JP5858686B2 - Process for producing sugar adducts of poorly water-soluble polyphenols - Google Patents
Process for producing sugar adducts of poorly water-soluble polyphenols Download PDFInfo
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- JP5858686B2 JP5858686B2 JP2011183331A JP2011183331A JP5858686B2 JP 5858686 B2 JP5858686 B2 JP 5858686B2 JP 2011183331 A JP2011183331 A JP 2011183331A JP 2011183331 A JP2011183331 A JP 2011183331A JP 5858686 B2 JP5858686 B2 JP 5858686B2
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- Prior art keywords
- poorly water
- sugar
- soluble
- heat treatment
- soluble polyphenols
- Prior art date
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- 150000008442 polyphenolic compounds Chemical class 0.000 title claims description 84
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- 238000000034 method Methods 0.000 title claims description 31
- 238000010438 heat treatment Methods 0.000 claims description 53
- 239000007788 liquid Substances 0.000 claims description 28
- 238000005918 transglycosylation reaction Methods 0.000 claims description 25
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- OVVGHDNPYGTYIT-BNXXONSGSA-N rutinose Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 OVVGHDNPYGTYIT-BNXXONSGSA-N 0.000 description 1
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Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、難水溶性ポリフェノール類の糖付加物を製造する方法に関する。 The present invention relates to a method for producing a sugar adduct of poorly water-soluble polyphenols.
昨今、生理機能を有する様々な素材が提案され、これらを含有する数多くの健康食品が上市されている。なかでも、ポリフェノールは、抗酸化力を有することが知られており、抗動脈硬化、抗アレルギー、血流増強等の効果が期待されるため、健康食品の重要な成分として認識されている。
しかしながら、ポリフェノールには難水溶性のものが多く、それらを清涼飲料等の水性食品へ使用することは難しい。
Recently, various materials having physiological functions have been proposed, and many health foods containing these have been put on the market. Among them, polyphenols are known to have an antioxidant power and are expected to have effects such as anti-arteriosclerosis, anti-allergy and blood flow enhancement, and thus are recognized as important ingredients in health foods.
However, many polyphenols have poor water solubility, and it is difficult to use them for aqueous foods such as soft drinks.
そこで、難水溶性のポリフェノールを水に可溶化させる技術が検討され、ポリフェノールにグルコース等の糖を付加させた糖付加物が提案されている。例えば、ヘスペリジンの２５℃における水への溶解度は僅かに０．０２ｍｇ／ｇであるが、ヘスペリジンにグルコースを結合させたα-グルコシルヘスペリジンでは、２５℃の水への溶解度が２００ｍｇ／ｇ以上と高くなる。更に、ポリフェノールの糖付加物は、ポリフェノールと同等の機能を発揮する等の利点がある。 Therefore, a technique for solubilizing poorly water-soluble polyphenols in water has been studied, and sugar adducts obtained by adding sugars such as glucose to polyphenols have been proposed. For example, the solubility of hesperidin in water at 25 ° C. is only 0.02 mg / g, but α-glucosyl hesperidin in which glucose is bound to hesperidin has a high solubility in water at 25 ° C. of 200 mg / g or more. Become. Furthermore, the sugar addition product of polyphenol has advantages such as exhibiting the same function as polyphenol.
ポリフェノールの糖付加物を製造する方法としては、例えば、懸濁状もしくはｐＨ７．０を超えるアルカリ側ｐＨで溶解させた溶液状のルチン又はヘスペリジンと澱粉部分加水分解物等を含有する溶液に糖転移酵素を作用させる方法（特許文献１、２）、フラボノイドをｐＨ８〜１０に調整した増粘多糖類溶液に溶解させ、サイクロデキストリン合成酵素を作用させるフラボノイド糖転移法（特許文献３）、フラボノイド類をｐＨ８以上のアルカリ域であるいは／およびサイクロデキストリンを加えて可溶化し、さらにアルカリ域でサイクロデキストリン合成酵素を作用させるフラボノイド配糖体の製造方法（特許文献４）等が報告されている。 As a method for producing a sugar addition product of polyphenol, for example, sugar transfer to a solution containing rutin or hesperidin in a suspended state or dissolved at an alkaline pH exceeding 7.0, and a partial hydrolyzate of starch, etc. A method of allowing an enzyme to act (Patent Documents 1 and 2), a flavonoid glycosyl transfer method (Patent Document 3) in which a flavonoid is dissolved in a thickened polysaccharide solution adjusted to pH 8 to 10 and a cyclodextrin synthase is allowed to act; There has been reported a flavonoid glycoside production method (Patent Document 4) in which the solubilization is performed in an alkaline region of pH 8 or higher and / or cyclodextrin is added, and a cyclodextrin synthase is allowed to act in the alkaline region.
しかしながら、特許文献１〜４のように、ポリフェノールをアルカリ性の水溶液に溶解した後、糖転移酵素と反応させる方法は、アルカリ域でのポリフェノールの安定性が低く分解され易い上に、アルカリの中和と脱塩の工程が必要で、製造プロセスの煩雑化が懸念される。また、特許文献１及び２のように、難水溶性のポリフェノールを懸濁した溶液に糖転移酵素を作用させる方法は、糖転移反応液中のポリフェノールの溶解量が少ないため十分な転化率が得られない場合がある。
したがって、本発明の課題は、難水溶性ポリフェノール類からその糖付加物を効率よく製造する方法を提供することにある。
However, as disclosed in Patent Documents 1 to 4, the method of reacting with a glycosyltransferase after dissolving polyphenol in an alkaline aqueous solution is low in stability of the polyphenol in the alkaline region and easily decomposed, and also neutralizes the alkali. And a desalting step is necessary, and there is a concern that the manufacturing process may become complicated. In addition, as in Patent Documents 1 and 2, the method of allowing glycosyltransferase to act on a solution in which a poorly water-soluble polyphenol is suspended has a sufficient conversion rate because the amount of polyphenol dissolved in the transglycosylation reaction solution is small. It may not be possible.
Accordingly, an object of the present invention is to provide a method for efficiently producing a sugar adduct from poorly water-soluble polyphenols.
