JP5585454B2 - Method for producing emulsified powder lipase preparation - Google Patents
Method for producing emulsified powder lipase preparation Download PDFInfo
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- JP5585454B2 JP5585454B2 JP2010549376A JP2010549376A JP5585454B2 JP 5585454 B2 JP5585454 B2 JP 5585454B2 JP 2010549376 A JP2010549376 A JP 2010549376A JP 2010549376 A JP2010549376 A JP 2010549376A JP 5585454 B2 JP5585454 B2 JP 5585454B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
Description
本発明は、各種エステル化反応、エステル交換反応等に好適に使用できる、高活性な乳化粉末リパーゼ製剤の製造方法に関する。 The present invention relates to a method for producing a highly active emulsified powder lipase preparation that can be suitably used for various esterification reactions, transesterification reactions, and the like.
リパーゼを触媒として使用した油脂のエステル交換反応による改質が広く行われている。これら油脂のエステル交換反応には、従来から何らかの担体に固定化した、固定化リパーゼが用いられることが多かった。しかし、固定化酵素を実用スケールで用いると、その担体に由来する水分などの反応系内への持込みによる、副反応が十分に抑制できず、反応物の品質を低下させてしまうという問題は、現在でも解消しきれていない。また、カラムを用いる装置が大掛かりとなり、少量多品種の生産には向いていない。 The reforming of fats and oils using lipase as a catalyst has been widely carried out by transesterification. In the transesterification reaction of these fats and oils, an immobilized lipase that has been conventionally immobilized on some carrier has often been used. However, when the immobilized enzyme is used on a practical scale, the side reaction due to the introduction of moisture derived from the carrier into the reaction system cannot be sufficiently suppressed, and the quality of the reaction product is reduced. It has not been solved even now. Moreover, the apparatus using a column becomes large and is not suitable for the production of a small variety of products.
最近は担体への固定化を行わず、リパーゼ製剤を粉末で利用する方法が提案されている。しかし、一般にカビ，酵母等の真菌に由来する市販の粉末リパーゼ製剤は、そのエステル交換活性は、非常に低い。一方、特許文献１などの細菌由来のものでは、比較的活性を発揮するものもあるが、まだ十分ではなく、安全性の観点からも、食品用途に広く使用できるものは少ない。 Recently, a method has been proposed in which a lipase preparation is used in powder form without being immobilized on a carrier. However, commercially available powder lipase preparations derived from fungi such as mold and yeast generally have very low transesterification activity. On the other hand, some bacteria-derived ones such as Patent Document 1 are relatively active, but are not yet sufficient, and few are widely usable for food applications from the viewpoint of safety.
カビリパーゼに獣乳を混合し、リパーゼの基質特異性を向上させる粉末リパーゼの製造方法（特許文献２）、大豆など他種の蛋白素材を混合して粉末リパーゼの酵素活性を発揮させる方法（特許文献３）も提案されているが、エステル交換活性としてはまだ十分ではない。 A method for producing powdered lipase that mixes animal milk with kablipase to improve the substrate specificity of lipase (Patent Document 2), a method for mixing other protein materials such as soybeans to exert enzyme activity of powdered lipase (Patent Document) 3) has also been proposed, but is still not sufficient as transesterification activity.
本発明の目的は、従来のような固定化担体を必要とせず、粉末状態にて高活性なエステル交換活性を発揮する乳化粉末リパーゼ製剤を提供することである。 An object of the present invention is to provide an emulsified powder lipase preparation that exhibits high transesterification activity in a powder state without requiring a conventional immobilization carrier.
本発明者らは、上記の課題に対して鋭意研究を重ねた結果、乳化機能のある多糖類および油脂で調製した水中油型乳化物と、リパーゼ活性を有する酵素を混合後に乾燥して得られる乳化粉末リパーゼ製剤は、従来のような固定化操作を行わずとも良好なエステル交換活性を発揮することを見出し、本発明を完成させた。
すなわち、本発明は、
（１）乳化性多糖類，油脂及びリパーゼ活性を有する酵素を含む、水中油型乳化物を乾燥させてなる、乳化粉末リパーゼ製剤の製造方法。
（２）乳化性多糖類が水溶性大豆多糖類，アラビアガム，オクテニルコハク酸澱粉から選ばれる１種以上である、（１）記載の乳化粉末リパーゼ製剤の製造方法。
（３）リパーゼ活性が、トリグリセリドの１,３位に特異的に作用するものである、（１）記載の乳化粉末リパーゼ製剤の製造方法。
（４）乳化粉末リパーゼ製剤がエステル交換反応またはエステル合成反応用である、請求項１に記載の乳化粉末リパーゼ製剤の製造方法。
である。As a result of intensive research on the above problems, the present inventors obtained an oil-in-water emulsion prepared with polysaccharides and fats and oils having an emulsifying function and an enzyme having lipase activity and then dried. The emulsified powder lipase preparation has been found to exhibit good transesterification activity without performing a conventional immobilization operation, and has completed the present invention.
That is, the present invention
(1) A method for producing an emulsified powder lipase preparation comprising drying an oil-in-water emulsion containing an emulsifiable polysaccharide, an oil and fat, and an enzyme having lipase activity.
(2) The method for producing an emulsified powder lipase preparation according to (1), wherein the emulsifiable polysaccharide is at least one selected from water-soluble soybean polysaccharide, gum arabic, and octenyl succinate starch.
(3) The method for producing an emulsified powder lipase preparation according to (1), wherein the lipase activity specifically acts on positions 1 and 3 of triglyceride.
(4) The method for producing an emulsified powder lipase preparation according to claim 1, wherein the emulsified powder lipase preparation is for transesterification or ester synthesis reaction.
It is.
