pl) * | 2004-01-26 | 2013-02-28 | Unilever Bcs Europe Bv | Enzymatyczne modyfikowanie tłuszczów triglicerydowych |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8911980B2 (en) | 2009-03-27 | 2014-12-16 | The Nisshin Oillio Group, Ltd. | Method for producing lipase powder compositions |
| TWI504745B (zh) * | 2009-03-27 | 2015-10-21 | Nisshin Oillio Group Ltd | 脂肪酶粉末組成物的製造方法 |
| CN111630161A (zh) * | 2017-11-24 | 2020-09-04 | 诺维信公司 | 用于酯交换的小的酶颗粒 |
| CN107805647A (zh) * | 2017-12-14 | 2018-03-16 | 广州白云山汉方现代药业有限公司 | 一种酶法合成中长链结构酯的方法 |
| CN113939589A (zh) * | 2019-05-29 | 2022-01-14 | 诺维信公司 | 用于酯化和酯交换的脂肪分解聚合物颗粒 |
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