本発明者らは、上記課題に鑑み鋭意検討したところ、難水溶性ポリフェノール類を特定の温度範囲で加熱処理した後に、特定の時間内に糖転移酵素を作用させると、難水溶性ポリフェノールの溶解濃度を大幅に高め、かつ高濃度を維持したまま糖転移反応が進行し、水溶性に優れたポリフェノール糖付加物を高収率で得られることを見出した。 The inventors of the present invention have intensively studied in view of the above-mentioned problems. After heat-treating poorly water-soluble polyphenols in a specific temperature range, if a glycosyltransferase is allowed to act within a specific time, dissolution of the poorly water-soluble polyphenols The present inventors have found that the transglycosylation reaction proceeds while maintaining the high concentration significantly while increasing the concentration, and a polyphenol sugar adduct excellent in water solubility can be obtained in a high yield.
すなわち、本発明は、次の工程（１）及び（２）：
（１）水性媒体の存在下、難水溶性ポリフェノール類（Ａ）を１００〜１８０℃で加熱処理し、加熱処理液を得る工程、
（２）得られた加熱処理液に糖供与体（Ｂ）及び糖転移酵素を添加し、加熱処理後３００分以内に糖転移反応を開始し、難水溶性ポリフェノール類（Ａ）の糖付加物を得る工程、
を含む難水溶性ポリフェノール類の糖付加物の製造方法を提供するものである。
That is, the present invention includes the following steps (1) and (2):
(1) A step of heat-treating the poorly water-soluble polyphenols (A) at 100 to 180 ° C. in the presence of an aqueous medium to obtain a heat-treated liquid,
(2) A sugar donor (B) and a glycosyltransferase are added to the obtained heat-treated liquid, and a sugar-transfer reaction is started within 300 minutes after the heat treatment, whereby a sugar adduct of a poorly water-soluble polyphenol (A) Obtaining a step,
The present invention provides a method for producing a sugar adduct of a poorly water-soluble polyphenol comprising
本発明によれば、糖転移反応の転化率を向上させることができ、高収率で溶解性に優れるポリフェノール類の糖付加物を製造することができる。 ADVANTAGE OF THE INVENTION According to this invention, the conversion rate of a transglycosylation reaction can be improved, and the sugar addition product of polyphenols which is excellent in solubility with a high yield can be manufactured.
本発明の方法における工程（１）は、水性媒体の存在下、難水溶性ポリフェノール類（Ａ）を１００〜１８０℃で加熱処理する工程である。
本明細書において「難水溶性ポリフェノール類」とは、ｌｏｇＰ値が−１．０〜４．０のポリフェノール類を云う。難水溶性ポリフェノール類は、ｌｏｇＰ値が−０．５〜３．５のものが好ましい。ｌｏｇＰ値は、１−オクタノール／水間の分配係数の常用対数をとった値で、有機化合物の疎水性を示す指標である。この値が正に大きい程疎水性が高いことを表す。ポリフェノールのｌｏｇＰ値は、日本工業規格 Ｚ７２６０−１０７記載のフラスコ振盪法により測定できる。詳細は実施例に記載した。
Step (1) in the method of the present invention is a step of heat-treating the poorly water-soluble polyphenols (A) at 100 to 180 ° C. in the presence of an aqueous medium.
In the present specification, “poorly water-soluble polyphenols” refers to polyphenols having a log P value of −1.0 to 4.0. The slightly water-soluble polyphenols preferably have a log P value of -0.5 to 3.5. The log P value is a value obtained by taking the common logarithm of the distribution coefficient between 1-octanol / water and is an index indicating the hydrophobicity of an organic compound. The larger the value, the higher the hydrophobicity. The log P value of polyphenol can be measured by a flask shaking method described in Japanese Industrial Standard Z7260-107. Details are described in the examples.
難水溶性ポリフェノール類（Ａ）としては、ベンゼン環にヒドロキシル基が１個以上、更に２個以上結合したフェノール性物質が好ましく適用できる。例えば、植物由来のフラボノイド、タンニン、フェノール酸等が挙げられる。より好ましく適用できる難水溶性ポリフェノール類としては、フラボノール類、フラバノン類、フラボン類、イソフラボン類、フェノールカルボン酸類等が挙げられる。
具体的には、ルチン、ケルシトリン、イソケルシトリン、ケルセチン、ミリシトリン、ダイゼイン、ダイジン、グリシテイン、グリシチン、ゲニステイン、ゲニスチン、ミリセチン、ヘスペリジン、ネオヘスペリジン、ヘスペレチン、ナリンギン、クルクミン、リンゲニン、プルニン、アストラガリン、ケンフェロール、レスベラトロール、アピイン、アピゲニン、デルフィニジン、デルフィン、ナスニン、ペオニジン、ペオニン、ペツニン、ペオニジン、マルビジン、マルビン、エニン、シアニジン、ロイコシアニジン、シアニン、クリサンテミン、ケラシアニン、イデイン、メコシアニン、ペラルゴニジン、カリステフィン、フェルラ酸、ｐ−クマル酸又はこれらの誘導体が挙げられる。上記誘導体としては、アセチル化物、マロニル化物、メチル化物が例示される。なかでも、原料の溶解性の低さから、フラボノール類及びフラバノン類が好ましく適用でき、フラバノン類がより好ましく、ルチン、ヘスペリジンが更に好ましい。難水溶性ポリフェノール類は、１種であっても、２種以上の混合物であってもよい。
As the poorly water-soluble polyphenols (A), phenolic substances in which one or more hydroxyl groups are further bonded to a benzene ring and two or more hydroxyl groups are preferably applicable. For example, plant-derived flavonoids, tannins, phenolic acids and the like can be mentioned. Examples of the poorly water-soluble polyphenols that can be more preferably applied include flavonols, flavanones, flavones, isoflavones, and phenolcarboxylic acids.
Specifically, rutin, quercitrin, isoquercitrin, quercetin, myricitrin, daidzein, daidzin, glycitein, glycitin, genistein, genistin, myricetin, hesperidin, neohesperidin, hesperetin, naringin, curcumin, ringenin, prnin, astragaline, Kaempferol, Resveratrol, Apine, Apigenin, Delphinidin, Delphin, Nasnin, Peonidin, Peonin, Petunin, Peonidin, Malvidin, Malvin, Enine, Cyanidine, Leucocyanidin, Cyanine, Chrysanthemin, Kerocyanine, Idein, Mecocyanine, Pelargonidin Examples include ferulic acid, p-coumaric acid, and derivatives thereof. Examples of the derivatives include acetylated products, malonylated products, and methylated products. Among these, flavonols and flavanones can be preferably applied because of low solubility of the raw materials, flavanones are more preferable, and rutin and hesperidin are more preferable. The poorly water-soluble polyphenols may be one kind or a mixture of two or more kinds.