本発明によれば、従来のような固定化担体を使用することなく、リパーゼ活性を有する酵素を、乳化性多糖類を含む水中油型乳化物と混合し、乾燥することで良好なエステル交換活性を発揮するリパーゼ製剤を得ることができるため、従来の固定化という煩雑な操作を必要としない。また、固定化担体が必要ないため、酵素活性に対する酵素製剤の体積を大幅に減少させることができ、反応設備をコンパクトにできる。更に、種々の低分子乳化剤や動植物由来の蛋白質である高分子乳化剤に比べ、本発明による乳化性多糖類を使用した方法では、エステル交換活性を従来の方法よりもさらに効率的に発揮させることが可能となり、エステル交換反応やエステル合成反応に好適に使用できる。 According to the present invention, an enzyme having lipase activity is mixed with an oil-in-water emulsion containing an emulsifying polysaccharide and dried without using a conventional immobilizing carrier, and good transesterification activity is achieved. Therefore, a complicated operation of conventional immobilization is not required. Further, since no immobilization carrier is required, the volume of the enzyme preparation for the enzyme activity can be greatly reduced, and the reaction equipment can be made compact. Furthermore, compared to various low molecular emulsifiers and high molecular emulsifiers that are proteins derived from animals and plants, the method using the emulsifiable polysaccharide according to the present invention can exhibit transesterification activity more efficiently than the conventional methods. It becomes possible and can be used suitably for transesterification and ester synthesis reaction.
（乳化性多糖類）
以下、本発明を具体的に説明する。本発明で用いる乳化性多糖類とは、単独で水中油型乳化物を形成できる乳化性をもつ多糖類であり、好ましくは水溶性大豆多糖類，アラビアガム，オクテニルコハク酸澱粉から選ばれる１種以上である。水溶性大豆多糖類とは、大豆若しくは大豆処理物から弱酸性で100℃を超える温度で抽出されたものを使用すると、エステル交換活性が強く維持され好ましく、抽出pHがpH2.4〜4.0である高乳化型の水溶性多糖類を使用すると更に好ましい。これら乳化性多糖類は、複数のものを同時に使用することもできる。ここでいう多糖類の乳化性は、例えば、１重量%の多糖類水溶液に４重量%に相当する精製パーム油を加え、ホモミキサーおよび高圧ホモゲナイザーで乳化した際に、レーザー粒度分布計で測定する平均粒径５μm以下の水中油型乳化物が調製できることでも確認を行える。(Emulsifying polysaccharide)
Hereinafter, the present invention will be specifically described. The emulsifiable polysaccharide used in the present invention is a polysaccharide having emulsifiability capable of forming an oil-in-water emulsion alone, and preferably one or more selected from water-soluble soybean polysaccharide, gum arabic, and octenyl succinate starch. It is. The water-soluble soybean polysaccharide is preferably weakly acidic and extracted at a temperature exceeding 100 ° C. from soybean or a processed soybean product, and the transesterification activity is strongly maintained, and the extraction pH is pH 2.4 to 4.0. It is more preferable to use a highly emulsified water-soluble polysaccharide. A plurality of these emulsifying polysaccharides can be used simultaneously. The emulsifiability of the polysaccharide here is measured with a laser particle size distribution meter when, for example, purified palm oil corresponding to 4% by weight is added to a 1% by weight polysaccharide aqueous solution and emulsified with a homomixer and a high-pressure homogenizer. It can also be confirmed that an oil-in-water emulsion having an average particle size of 5 μm or less can be prepared.
（油脂）
本発明の水中油型乳化物の調製に用いる油脂としては、例えば、大豆油，ヤシ油，パーム油，パーム核油，なたね油，ひまわり油，ピーナッツ油，オリーブ油，コメ油，シア脂，サル脂，綿実油，カカオ脂，オリーブ油，ゴマ油，コムギ胚芽油，イリッペ脂，ベニバナ油，トウモロコシ油，乳脂，羊脂，山羊脂，馬脂，卵黄脂，イワシ油，鯨油等の動植物油脂、またはこれら油脂類の分別品，水素添加品やエステル交換品等を使用することができる。また、前述した乳化性高分子および油脂に、各種ショ糖脂肪酸エステルに代表されるような低分子乳化剤、または大豆や乳等に由来する蛋白質系の高分子乳化剤を、リパーゼの活性に影響のない範囲で併用すると、乳化の安定性を向上させ、粉末活性の安定的発現に寄与させることができ、好ましい。(Oil and fat)
Examples of the oil used in the preparation of the oil-in-water emulsion of the present invention include soybean oil, coconut oil, palm oil, palm kernel oil, rapeseed oil, sunflower oil, peanut oil, olive oil, rice oil, shea fat, monkey fat, Animal oil such as cottonseed oil, cacao butter, olive oil, sesame oil, wheat germ oil, iripe oil, safflower oil, corn oil, milk fat, sheep fat, goat fat, horse fat, egg yolk fat, sardine oil, whale oil, etc. Sorted products, hydrogenated products, transesterified products, etc. can be used. In addition, low molecular weight emulsifiers such as various sucrose fatty acid esters or protein-based high molecular weight emulsifiers derived from soybeans, milk, etc., have no effect on the activity of lipase. Use in combination within the range is preferable because it can improve the stability of emulsification and contribute to stable expression of powder activity.
（リパーゼ活性を有する酵素）
本発明で用いられるリパーゼ活性を有する酵素としては、酵母，糸状菌等の真菌類，細菌，放線菌，動植物等に由来する、広い起源のものが使用できるが、真菌類特に酵母，カビ由来の酵素の粉末活性化には、特に有効に適用できる。具体的には、リゾプス属（Rhizopus sp.），アスペルギルス属（Aspergillus sp.），キャンディダ属（Candida sp.），ムコール属（Mucor sp.），ペニシリウム属（Penicillum sp.）等が好ましく、シュードモナス属（Psudomonas sp.），アルカリゲネス属（Alcaligenes sp.），アスロバクター属（Arthrobacter sp.），スタフィロコッカス属（Staphylococcus sp.），ジオトリカム属（Geotrichum sp.）に加え、膵臓リパーゼや米糠リパーゼ等動植物由来のものも使用することができる。これらのリパーゼ酵素には、基質となるトリグリセライドの１,３位に特異的に作用するもの、位置特異性なく作用するものがあるが、本方法においてはそれら特異性に変化を与えることはないが、トリグリセライドの１,３位に特異的に作用するリパーゼを用いると、本発明が効果的に発現され好ましい。
本発明に於いては、これらリパーゼ活性を有する酵素を製剤化したものであるリパーゼ製剤を用いる場合が一般的であるが、製剤化していない各種の純度のリパーゼ蛋白質を用いることも可能である。(Enzyme having lipase activity)
As the enzyme having lipase activity used in the present invention, those derived from fungi such as yeast and filamentous fungi, bacteria, actinomycetes, animals and plants, and the like can be used, but those derived from fungi, particularly yeasts and molds, can be used. It can be particularly effectively applied to powder activation of enzymes. Specifically, Rhizopus sp., Aspergillus sp., Candida sp., Mucor sp., Penicillium sp., Etc. are preferred, Pseudomonas sp. In addition to the genus (Psudomonas sp.), Alcaligenes sp., Arthrobacter sp., Staphylococcus sp., Geotrichum sp., Pancreatic lipase, rice bran lipase, etc. The thing derived from animals and plants can also be used. Some of these lipase enzymes act specifically at positions 1 and 3 of triglyceride as a substrate, and others act without positional specificity. However, in this method, the specificity is not changed. The use of a lipase that specifically acts on positions 1 and 3 of triglyceride is preferred because the present invention is effectively expressed.
In the present invention, it is common to use a lipase preparation obtained by formulating these enzymes having lipase activity, but it is also possible to use lipase proteins of various purity that are not formulated.
（水中油型乳化物）
リパーゼ活性を有する酵素を含む水中油型乳化物（リパーゼ乳化物）の調製において、乳化工程時にリパーゼ製剤を添加し乳化させても良いし、リパーゼ活性を有しない乳化物を調製し、リパーゼ製剤またはその溶液と混合しても良い。リパーゼを含まない乳化物にリパーゼ製剤を添加する方が、リパーゼを有する酵素分子が乳化処理を受けないために、活性を維持し易く好ましい。水中油型乳化物は、先に説明した乳化性多糖類，油脂，酵素製剤等を含むが、これらの乾燥重量を合計した固形分は、リパーゼ乳化物中の濃度として２重量%以上且つ60重量%以下に調製するのが好ましく、３重量%〜25重量%とするのが更に好ましい。リパーゼ乳化物中の固形分が多いと乳化が安定しにくく、少ないとリパーゼ乳化物の乾燥効率が上がりにくい。(Oil-in-water emulsion)
In the preparation of an oil-in-water emulsion (lipase emulsion) containing an enzyme having lipase activity, a lipase preparation may be added and emulsified during the emulsification step, or an emulsion having no lipase activity may be prepared, You may mix with the solution. It is preferable to add a lipase preparation to an emulsion containing no lipase because the enzyme molecules having the lipase are not subjected to an emulsification treatment, so that the activity is easily maintained. The oil-in-water emulsion includes the above-described emulsifiable polysaccharides, fats and oils, enzyme preparations, etc., and the total solid content of these dry weights is 2% by weight or more and 60% by weight as the concentration in the lipase emulsion. It is preferable to adjust to 3% by weight or less, and more preferably 3 to 25% by weight. If the solid content in the lipase emulsion is large, the emulsification is difficult to stabilize, and if it is small, the drying efficiency of the lipase emulsion is difficult to increase.
またリパーゼ乳化物中の油脂の混合量は、リパーゼ乳化物中のリパーゼ製剤に対し、その１重量%以上混合することが好ましく、６重量%以上混合することがさらに好ましい。また200重量%以下であることが望ましい。リパーゼ製剤に対する油脂量が少ないと本発明の効果が弱く、多いと粉体重量に対する酵素活性が低下する。乳化性多糖類の混合量としては、調製される水中油型乳化物が安定性を保つ範囲であれば特に制限はないが、リパーゼ乳化物中の油脂に対して0.5重量%〜50重量%であると好ましく、５重量%〜40重量%であると更に好ましい。油脂に対する乳化性多糖類が少ないと乳化が安定しくく、多いと粉体重量に対する酵素活性が低下する。 The amount of fats and oils in the lipase emulsion is preferably 1% by weight or more, more preferably 6% by weight or more, based on the lipase preparation in the lipase emulsion. Moreover, it is desirable that it is 200 weight% or less. When the amount of fats and oils for the lipase preparation is small, the effect of the present invention is weak, and when it is large, the enzyme activity with respect to the powder weight is lowered. The mixing amount of the emulsifying polysaccharide is not particularly limited as long as the prepared oil-in-water emulsion maintains stability, but it is 0.5 to 50% by weight with respect to the fats and oils in the lipase emulsion. Preferably, it is 5 to 40% by weight. If there are few emulsifiable polysaccharides with respect to fats and oils, emulsification will be stable, and if there are many, the enzyme activity with respect to a powder weight will fall.