なお、本発明の難水溶性ポリフェノール類（Ａ）の中には、前述の定義を満たす限り、アグリコンのみならずアグリコンに糖が結合した配糖体が含まれる。
例えば、ヘスペレチン（５，７，３'−トリヒドロキシ−４'−メトキシフラバノン）の７位の水酸基にルチノース（Ｌ−ラムノシル−（α１→６）−Ｄ−グルコース）がβ結合した配糖体であるヘスペリジン、アピゲニンにアピオース及びグルコースが結合したアピイン、ケルセチンにルチノースが結合したルチン、ケルセチンにラムノースが結合したケルシトリン等が挙げられる。
The poorly water-soluble polyphenols (A) of the present invention include not only aglycones but also glycosides in which sugars are bound to aglycones as long as the above definition is satisfied.
For example, a glycoside in which rutinose (L-rhamnosyl- (α1 → 6) -D-glucose) is β-bonded to the hydroxyl group at position 7 of hesperetin (5,7,3′-trihydroxy-4′-methoxyflavanone). Examples include hesperidin, apiin in which apiose and glucose are bound to apigenin, rutin in which rutinose is bound to quercetin, and quercitrin in which rhamnose is bound to quercetin.
本明細書において水性媒体とは、水、及び有機溶媒の水溶液を云う。水としては、水道水、蒸留水、イオン交換水、精製水が例示される。有機溶媒としては、水と均一に混合するものであれば特に限定されない。有機溶媒としては炭素数４以下のアルコールが好ましく、メタノール及びエタノールがより好ましく、食品に適用可能であるという観点よりエタノールが更に好ましい。水性媒体として有機溶媒の水溶液を用いる場合は、前記水溶液中の有機溶媒の濃度は、０．１〜８０質量％（以下、単に「％」とする）が好ましく、１〜７０％がより好ましく、５〜６０％が更に好ましい。 In the present specification, the aqueous medium refers to water and an aqueous solution of an organic solvent. Examples of water include tap water, distilled water, ion exchange water, and purified water. The organic solvent is not particularly limited as long as it is uniformly mixed with water. As the organic solvent, alcohol having 4 or less carbon atoms is preferable, methanol and ethanol are more preferable, and ethanol is more preferable from the viewpoint of being applicable to foods. When an aqueous solution of an organic solvent is used as the aqueous medium, the concentration of the organic solvent in the aqueous solution is preferably 0.1 to 80% by mass (hereinafter simply referred to as “%”), more preferably 1 to 70%, 5 to 60% is more preferable.
水性媒体のｐＨは、難水溶性ポリフェノール類の安定性の観点より、３以上７未満が好ましく、３．５〜６．９がより好ましく、４〜６．８が更に好ましい。 From the viewpoint of the stability of the poorly water-soluble polyphenols, the pH of the aqueous medium is preferably 3 or more and less than 7, more preferably 3.5 to 6.9, and still more preferably 4 to 6.8.
難水溶性ポリフェノール類（Ａ）は水への溶解度が低いため、水性媒体へ分散させ、スラリーの状態で存在させるのが好ましい。水性媒体中の難水溶性ポリフェノール類（Ａ）の含有量は、難水溶性ポリフェノール類の種類によって異なるが、スラリーの流動性の点から、０．１〜１００ｇ／Ｌが好ましく、０．５〜５０ｇ／Ｌがより好ましく、０．７〜２０ｇ／Ｌが更に好ましく、０．７〜１０ｇ／Ｌが更に好ましい。 Since the poorly water-soluble polyphenols (A) have low solubility in water, it is preferable that they be dispersed in an aqueous medium and exist in a slurry state. The content of the poorly water-soluble polyphenols (A) in the aqueous medium varies depending on the kind of the poorly water-soluble polyphenols, but is preferably 0.1 to 100 g / L from the viewpoint of the fluidity of the slurry, 0.5 to 50 g / L is more preferable, 0.7 to 20 g / L is still more preferable, and 0.7 to 10 g / L is still more preferable.
水性媒体の存在下、難水溶性ポリフェノール類（Ａ）を加熱処理する方法は、特に制限されず、公知の方法を適用できる。
加熱処理の温度は、１００〜１８０℃であるが、１１０〜１７０℃がより好ましく、１２０〜１６０℃が更に好ましく、１２０〜１５０℃が更に好ましい。１００℃以上において大幅な溶解性の向上が達成され、また、１８０℃以下において難水溶性ポリフェノール類の安定性が確保される。加熱の手段は、例えば、水蒸気、電気が挙げられる。
The method for heat-treating the poorly water-soluble polyphenols (A) in the presence of an aqueous medium is not particularly limited, and known methods can be applied.
Although the temperature of heat processing is 100-180 degreeC, 110-170 degreeC is more preferable, 120-160 degreeC is still more preferable, 120-150 degreeC is still more preferable. A significant improvement in solubility is achieved at 100 ° C. or higher, and stability of poorly water-soluble polyphenols is ensured at 180 ° C. or lower. Examples of the heating means include water vapor and electricity.
加熱処理時の圧力は、ゲージ圧で０〜１０ＭＰａが好ましく、０．１〜８ＭＰａがより好ましく、０．１〜６ＭＰａが更に好ましく、０．２〜６ＭＰａが更に好ましく、０．２〜４ＭＰａが更に好ましく、０．２５〜２ＭＰａが更に好ましく、０．３〜１．５ＭＰａが更に好ましく、０．３〜０．６ＭＰａが更に好ましい。また、水の飽和蒸気圧以上に設定するのが好ましい。飽和蒸気圧以上の加圧は、背圧弁により調整しても良く、また、ガスを用いてもよい。用いられるガスとしては、例えば、不活性ガスが好ましく、窒素ガス、ヘリウムガスがより好ましい。 The pressure during the heat treatment is preferably 0 to 10 MPa, more preferably 0.1 to 8 MPa, further preferably 0.1 to 6 MPa, further preferably 0.2 to 6 MPa, and further preferably 0.2 to 4 MPa in terms of gauge pressure. Preferably, 0.25 to 2 MPa is more preferable, 0.3 to 1.5 MPa is further preferable, and 0.3 to 0.6 MPa is further preferable. Moreover, it is preferable to set it more than the saturated vapor pressure of water. Pressurization higher than the saturated vapor pressure may be adjusted by a back pressure valve, or gas may be used. As the gas used, for example, an inert gas is preferable, and nitrogen gas and helium gas are more preferable.