乳化性多糖類と油脂を含む混合液は、適当な乳化処理を施して水中油型乳化物とする。乳化にはホモミキサー，ホモゲナイザーなどの乳化装置や、「ナノマイザー」（ナノマイザー株式会社製）等の超微粒化装置を用いて適切な圧力を設定して行うことができるし、膜乳化や超音波を用いて乳化することもできる。使用する水中油型乳化物は、その平均粒子径を20μm以下にすることが好ましく、5μm以下にすることが、リパーゼ分子が有効に作用することのできる表面積を広げる意味で更に好ましい。また、平均粒子径0.5μm未満への調製は効率が悪く、これ以上の粒子径での使用が一般的である。油脂と乳化性高分子溶液の乳化処理前にリパーゼ製剤を添加するか、乳化処理後の油脂と乳化性高分子溶液の水中油型乳化物にリパーゼ製剤を添加して、リパーゼ活性を有する水中油型乳化物である、リパーゼ乳化物とする。 The mixed liquid containing the emulsifiable polysaccharide and the fat is subjected to an appropriate emulsification treatment to obtain an oil-in-water emulsion. Emulsification can be performed by setting an appropriate pressure using an emulsifier such as a homomixer or homogenizer, or an ultrafine atomizer such as “Nanomizer” (manufactured by Nanomizer Co., Ltd.). And can be emulsified. The oil-in-water emulsion to be used preferably has an average particle size of 20 μm or less, and more preferably 5 μm or less in terms of increasing the surface area on which lipase molecules can effectively act. In addition, preparation to an average particle size of less than 0.5 μm is inefficient, and use with particle sizes larger than this is common. Oil-in-water having lipase activity by adding lipase preparation before emulsification treatment of oil and fat and emulsifiable polymer solution, or adding lipase preparation to oil-in-water emulsion of oil and emulsification polymer solution after emulsification treatment Let it be a lipase emulsion which is a type emulsion.
（乾燥）
上記に記載したリパーゼ活性を有する酵素を含む水中油型乳化物、すなわちリパーゼ乳化物を乾燥させたものが、本発明である乳化粉末リパーゼ製剤である。乾燥前に、リパーゼ乳化物を、リパーゼが活性を発揮できる温度域で、30分から12時間ほど保持することで、より高い活性を得ることができる。乾燥方法としては、スプレードライヤーや凍結乾燥機，真空乾燥機などを用いるのが良い。スプレードライは、例えば、ノズル向流式，ノズル 並流式，ディスク並流式等の噴霧乾燥機を用いて行うのがよい。噴霧乾燥の乾燥条件としては、例えば熱風温度100℃〜185℃、排風温度40℃〜100℃にて行うことができる。凍結乾燥機は、例えば棚段式凍結乾燥機を使用するのが良い。また、真空乾燥においては、リパーゼ乳化物を有機溶剤にて沈殿させ、有機溶剤と共に棚式もしくはドラム式の真空乾燥を行うのが良い。得られる乳化粉末リパーゼ製剤の水分含量は、10重量%以下に抑えるのが好ましく、特に水分の低い系で使用する場合には水分５重量%以下等、さらに低水分まで乾燥させることが好ましい。(Dry)
The oil-in-water emulsion containing the enzyme having the lipase activity described above, that is, the lipase emulsion dried is the emulsified powder lipase preparation of the present invention. Higher activity can be obtained by holding the lipase emulsion for about 30 minutes to 12 hours in a temperature range where the lipase can exhibit activity before drying. As a drying method, a spray dryer, a freeze dryer, a vacuum dryer or the like is preferably used. Spray drying is preferably performed using, for example, a spray dryer such as a nozzle countercurrent type, a nozzle cocurrent type, or a disk cocurrent type. As drying conditions for spray drying, for example, a hot air temperature of 100 ° C to 185 ° C and an exhaust air temperature of 40 ° C to 100 ° C can be used. As the freeze dryer, for example, a shelf type freeze dryer may be used. In vacuum drying, it is preferable to precipitate the lipase emulsion with an organic solvent and perform shelf-type or drum-type vacuum drying together with the organic solvent. The water content of the obtained emulsified powder lipase preparation is preferably suppressed to 10% by weight or less, and when used in a system with low water content, it is preferable to further dry to a low water content such as 5% by weight or less.
この様に調製された乳化粉末リパーゼ製剤は、高いエステル交換活性を維持しているために、エステル交換反応やエステル合成反応を行う事で、油脂の改質等に用いることができる。その形態は、バッチ方式やカラムを用いた連続方式等、種々の方法に用いることができる。バッチ方式としては、例えば攪拌機付きのタンクにて目的の基質油脂に本発明の乳化粉末リパーゼ製剤を添加し、所定の反応率に達した反応液を遠心分離機や濾過機などで分離する方法が良い。連続式としては、乳化粉末リパーゼ製剤を充填した反応カラムや反応容器に、濾過機や連続式の遠心分離機を組み合わせることで、単カラム反応や多段カラム反応をするのが良い。 Since the emulsified powder lipase preparation thus prepared maintains a high transesterification activity, it can be used for the modification of fats and oils by performing the transesterification reaction or the ester synthesis reaction. The form can be used for various methods such as a batch method and a continuous method using a column. As a batch system, for example, there is a method in which the emulsified powder lipase preparation of the present invention is added to the target substrate fat in a tank equipped with a stirrer, and the reaction solution reaching a predetermined reaction rate is separated by a centrifuge or a filter. good. As a continuous type, it is preferable to perform a single column reaction or a multistage column reaction by combining a reaction column or reaction vessel filled with an emulsified powder lipase preparation with a filter or a continuous centrifuge.
以下に実施例を記載することで本発明を説明する。 The invention is illustrated by the following examples.
（実施例１）
（水溶性大豆多糖類を用いた１,３位特異的酵素粉末の調製）
分離大豆蛋白製造工程において得られた生オカラに２倍量の水を加え、塩酸にてpHを3.0に調整し、120℃ で1.5時間加熱抽出した。冷却後の加熱抽出スラリーpHは2.98であった。回収したスラリーのpHを5.0に調整した後に遠心分離し（10,000×g, 30分）、上澄と沈澱部に分離した。こうして分離した沈澱部を更に等重量の水で水洗し、遠心分離し、この上澄を先の上澄と一緒にしてから電気透析による脱塩処理を行い、その後に乾燥して水溶性大豆多糖類αを得た。水95gに水溶性大豆多糖類α１gを分散させ、これに精製パーム油４gを加え、ホモミキサー（特殊機化工業社製）で予備乳化し、その後高圧ホモゲナイザー（APV社製）にて150psiにて通液し乳化させ、水中油型乳化物Ａを調製した。乳化物の粒子径は、レーザー回折式粒度分布計（島津製作所製SALD2200）を用いて測定し、平均粒子径は1.8μmであった。天野製薬製市販粉末リパーゼ製剤（リゾプス起源、加水分解活性10,000unit/g、１,３位特異的）５gを氷冷したイオン交換水95gに分散，溶解させた。この酵素溶液100gと乳化物Ａ ２gを混合し、乳化物混合酵素液（リパーゼ乳化物）を得た。この混合液を、東京理化器械製スプレードライヤーSD1000にて、熱風温度90〜100℃、排風温度60〜80℃、排気流量0.77〜0.88ｍ3/hr，噴霧圧力11kpaにて、噴霧乾燥し、水分５重量%の乳化粉末リパーゼ製剤を得た。得られた乳化粉末リパーゼ製剤を、以下の方法にて、１,３位特異的エステル交換活性を評価した。Example 1
(Preparation of 1,3-position specific enzyme powder using water-soluble soybean polysaccharide)
Two times the amount of water was added to the raw okara obtained in the process of producing isolated soybean protein, the pH was adjusted to 3.0 with hydrochloric acid, and the mixture was extracted by heating at 120 ° C for 1.5 hours. The heated extraction slurry pH after cooling was 2.98. The pH of the recovered slurry was adjusted to 5.0 and then centrifuged (10,000 × g, 30 minutes) to separate the supernatant and the precipitate. The separated precipitate is further washed with an equal weight of water, centrifuged, and the supernatant is combined with the previous supernatant, followed by desalting by electrodialysis, followed by drying to remove water-soluble soybean rich Saccharide α was obtained. Disperse α1g of water-soluble soybean polysaccharide in 95g of water, add 4g of refined palm oil, pre-emulsify with homomixer (made by Tokushu Kika Kogyo Co., Ltd.), and then use high-pressure homogenizer (made by APV) at 150psi The mixture was passed through and emulsified to prepare an oil-in-water emulsion A. The particle size of the emulsion was measured using a laser diffraction particle size distribution analyzer (SALD2200, manufactured by Shimadzu Corporation), and the average particle size was 1.8 μm. 5 g of Amano Pharmaceutical's commercially available powder lipase preparation (Rhizopus origin, hydrolysis activity 10,000 unit / g, specific for position 1 and 3) was dispersed and dissolved in 95 g of ice-cooled ion-exchanged water. 100 g of this enzyme solution and 2 g of Emulsion A were mixed to obtain an emulsion mixed enzyme solution (lipase emulsion). This mixture is spray dried with a spray dryer SD1000 manufactured by Tokyo Rika Instrument Co., Ltd. at a hot air temperature of 90-100 ° C., an exhaust air temperature of 60-80 ° C., an exhaust flow rate of 0.77-0.88 m 3 / hr, and a spray pressure of 11 kpa. An emulsified powder lipase preparation having a water content of 5% by weight was obtained. The emulsified powder lipase preparation thus obtained was evaluated for 1,3-position specific transesterification activity by the following method.
（１,３位特異的エステル交換活性測定方法）
反応基質として、パーム中融点画分とステアリン酸エチルを１：１に配合したものを用いた（基質水分＜0.1%）。本基質100gに１gの各粉末リパーゼ製剤を加え、40℃にて反応した。反応物を24時間後にサンプリングし、ヘキサンにて希釈したものをGC（ガスクロマトグラフィー）を用い、全脂肪酸エチルエステル中のC16エチルエステルの量を分析し、以下の式によりエステル交換活性を求めた。
１．反応率＝（反応物のC16:0Et含量−反応基質のC16:0Et含量）/（反応平衡物のC16:0Et含量−反応基質のC16:0Et含量）（Et：エチルエステル）
２．初期反応速度定数k＝ln[1/（１−反応24時間後の反応率）]
３．エステル交換活性値＝k×（基質油脂量/粉末リパーゼ製剤）×100(Method for measuring transesterification activity specific to 1,3-position)
As a reaction substrate, a mixture of a palm melting point fraction and ethyl stearate 1: 1 was used (substrate moisture <0.1%). 1 g of each powdered lipase preparation was added to 100 g of this substrate and reacted at 40 ° C. The reaction product was sampled 24 hours later, and diluted with hexane to analyze the amount of C16 ethyl ester in the total fatty acid ethyl ester using GC (gas chromatography), and the transesterification activity was determined by the following formula. .
1. Reaction rate = (C16: 0Et content of reaction product−C16: 0Et content of reaction substrate) / (C16: 0Et content of reaction equilibrium product−C16: 0Et content of reaction substrate) (Et: ethyl ester)
2. Initial reaction rate constant k = ln [1 / (1-reaction rate after 24 hours of reaction)]
3. Transesterification activity value = k × (Substance oil / fat amount / powder lipase preparation) × 100
（実施例２）
実施例１において乳化物Ａの混合量を５gとした以外は、実施例１と同様に粉末酵素を調製し、水分５重量%の乳化粉末リパーゼ製剤を得た。得られた乳化粉末リパーゼ製剤は、実施例１と同様に１,３位特異的エステル交換活性を評価した。(Example 2)
A powder enzyme was prepared in the same manner as in Example 1 except that the amount of Emulsion A mixed in Example 1 was changed to 5 g to obtain an emulsified powder lipase preparation having a water content of 5% by weight. The obtained emulsified powder lipase preparation was evaluated for 1,3-position specific transesterification activity in the same manner as in Example 1.
（実施例３）
実施例１において乳化物Ａの混合量を10gとした以外は、実施例１と同様に粉末酵素を調製し、水分4.6重量%の乳化粉末リパーゼ製剤を得た。得られた乳化粉末リパーゼ製剤は、実施例１と同様に１,３位特異的エステル交換活性を評価した。(Example 3)
A powder enzyme was prepared in the same manner as in Example 1 except that the amount of Emulsion A in Example 1 was changed to 10 g to obtain an emulsified powder lipase preparation having a water content of 4.6% by weight. The obtained emulsified powder lipase preparation was evaluated for 1,3-position specific transesterification activity in the same manner as in Example 1.