加熱処理は、例えば、回分法、半回分法、流通式処理方法等いずれの方法によっても実施できる。なかでも、流通式処理方法は、処理時間の制御が容易である点で好ましい。 The heat treatment can be performed by any method such as a batch method, a semi-batch method, and a flow-type treatment method. Among these, the flow-type processing method is preferable in that the processing time can be easily controlled.
加熱処理の時間は、難水溶性ポリフェノール類の溶解性向上と熱安定性の点から、上記の加熱処理の温度範囲にある時間が０．１〜３０分が好ましく、更に０．２〜１５分、更に０．５〜８分が好ましい。
流通式処理方式で行う場合、加熱処理の時間は、処理器の高温高圧部の体積を水性媒体の供給速度で割ることにより算出される平均滞留時間を用いる。
The heat treatment time is preferably from 0.1 to 30 minutes, more preferably from 0.2 to 15 minutes, in the temperature range of the above heat treatment from the viewpoint of improvement in solubility of the poorly water-soluble polyphenols and heat stability. Further, 0.5 to 8 minutes is preferable.
In the case of using the flow treatment method, the heat treatment time is an average residence time calculated by dividing the volume of the high-temperature high-pressure part of the processor by the supply rate of the aqueous medium.
加熱処理後、得られた加熱処理液を９０℃以下、好ましくは５０℃以下、更に好ましくは３０℃以下に冷却する工程を行うのが、ポリフェノールの熱劣化防止の点から好ましい。冷却時に、加熱処理液を混合攪拌してもよい。 After the heat treatment, it is preferable from the viewpoint of preventing thermal degradation of the polyphenol that the obtained heat treatment liquid is cooled to 90 ° C. or lower, preferably 50 ° C. or lower, more preferably 30 ° C. or lower. When cooling, the heat treatment liquid may be mixed and stirred.
加熱処理温度から９０℃まで低下するのに要した時間から算出される加熱処理液の冷却速度は０．２℃／ｓ以上、更に０．５℃／ｓ以上、１℃／ｓ以上、更に３℃／ｓ以上、更に５℃／ｓ以上が好ましい。冷却速度が大きいほど難水溶性ポリフェノール類の溶解量を向上することができる。このため、冷却速度の上限は特に定めないが、例えば１００℃／ｓ以下、更に５０℃／ｓ以下が好ましい。 The cooling rate of the heat treatment liquid calculated from the time required to decrease from the heat treatment temperature to 90 ° C. is 0.2 ° C./s or more, further 0.5 ° C./s or more, 1 ° C./s or more, and further 3 It is preferably at least 5 ° C / s, more preferably at least 5 ° C / s. The higher the cooling rate, the more the amount of poorly water-soluble polyphenols dissolved can be improved. For this reason, the upper limit of the cooling rate is not particularly defined, but is preferably 100 ° C./s or less, and more preferably 50 ° C./s or less.
更に、加熱処理液から、溶解せずに残留する固体部を除去する工程を行うのが、得られる難水溶性ポリフェノール類の糖付加物の溶解性を高める点から好ましい。固体部を除去する方法としては、特に制限されず、例えば遠心分離やデカンテーション、ろ過により行うことができる。 Further, it is preferable to perform a step of removing the solid part remaining without being dissolved from the heat treatment liquid, from the viewpoint of increasing the solubility of the resulting poorly water-soluble polyphenols sugar adduct. The method for removing the solid part is not particularly limited, and can be performed, for example, by centrifugation, decantation, or filtration.
本発明の方法において、加熱処理は、可溶化剤（Ｃ）の存在下に行ってもよい。加熱処理は、難水溶性ポリフェノール類（Ａ）と可溶化剤（Ｃ）の合計に対する可溶化剤（Ｃ）の質量比［（Ｃ）／（（Ａ）＋（Ｃ））］が０．１未満である条件で行うのが、難水溶性ポリフェノール類（Ａ）から糖付加物への転化率向上の点、反応生成物の精製負荷軽減の点から好ましい。
すなわち、難水溶性ポリフェノール類（Ａ）と可溶化剤（Ｃ）の関係は、
０≦（Ｃ）／（（Ａ）＋（Ｃ））＜０．１
と表すことができる。
反応生成物の精製負荷軽減の点という観点からは、上記質量比は０．０９以下が好ましく、０．０７以下がより好ましく、０．０５以下が更に好ましく、０．０４以下が更に好ましく、０（可溶化剤（Ｃ）が存在しない条件）が殊更好ましい。
In the method of the present invention, the heat treatment may be performed in the presence of the solubilizer (C). In the heat treatment, the mass ratio [(C) / ((A) + (C))] of the solubilizer (C) to the total of the poorly water-soluble polyphenols (A) and the solubilizer (C) is 0.1. It is preferable to carry out under the condition of less than the viewpoint of improving the conversion rate of the poorly water-soluble polyphenols (A) to the sugar adduct and reducing the purification load of the reaction product.
That is, the relationship between the poorly water-soluble polyphenols (A) and the solubilizer (C) is
0 ≦ (C) / ((A) + (C)) <0.1
It can be expressed as.
From the viewpoint of reducing the purification load of the reaction product, the mass ratio is preferably 0.09 or less, more preferably 0.07 or less, further preferably 0.05 or less, further preferably 0.04 or less, 0 (Conditions where no solubilizer (C) is present) are particularly preferred.
可溶化剤（Ｃ）としては、原料である難水溶性ポリフェノール類（Ａ）より水溶性が高いものが好ましく、難水溶性ポリフェノール類（Ａ）の糖付加物が好ましく、ヘスペリジン糖付加物がより好適に用いられる。 As the solubilizer (C), those having higher water solubility than the poorly water-soluble polyphenols (A) as raw materials are preferred, sugar adducts of the poorly water-soluble polyphenols (A) are preferred, and hesperidin sugar adducts are more preferred. Preferably used.