（実施例４）
実施例１において乳化物Ａの混合量を50gとした以外は、実施例１と同様に粉末酵素を調製し、水分3.0重量%の乳化粉末リパーゼ製剤を得た。得られた乳化粉末リパーゼは、実施例１と同様に１,３位特異的エステル交換活性を評価した。Example 4
A powder enzyme was prepared in the same manner as in Example 1 except that the amount of Emulsion A in Example 1 was changed to 50 g to obtain an emulsified powder lipase preparation having a water content of 3.0% by weight. The obtained emulsified powder lipase was evaluated for the 1,3-position specific transesterification activity in the same manner as in Example 1.
（実施例５）
実施例１において乳化物Ａの混合量を100gとした以外は、実施例１と同様に粉末酵素を調製し、水分3.0重量%の乳化粉末リパーゼ製剤を得た。得られた乳化粉末リパーゼは、実施例１と同様に１,３位特異的エステル交換活性を評価した。(Example 5)
A powder enzyme was prepared in the same manner as in Example 1 except that the amount of Emulsion A mixed in Example 1 was 100 g to obtain an emulsified powder lipase preparation having a water content of 3.0% by weight. The obtained emulsified powder lipase was evaluated for the 1,3-position specific transesterification activity in the same manner as in Example 1.
（比較例１）
（油脂無添加）
天野製薬製市販粉末リパーゼ製剤（リゾプス起源、加水分解活性10,000unit/g、１,３位特異的）５gを氷冷したイオン交換水95gに分散，溶解させた。この溶液を凍結乾燥し、粉末リパーゼ製剤を得た。得られた粉末リパーゼ製剤は実施例１と同様に１,３位特異的エステル交換活性を評価した。(Comparative Example 1)
(No fat added)
5 g of Amano Pharmaceutical's commercially available powder lipase preparation (Rhizopus origin, hydrolysis activity 10,000 unit / g, specific for position 1 and 3) was dispersed and dissolved in 95 g of ice-cooled ion-exchanged water. This solution was freeze-dried to obtain a powder lipase preparation. The obtained powder lipase preparation was evaluated for 1,3-position specific transesterification activity in the same manner as in Example 1.
（表１）各粉末リパーゼ製剤のエステル交換活性（１,３位特異的）
実施例１〜５および比較例１で調製した各粉末リパーゼ製剤のエステル交換活性を表１に示した。比較例に比べ、実施例は全てでエステル交換活性が上昇している。特に酵素製剤に対して油脂を８重量%以上加えた場合に於いて、その効果は高いことが判った。 Table 1 shows the transesterification activity of each powder lipase preparation prepared in Examples 1 to 5 and Comparative Example 1. Compared with the comparative example, the transesterification activity is increased in all of the examples. In particular, when oils and fats were added to the enzyme preparation in an amount of 8% by weight or more, it was found that the effect was high.
（実施例６）
（オクテニルコハク酸澱粉による調製）
水95gにオクテニルコハク酸澱粉（エマルスター、松谷化学工業製）１gを分散させ、精製パーム油４gを加え実施例１と同様の方法で乳化物を調製した。得られた乳化物の平均粒子径は2.0μmであった。その後この乳化物100gに、実施例１と同様に調製した酵素溶液100gを混合し、リパーゼ乳化物を得た。実施例１同様に乳化粉末リパーゼ製剤を調製し、１,３位特異的エステル交換活性を評価した。(Example 6)
(Preparation with octenyl succinate starch)
1 g of octenyl succinic acid starch (Emulstar, manufactured by Matsutani Chemical Industry) was dispersed in 95 g of water, 4 g of purified palm oil was added, and an emulsion was prepared in the same manner as in Example 1. The average particle size of the obtained emulsion was 2.0 μm. Thereafter, 100 g of the enzyme solution prepared in the same manner as in Example 1 was mixed with 100 g of this emulsion to obtain a lipase emulsion. An emulsified powder lipase preparation was prepared in the same manner as in Example 1, and the 1,3-position specific transesterification activity was evaluated.
（実施例７）
（アラビアガムによる調製）
乳化物の調製において、オクテニルコハク酸澱粉の代わりにアラビアガム（ワコー純薬製試薬）を使用し、実施例６と同様に調製を行った。得られた乳化物の平均粒子径は1.7μmであった。この乳化物を使用して、実施例６と同様の方法で乳化粉末リパーゼ製剤を調製し、同様に評価を行った。(Example 7)
(Preparation with gum arabic)
In the preparation of the emulsion, gum arabic (a reagent manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of octenyl succinate starch, and the preparation was performed in the same manner as in Example 6. The average particle size of the obtained emulsion was 1.7 μm. Using this emulsion, an emulsified powder lipase preparation was prepared in the same manner as in Example 6 and evaluated in the same manner.
（比較例２）
（分離大豆蛋白質による調製）
乳化物の調製において、オクテニルコハク酸澱粉の代わりに分離大豆蛋白質（フジプロＲ：不二製油製）を使用し、実施例６と同様に調製を行った。得られた乳化物の平均粒子径は2.3μmであった。この乳化物を使用して、実施例６と同様の方法で乳化粉末リパーゼ製剤を調製し、同様に評価を行った。(Comparative Example 2)
(Preparation with isolated soy protein)
In the preparation of an emulsion, preparation was carried out in the same manner as in Example 6 using separated soybean protein (Fujipro R: manufactured by Fuji Oil Co., Ltd.) instead of octenyl succinate starch. The average particle diameter of the obtained emulsion was 2.3 μm. Using this emulsion, an emulsified powder lipase preparation was prepared in the same manner as in Example 6 and evaluated in the same manner.