本発明の方法における工程（２）は、得られた加熱処理液に糖供与体（Ｂ）及び糖転移酵素を添加し、加熱処理後３００分以内に糖転移反応を開始し、難水溶性ポリフェノール類（Ａ）の糖付加物を得る工程である。
本明細書において、加熱処理後「３００分以内」とは、加熱処理が終了した時点、すなわち加熱処理液が１００℃未満に下がった時点から、糖転移反応の開始までの時間である。加熱処理後から糖転移反応の開始までの時間は、転化率向上の点から、０．１〜３００分が好ましく、更に０．１〜１８０分が好ましく、更に１〜１２０分が好ましく、１〜６０分が好ましい。
In step (2) in the method of the present invention, a sugar donor (B) and a glycosyltransferase are added to the obtained heat-treated liquid, and a glycosyltransferase reaction is started within 300 minutes after the heat treatment. In this step, a sugar adduct of class (A) is obtained.
In this specification, “within 300 minutes” after the heat treatment refers to the time from the time when the heat treatment is completed, that is, the time when the heat treatment liquid is lowered to less than 100 ° C. to the start of the transglycosylation reaction. The time from the heat treatment to the start of the transglycosylation reaction is preferably from 0.1 to 300 minutes, more preferably from 0.1 to 180 minutes, further preferably from 1 to 120 minutes, from the viewpoint of improving the conversion rate. 60 minutes is preferred.
本発明の方法で用いられる糖供与体（Ｂ）としては、難水溶性ポリフェノール類（Ａ）に後述する糖を供与できるものであれば特に制限はない。
糖供与体（Ｂ）の具体例としては、澱粉、デキストリン、シクロデキストリン、マルトオリゴ糖等の澱粉部分加水分解物、キシロオリゴ糖、フラクタン、アラビノガラクタン、プルラン、ラフィノース、又はこれらの含有物等が挙げられる。
The sugar donor (B) used in the method of the present invention is not particularly limited as long as it can donate the sugar described later to the poorly water-soluble polyphenols (A).
Specific examples of the sugar donor (B) include starch, dextrin, cyclodextrin, starch partial hydrolyzate such as maltooligosaccharide, xylo-oligosaccharide, fructan, arabinogalactan, pullulan, raffinose, or contents thereof. It is done.
糖供与体（Ｂ）の使用量は、難水溶性ポリフェノール類（Ａ）の転化率向上の点から、糖転移反応の反応開始時における難水溶性ポリフェノール類（Ａ）に対する質量比（Ｂ）／（Ａ）として１〜３０が好ましく、更に１．２〜１５が好ましい。 The amount of the sugar donor (B) used is the mass ratio (B) / mass to the poorly water-soluble polyphenols (A) at the start of the transglycosylation reaction from the viewpoint of improving the conversion rate of the poorly water-soluble polyphenols (A). (A) is preferably from 1 to 30, and more preferably from 1.2 to 15.
本発明の方法で用いられる糖転移酵素としては、難水溶性ポリフェノール類（Ａ）に対して糖の転移活性を有する酵素であれば特に制限はないが、用いる糖供与体（Ｂ）の種類に応じて適宜選択し得る。
糖転移酵素の具体例としては、シクロマルトデキストリングルカノトランスフェラーゼ、デキストリンデキストラナーゼ、アミロスクラーゼ、デキストランスクラーゼ、α−グルコシダーゼ、β−グルコシダーゼ、α−ガラクトシダーゼ、β−ガラクトシダーゼ、α−アミラーゼ、キシラナーゼ、プルラナーゼ、アラビノフラノシダーゼ等が挙げられる。
The glycosyltransferase used in the method of the present invention is not particularly limited as long as it is an enzyme having a sugar transfer activity with respect to poorly water-soluble polyphenols (A), but the type of sugar donor (B) to be used is not limited. It can be appropriately selected depending on the case.
Specific examples of glycosyltransferases include cyclomaltodextrin glucanotransferase, dextrin dextranase, amylosucrase, dextransucrase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, α-amylase, xylanase, pullulanase And arabinofuranosidase.
糖転移酵素は、その起源に限定はなく、動物由来、植物由来、微生物由来等のすべての由来のものを使用することができる。さらに、遺伝子組み換え技術、部分加水分解等による人工酵素であってもよい。
また、糖転移酵素の形態は特に限定されず、酵素蛋白質の乾燥物、酵素蛋白質を含む粒子、及び酵素蛋白質を含む液体等を用いることができる。
The origin of the glycosyltransferase is not limited, and those derived from all origins such as those derived from animals, plants, and microorganisms can be used. Further, it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
The form of glycosyltransferase is not particularly limited, and a dried product of enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.
糖転移酵素の使用量は、糖転移反応の条件、糖の種類等によって異なるが、例えば、シクロマルトデキストリングルカノトランスフェラーゼの場合、１０〜１０，０００Ｕ／ｍＬが好ましい。ここで活性は以下に示す方法により測定される。
５ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝溶液（ｐＨ７．５）、０．０５％（ｗ／ｖ）アミロース、および酵素を含む液１００μＬを３０℃、３０分間反応させた後、ヨウ素溶液（１ｍｇ／ｍＬ ＫＩ，０．１ｍｇ／ｍＬ Ｉ2 ，３．８ｍＭ ＨＣｌを含む）２ｍＬを添加して反応を停止し、波長６６０ｎｍにおける吸光度を測定して定量する。１分間に吸光度を１％低下させる酵素量を１ユニット（Ｕ）とする。
The amount of glycosyltransferase used varies depending on the conditions of the glycosyltransferase reaction, the type of sugar, and the like. For example, in the case of cyclomaltodextrin glucanotransferase, 10 to 10,000 U / mL is preferable. Here, the activity is measured by the following method.
After reacting 100 μL of a solution containing 5 mM Tris-HCl buffer solution (pH 7.5), 0.05% (w / v) amylose and enzyme at 30 ° C. for 30 minutes, iodine solution (1 mg / mL KI, 0. The reaction is stopped by adding 2 mL (containing 1 mg / mL I 2 , 3.8 mM HCl), and the absorbance at a wavelength of 660 nm is measured and quantified. The amount of enzyme that decreases the absorbance by 1% per minute is defined as 1 unit (U).
工程（２）において糖転移反応の条件は、使用する酵素の特性に合わせて反応温度や反応液のｐＨを選択することが可能であるが、例えば、シクロマルトデキストリングルカノトランスフェラーゼを用いる場合、ｐＨ３〜７とすることが好ましく、更に４〜６．５とすることが好ましい。 In the step (2), it is possible to select the reaction temperature and the pH of the reaction solution as the conditions for the glycosyltransferase reaction in accordance with the characteristics of the enzyme used. For example, when cyclomaltodextrin glucanotransferase is used, the pH is 3 It is preferable to set it to -7, and also it is preferable to set it as 4-6.5.
また、反応温度は、糖転移酵素の安定性の点、糖転移反応の反応速度向上の点から、１０〜８０℃、更に２０〜６０℃、更に３５〜５５℃とすることが好ましい。 In addition, the reaction temperature is preferably 10 to 80 ° C., more preferably 20 to 60 ° C., and further preferably 35 to 55 ° C. from the viewpoint of the stability of glycosyltransferase and the improvement of the reaction rate of the glycosyltransferase reaction.
反応時間は、糖転移酵素の種類等によって異なるが、難水溶性ポリフェノール類（Ａ）転化率向上の点、難水溶性ポリフェノール類（Ａ）の糖付加物の生産性向上の点から、例えば、０．５〜１２０時間が好ましく、更に１〜１００時間、更に２〜２０時間が好ましい。 Although the reaction time varies depending on the type of glycosyltransferase, etc., from the viewpoint of improving the conversion rate of poorly water-soluble polyphenols (A) and improving the productivity of sugar adducts of poorly water-soluble polyphenols (A), for example, 0.5 to 120 hours are preferable, further 1 to 100 hours, and further 2 to 20 hours are preferable.
本発明の方法によれば、４０〜１００％、更に４５〜９５％の転化率で、難水溶性ポリフェノール類（Ａ）から難水溶性ポリフェノール類の糖付加物を製造することができる。なお、転化率は、後記実施例に記載した式（１）により算出できる。 According to the method of the present invention, a sugar adduct of a hardly water-soluble polyphenol can be produced from the hardly water-soluble polyphenol (A) at a conversion rate of 40 to 100%, further 45 to 95%. The conversion rate can be calculated by the formula (1) described in Examples below.
本発明の方法により得られる難水溶性ポリフェノール類の糖付加物は、原料である難水溶性ポリフェノール類（Ａ）に少なくとも１個の糖が結合した化合物である。難水溶性ポリフェノール類（Ａ）に結合する糖の種類は、特に制限されないが、グルコース、ガラクトース、フルクトース、ラムノース、キシロース、アラビノース、エリトロース等の４〜６炭糖から選ばれる少なくとも１種以上が好ましい。また、糖の結合数は、好ましくは１個〜１０個、より好ましくは１個〜６個である。難水溶性ポリフェノール類（Ａ）への糖の結合部位は、フェノール性水酸基又は配糖体の糖残基である。難水溶性ポリフェノール類（Ａ）とこれら糖との結合様式はα−結合又はβ−結合のいずれであってもよい。 The sugar adduct of the poorly water-soluble polyphenols obtained by the method of the present invention is a compound in which at least one sugar is bound to the poorly water-soluble polyphenols (A) as a raw material. The type of sugar that binds to the poorly water-soluble polyphenols (A) is not particularly limited, but is preferably at least one selected from 4 to 6 carbon sugars such as glucose, galactose, fructose, rhamnose, xylose, arabinose, and erythrose. . The number of sugar bonds is preferably 1 to 10, more preferably 1 to 6. The sugar binding site to the poorly water-soluble polyphenols (A) is a phenolic hydroxyl group or a sugar residue of a glycoside. The binding mode between the poorly water-soluble polyphenols (A) and these sugars may be α-bond or β-bond.
本発明の方法により得られる難水溶性ポリフェノール類の糖付加物の溶解量は、好ましくは１ｇ／Ｌ以上であり、より好ましくは２ｇ／Ｌ以上であり、更に好ましくは５ｇ／Ｌ以上であり、更に好ましくは１０ｇ／Ｌ以上である。本明細書における溶解量は、水に対する２５℃での溶解量である。 The dissolution amount of the sugar adduct of the poorly water-soluble polyphenols obtained by the method of the present invention is preferably 1 g / L or more, more preferably 2 g / L or more, still more preferably 5 g / L or more, More preferably, it is 10 g / L or more. The amount dissolved in this specification is the amount dissolved in water at 25 ° C.
本発明の製造方法で得られるポリフェノール類の糖付加物は、様々な飲食品や医薬品等に使用することができる。例えば、飲食品としては、飲料、パン類、麺類、クッキー等の菓子類、スナック類、ゼリー類、乳製品、冷凍食品、粉末コーヒー等のインスタント食品、でんぷん加工製品、加工肉製品、その他加工食品、調味料、栄養補助食品等の液状、固形状又は半固形状の飲食品が挙げられる。とりわけ、容器詰飲料に利用するのが有用である。容器詰飲料としては、緑茶等の茶系飲料や、スポーツ飲料、アイソトニック飲料、ニアウォーター等の非茶系飲料が挙げられる。 The sugar addition product of polyphenols obtained by the production method of the present invention can be used for various foods and beverages, pharmaceuticals and the like. For example, as food and drink, confectionery such as beverages, breads, noodles, cookies, snacks, jelly, dairy products, frozen foods, instant foods such as powdered coffee, starch processed products, processed meat products, and other processed foods , Liquid, solid or semi-solid foods and beverages such as seasonings and dietary supplements. In particular, it is useful to be used for a packaged beverage. Examples of the packaged beverage include tea-based beverages such as green tea, and non-tea beverages such as sports beverages, isotonic beverages, and near water.