（比較例３）
（ショ糖脂肪酸エステルによる調製１）
乳化物の調製において、オクテニルコハク酸澱粉の代わりに市販ショ糖脂肪酸エステル（F110 ：第一工業製薬製）を使用し、実施例６と同様に行った。得られた乳化物の平均粒子径は1.8μmであった。この乳化物を使用して、実施例６と同様の方法で乳化粉末リパーゼ製剤を調製し、同様に評価を行った。(Comparative Example 3)
(Preparation with sucrose fatty acid ester 1)
In the preparation of the emulsion, a commercially available sucrose fatty acid ester (F110: manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) was used instead of octenyl succinate starch, and the same procedure as in Example 6 was performed. The average particle size of the obtained emulsion was 1.8 μm. Using this emulsion, an emulsified powder lipase preparation was prepared in the same manner as in Example 6 and evaluated in the same manner.
（比較例４）
（ショ糖脂肪酸エステルによる調製２）
乳化物の調製において、オクテニルコハク酸澱粉の代わりに市販ショ糖脂肪酸エステル（F160 ：第一工業製薬製）を使用し、実施例６と同様に行った。得られた乳化物の平均粒子径は1.9μmであった。この乳化物を使用して、実施例６と同様の方法で乳化粉末リパーゼ製剤を調製し、同様に評価を行った。(Comparative Example 4)
(Preparation with sucrose fatty acid ester 2)
In the preparation of the emulsion, a commercially available sucrose fatty acid ester (F160: manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) was used instead of octenyl succinate starch, and the same procedure as in Example 6 was performed. The average particle size of the obtained emulsion was 1.9 μm. Using this emulsion, an emulsified powder lipase preparation was prepared in the same manner as in Example 6 and evaluated in the same manner.
（表２）各粉末リパーゼ製剤のエステル交換活性（１,３位特異的）
実施例５〜７および比較例２〜４で調製した各粉末リパーゼ製剤のエステル交換活性を表２に示した。乳化素材が低分子乳化剤または蛋白質である比較例に比べ、乳化性多糖類である実施例は全てでエステル交換活性が上昇していた。 Table 2 shows the transesterification activity of each powder lipase preparation prepared in Examples 5 to 7 and Comparative Examples 2 to 4. Compared with the comparative example whose emulsification raw material is a low molecular emulsifier or protein, all the examples which are emulsifiable polysaccharides had increased transesterification activity.
（実施例８）
（水溶性大豆多糖類を用いた非特異的酵素粉末の調製）
水95gに水溶性大豆多糖類α １gを分散させ、これに精製パーム油４gを加え、ホモミキサー（特殊機化工業社製）で予備乳化し、その後高圧ホモゲナイザー（APV社製）にて150psiにて通液し乳化させ、水中油型乳化物Ｂを調製した。得られた乳化物の平均粒子径は1.9μmであった。天野製薬製市販粉末リパーゼ製剤（ペニシリウム起源、加水分解活性1,000unit/g、非選択的分解特性）５gを氷冷したイオン交換水95gに分散、溶解させた。この酵素溶液100gと乳化物Ｂ100gを混合し、乳化物混合酵素液（リパーゼ乳化物）を得た。この混合液を凍結乾燥し水分２重量%の乳化粉末リパーゼ製剤を得た。(Example 8)
(Preparation of non-specific enzyme powder using water-soluble soybean polysaccharide)
Disperse 1 g of water-soluble soybean polysaccharide α in 95 g of water, add 4 g of purified palm oil to this, pre-emulsify with homomixer (made by Tokushu Kika Kogyo Co., Ltd.), and then adjust to 150 psi with high-pressure homogenizer (made by APV) And then emulsified to prepare an oil-in-water emulsion B. The average particle size of the obtained emulsion was 1.9 μm. 5 g of Amano Pharmaceutical's commercially available powder lipase formulation (originating from Penicillium, hydrolytic activity 1,000 unit / g, non-selective degradation characteristics) was dispersed and dissolved in 95 g of ice-cooled ion-exchanged water. 100 g of this enzyme solution and 100 g of emulsion B were mixed to obtain an emulsion mixed enzyme solution (lipase emulsion). This mixed solution was freeze-dried to obtain an emulsified powder lipase preparation having a water content of 2% by weight.
（非特異的酵素の活性測定方法）
反応基質として、パーム中融点画分とパーム低融点画分を６：４に配合したものを用いた（基質水分＜0.1%）。本基質100gに、１gの各粉末リパーゼ製剤を加え、60℃にて反応した。反応物を24時間後にサンプリングし、ヘキサンにて希釈したものをトリグリセリド分析用GC（ガスクロマトグラフィー）を用い、構成脂肪酸の合計が48である、C48のトリグリセリドを分析し、全トリグリセリド中のC48相対含量から、以下の式によりエステル交換活性を求めた。
１．反応率＝（反応物のC48含量‐反応基質のC48含量）/（反応平衡物のC48含量‐反応基質のC48含量）
２．初期反応速度定数k＝ln[1/（１−反応24時間後の反応率）]
３．エステル交換活性値＝k×（基質油脂量/粉末リパーゼ製剤）×10(Non-specific enzyme activity measurement method)
As a reaction substrate, a 6: 4 mixture of a palm melting point fraction and a palm low melting point fraction was used (substrate moisture <0.1%). To 100 g of this substrate, 1 g of each powder lipase preparation was added and reacted at 60 ° C. The reaction product was sampled 24 hours later, diluted with hexane, and analyzed using triglyceride analysis GC (gas chromatography) for the analysis of C48 triglycerides with a total of 48 constituent fatty acids. The transesterification activity was determined from the content according to the following formula.
1. Reaction rate = (C48 content of reactant-C48 content of reaction substrate) / (C48 content of reaction equilibrium-C48 content of reaction substrate)
2. Initial reaction rate constant k = ln [1 / (1-reaction rate after 24 hours of reaction)]
3. Transesterification activity value = k × (Substance oil / fat amount / powder lipase preparation) × 10
（実施例９）
（アラビアガムによる調製）
乳化物の調製において、水溶性大豆多糖類αの代わりにアラビアガム（ワコー純薬製試薬）を使用し、実施例８と同様に調製を行った。得られた乳化物の平均粒子径は2.0μmであった。この乳化物を使用して、実施例８と同様の方法で乳化粉末リパーゼ製剤を調製し、同様に評価を行った。Example 9
(Preparation with gum arabic)
In the preparation of the emulsion, gum arabic (Wako Pure Chemicals Reagent) was used instead of the water-soluble soybean polysaccharide α, and the preparation was performed in the same manner as in Example 8. The average particle size of the obtained emulsion was 2.0 μm. Using this emulsion, an emulsified powder lipase preparation was prepared in the same manner as in Example 8, and evaluated in the same manner.