［難水溶性ポリフェノール類の測定］
難水溶性ポリフェノール類の測定は、日立製作所製高速液体クロマトグラフを用い、インタクト社製カラムＣａｄｅｎｚａ ＣＤ−Ｃ１８ （４．６ｍｍφ×１５０ｍｍ、 ３μｍ）を装着し、カラム温度４０℃でグラジエント法により行った。移動相Ａ液は０．０５ｍｏｌ／Ｌ酢酸水溶液、Ｂ液はアセトニトリルとし、１．０ｍＬ／分で送液した。グラジエント条件は以下のとおりである。
時間（分） Ａ液（％） Ｂ液（％）
０ ８５ １５
２０ ８０ ２０
３５ １０ ９０
５０ １０ ９０
４０．１ ８５ １５
６０ ８５ １５
試料注入量は１０μＬ、検出はルチンは波長３６０nm、ヘスペリジンは波長２８３nmの吸光度により定量した。
[Measurement of poorly water-soluble polyphenols]
Measurement of poorly water-soluble polyphenols was carried out by a gradient method using a high-performance liquid chromatograph manufactured by Hitachi, equipped with an intact column Cadenza CD-C18 (4.6 mmφ × 150 mm, 3 μm) and a column temperature of 40 ° C. . The mobile phase A solution was 0.05 mol / L acetic acid aqueous solution, the B solution was acetonitrile, and the solution was fed at 1.0 mL / min. The gradient conditions are as follows.
Time (min) A liquid (%) B liquid (%)
0 85 15
20 80 20
35 10 90
50 10 90
40.1 85 15
60 85 15
The sample injection amount was 10 μL, the detection was quantified by the absorbance of rutin for a wavelength of 360 nm, and hesperidin for a wavelength of 283 nm.
［難水溶性ポリフェノール類のｌｏｇＰ値の測定］
日本工業規格 Ｚ７２６０−１０７記載のフラスコ振盪法に従って測定した。まず１−オクタノールと蒸留水を２５℃で２４時間振とうして平衡化させた。次いで蓋付きガラス瓶にポリフェノール１０ｍｇを量りとり、平衡化させた１−オクタノールと蒸留水をそれぞれ４ｍＬずつ加え、２５℃で４日間振とうした。遠心分離により１−オクタノール相と水相を分け、上記［難水溶性ポリフェノールの測定］と同様にしてＨＰＬＣにより各相のポリフェノール類の濃度を測定した。２相間の分配係数の常用対数を取った値をｌｏｇＰ値とした。
[Measurement of log P value of poorly water-soluble polyphenols]
It was measured according to the flask shaking method described in Japanese Industrial Standard Z7260-107. First, 1-octanol and distilled water were equilibrated by shaking at 25 ° C. for 24 hours. Next, 10 mg of polyphenol was weighed into a glass bottle with a lid, 4 mL each of 1-octanol and distilled water that had been equilibrated were added, and the mixture was shaken at 25 ° C. for 4 days. The 1-octanol phase and the aqueous phase were separated by centrifugation, and the concentration of polyphenols in each phase was measured by HPLC in the same manner as in the above [Measurement of poorly water-soluble polyphenol]. The value obtained by taking the common logarithm of the distribution coefficient between the two phases was defined as the logP value.
［水性媒体及び糖転移反応液のｐＨの測定］
蒸留水と糖転移反応終了後の反応液のｐＨを２５℃にて測定した。
[Measurement of pH of aqueous medium and transglycosylation reaction solution]
The pH of the reaction solution after completion of the sugar transfer reaction with distilled water was measured at 25 ° C.
［転化率の算出］
糖転移反応開始から７時間後に転化率を求めた。転化率は次式（１）により算出した。
転化率（％）＝｛（１−糖転移反応終了時に反応液中に残存している難水溶性ポリフェノール類濃度）／（糖転移反応開始時の反応液中の難水溶性ポリフェノール類濃度）｝×１００ （１）
[Calculation of conversion rate]
The conversion was determined 7 hours after the start of the transglycosylation reaction. The conversion rate was calculated by the following formula (1).
Conversion rate (%) = {(1-concentration of poorly water-soluble polyphenols remaining in the reaction liquid at the end of the transglycosylation reaction) / (concentration of poorly water-soluble polyphenols in the reaction liquid at the start of the transglycosylation reaction)} × 100 (1)
実施例１
ルチン製剤（（株）常盤植物化学研究所製、ルチン含有量１００％、以下、同じ）を蒸留水（ｐＨ６．５）に５ｇ／Ｌの濃度で分散し、スラリー供給タンク内で均一攪拌した。内容積１００ｍＬのステンレス製流通式処理器（日東高圧（株）製）に、スラリー供給タンク内の液を１００ｍＬ／分で供給し、１５０℃で加熱処理を行った（平均滞留時間１分）。処理器内の圧力は出口バルブにより０．６ＭＰａに調整した。処理器出口から加熱処理液を抜き出し、熱交換器により２５℃まで冷却し、孔径７μｍの金属焼結フィルターを通した後、出口バルブで圧力を大気圧に戻して加熱処理液を得た。１５０℃から９０℃までの冷却時間から求めた冷却速度は８℃／ｓであった。
加熱処理終了時点から５分後に、得られた加熱処理液１０ｍＬをガラス瓶に入れ、γ-シクロデキストリン（γ−ＣＤ、（株）シクロケム製、以下同じ）０．２ｇと糖転移酵素（天野エンザイム（株）製コンチザイム、以下同じ）０．５ｍＬ（糖転移反応液１ｍＬあたりの添加は５００Ｕ）を添加した。糖転移反応開始時の反応液中のルチン濃度は５．０ｇ／Ｌであり、２５℃における水への溶解度（０．０３ｇ／Ｌ）よりも顕著に高い過飽和溶解状態であった。反応液を４５℃においてロータリーシェーカーで７０ｒ／ｍｉｎで７時間振盪した。ガラス瓶を沸騰水中に１分間浸漬させることにより酵素を失活させて反応を停止させ、ルチンの糖付加物を得た。加熱処理条件、糖転移反応条件、及びルチンの転化率を表１に示す（以下同じ）。
Example 1
A rutin preparation (manufactured by Tokiwa Plant Chemical Research Co., Ltd., rutin content 100%, hereinafter the same) was dispersed in distilled water (pH 6.5) at a concentration of 5 g / L, and uniformly stirred in a slurry supply tank. The liquid in the slurry supply tank was supplied at a rate of 100 mL / min to a stainless steel flow-type processing device (manufactured by Nitto Koatsu Co., Ltd.) having an internal volume of 100 mL, and heat treatment was performed at 150 ° C. (average residence time 1 minute). The pressure in the processor was adjusted to 0.6 MPa by an outlet valve. The heat treatment liquid was extracted from the outlet of the processor, cooled to 25 ° C. with a heat exchanger, passed through a sintered metal filter having a pore diameter of 7 μm, and then the pressure was returned to atmospheric pressure with an outlet valve to obtain a heat treatment liquid. The cooling rate obtained from the cooling time from 150 ° C. to 90 ° C. was 8 ° C./s.