（比較例５）
（ショ糖脂肪酸エステルによる調製）
乳化物の調製において、水溶性大豆多糖類αの代わりに市販ショ糖脂肪酸エステル（F160：第一工業製薬製）を使用し、実施例８と同様に調製を行った。得られた乳化物の平均粒子径は2.4μmであったこの乳化物を使用して、実施例８と同様の方法で乳化粉末リパーゼ製剤を調製し、同様に評価を行った。(Comparative Example 5)
(Preparation with sucrose fatty acid ester)
In the preparation of the emulsion, a commercially available sucrose fatty acid ester (F160: manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) was used instead of the water-soluble soybean polysaccharide α, and the preparation was carried out in the same manner as in Example 8. An emulsion powder lipase preparation was prepared in the same manner as in Example 8 using this emulsion having an average particle size of 2.4 μm, and evaluated in the same manner.
（比較例６）
（牛乳を用いた調製）
実施例８における乳化物として、市販牛乳（総固形分12.7重量%，油分3.0重量%）を用いた。すなわち、実施例８の酵素溶液100g，市販牛乳39.4gおよび水60.6gを混合し、混合乳化物酵素液を得た。得られた乳化物混合酵素を実施例８と同様の方法で乾燥して乳化粉末リパーゼ製剤を調製し、同様に評価を行った。(Comparative Example 6)
(Preparation using milk)
As the emulsion in Example 8, commercially available milk (total solid content 12.7% by weight, oil content 3.0% by weight) was used. That is, 100 g of the enzyme solution of Example 8, 39.4 g of commercial milk, and 60.6 g of water were mixed to obtain a mixed emulsion enzyme solution. The obtained emulsion mixed enzyme was dried in the same manner as in Example 8 to prepare an emulsified powder lipase preparation, and evaluated in the same manner.
（比較例１０）
（油脂無添加）
天野製薬製市販粉末リパーゼ製剤（ペニシリウム起源、加水分解活性1,000unit/g、非特異的）５gを氷冷したイオン交換水95gに分散、溶解させた。この溶液を凍結乾燥し、実施例８と同様に活性評価を行った。(Comparative Example 10)
(No fat added)
5 g of Amano Pharmaceutical's commercially available powder lipase preparation (originating from Penicillium, hydrolytic activity 1,000 unit / g, non-specific) was dispersed and dissolved in 95 g of ion-exchanged water cooled with ice. This solution was freeze-dried and the activity was evaluated in the same manner as in Example 8.
（表３）各粉末リパーゼ製剤のエステル交換活性（非特異的）
実施例８〜９および比較例５〜７で調製した各粉末リパーゼ製剤のエステル交換活性を表３に示した。非特異的リパーゼについても、低分子乳化剤および蛋白質である比較例に比べ、乳化性多糖類である実施例はエステル交換活性が上昇していた。また、低分子乳化剤に比較した活性は、１,３特異的酵素の場合より若干少ないものだった。 Table 3 shows the transesterification activity of each powder lipase preparation prepared in Examples 8 to 9 and Comparative Examples 5 to 7. Regarding non-specific lipase, the transesterification activity was higher in the example of the emulsifying polysaccharide than in the comparative example of the low molecular weight emulsifier and the protein. The activity compared to the low molecular emulsifier was slightly less than that of the 1,3-specific enzyme.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09322770A (en) * | 1996-05-31 | 1997-12-16 | Ichibiki Kk | Exogenous enzyme composition stably dispersed in oil and its production and usage |
| JP2000106873A (en) * | 1998-10-06 | 2000-04-18 | Nisshin Oil Mills Ltd:The | Thermally stable enzyme and production thereof |
| JP2007068426A (en) * | 2005-09-05 | 2007-03-22 | Nisshin Oillio Group Ltd | Lipase powder preparation, method for producing the same and use of the same |
| JP2007534314A (en) * | 2004-01-28 | 2007-11-29 | シーエスアイアール | Enzyme stabilization |
| WO2008114656A1 (en) * | 2007-03-16 | 2008-09-25 | The Nisshin Oillio Group, Ltd. | Powdery lipase preparation, method for production thereof, and use thereof |
| WO2009017087A1 (en) * | 2007-07-31 | 2009-02-05 | Fuji Oil Company, Limited | Immobilized lipase and method for producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09322770A (en) * | 1996-05-31 | 1997-12-16 | Ichibiki Kk | Exogenous enzyme composition stably dispersed in oil and its production and usage |
| JP2000106873A (en) * | 1998-10-06 | 2000-04-18 | Nisshin Oil Mills Ltd:The | Thermally stable enzyme and production thereof |
| JP2007534314A (en) * | 2004-01-28 | 2007-11-29 | シーエスアイアール | Enzyme stabilization |
| JP2007068426A (en) * | 2005-09-05 | 2007-03-22 | Nisshin Oillio Group Ltd | Lipase powder preparation, method for producing the same and use of the same |
| WO2008114656A1 (en) * | 2007-03-16 | 2008-09-25 | The Nisshin Oillio Group, Ltd. | Powdery lipase preparation, method for production thereof, and use thereof |
| WO2009017087A1 (en) * | 2007-07-31 | 2009-02-05 | Fuji Oil Company, Limited | Immobilized lipase and method for producing the same |
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| WO2010089967A1 (en) | 2010-08-12 |
| JPWO2010089967A1 (en) | 2012-08-09 |
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