Five minutes after the end of the heat treatment, 10 mL of the obtained heat treatment solution was put in a glass bottle, 0.2 g of γ-cyclodextrin (γ-CD, manufactured by Cyclochem Co., Ltd., hereinafter the same) and glycosyltransferase (Amano Enzyme ( 0.5 mL (addition of 500 U per 1 mL of the sugar transfer reaction solution) was added. The rutin concentration in the reaction solution at the start of the transglycosylation reaction was 5.0 g / L, which was a supersaturated dissolution state significantly higher than the solubility in water at 25 ° C. (0.03 g / L). The reaction solution was shaken at 45 rpm with a rotary shaker at 70 r / min for 7 hours. The reaction was stopped by inactivating the enzyme by immersing the glass bottle in boiling water for 1 minute to obtain a rutin sugar adduct. The heat treatment conditions, the transglycosylation reaction conditions, and the conversion rate of rutin are shown in Table 1 (the same applies hereinafter).
実施例２
実施例１と同様にルチン製剤を加熱処理し、金属焼結フィルターを通した後、加熱処理液１０ｍＬをガラス瓶に入れて静置した。加熱処理終了時点から１２０分後に実施例１と同様に糖転移反応を行い、ルチンの糖付加物を得た。糖転移反応開始時の反応液中のルチン濃度は５．０ｇ／Ｌであるがやや懸濁した状態であった。
Example 2
The rutin preparation was heat-treated in the same manner as in Example 1, and after passing through a sintered metal filter, 10 mL of the heat-treated solution was placed in a glass bottle and allowed to stand. 120 minutes after the end of the heat treatment, a sugar transfer reaction was carried out in the same manner as in Example 1 to obtain a rutin sugar adduct. The rutin concentration in the reaction solution at the start of the transglycosylation reaction was 5.0 g / L, but it was in a slightly suspended state.
比較例１
実施例１と同様にしてルチン製剤を加熱処理し、金属焼結フィルターを通した後、加熱処理液１０ｍＬをガラス瓶に入れて静置した。加熱処理終了時点から１４４０分後に実施例１と同様に糖転移反応を行い、ルチンの糖付加物を得た。糖転移反応開始時の反応液中のルチン濃度は５．０ｇ／Ｌであるが懸濁状態であった。
Comparative Example 1
The rutin preparation was heat-treated in the same manner as in Example 1, and after passing through a sintered metal filter, 10 mL of the heat-treatment liquid was placed in a glass bottle and allowed to stand. After 1440 minutes from the end of the heat treatment, a sugar transfer reaction was performed in the same manner as in Example 1 to obtain a rutin sugar adduct. Although the rutin concentration in the reaction solution at the start of the transglycosylation reaction was 5.0 g / L, it was in a suspended state.
比較例２
ルチン製剤を０．０５ｇ、γ-シクロデキストリンを０．２ｇと蒸留水１０ｍＬをガラス瓶に入れた。ルチン濃度は５．０ｇ／Ｌであるが懸濁状態であった。糖転移酵素を０．５ｍＬ添加し、実施例１と同様に糖転移反応を行い、ルチンの糖付加物を得た。
Comparative Example 2
0.05 g of the rutin preparation, 0.2 g of γ-cyclodextrin and 10 mL of distilled water were placed in a glass bottle. Although the rutin concentration was 5.0 g / L, it was in a suspended state. 0.5 mL of glycosyltransferase was added and a glycosyltransferase reaction was performed in the same manner as in Example 1 to obtain a rutin sugar adduct.
実施例３
ルチン製剤に代えてヘスペリジン製剤（ヘスペリジン「ハマリ」（商品名）、浜理薬品工業（株）製、ヘスペリジン（ＨＥＳ）含有量９０％、以下同じ）を５ｇ／Ｌで蒸留水（ｐＨ６．５）に分散して用いた以外は実施例１と同様にして加熱処理を行い、金属焼結フィルターを通し、加熱処理液を得た。
得られた加熱処理液を用いて実施例１と同様に糖転移反応を行い、ヘスペリジンの糖付加物を得た。糖転移反応開始時の反応液中のヘスペリジン濃度は２．５ｇ／Ｌであり、２５℃における水への溶解度（０．０２ｇ／Ｌ）よりも顕著に高い過飽和溶解状態であった。加熱処理条件、糖転移反応条件、及びヘスペリジンの転化率を表２に示す（以下同じ）。
Example 3
Hesperidin preparation (Hesperidin “Hamari” (trade name), manufactured by Hamari Pharmaceutical Co., Ltd., hesperidin (HES) content 90%, the same applies hereinafter) instead of rutin preparation at 5 g / L in distilled water (pH 6.5) A heat treatment was performed in the same manner as in Example 1 except that the heat treatment liquid was used after being dispersed in a metal sintered filter.
The resulting heat treatment solution was used for transglycosylation in the same manner as in Example 1 to obtain a hesperidin sugar adduct. The hesperidin concentration in the reaction solution at the start of the transglycosylation reaction was 2.5 g / L, which was a supersaturated dissolution state significantly higher than the solubility in water at 25 ° C. (0.02 g / L). Table 2 shows the heat treatment conditions, the sugar transfer reaction conditions, and the conversion rate of hesperidin (the same applies hereinafter).
実施例４
加熱温度を１２０℃、圧力を０．３ＭＰａとした以外は実施例３と同様にヘスペリジン製剤を加熱処理し、金属焼結フィルターを通し、加熱処理液を得た。得られた加熱処理液を用いて実施例１と同様に糖転移反応を行い、ヘスペリジンの糖付加物を得た。糖転移反応開始時の反応液中のヘスペリジン濃度は１．９ｇ／Ｌであり、２５℃における溶解度（０．０２ｇ／Ｌ）よりも顕著に高い過飽和溶解状態であった。
Example 4
The hesperidin preparation was heat-treated in the same manner as in Example 3 except that the heating temperature was 120 ° C. and the pressure was 0.3 MPa, and a heat treatment liquid was obtained through a metal sintered filter. The resulting heat treatment solution was used for transglycosylation in the same manner as in Example 1 to obtain a hesperidin sugar adduct. The hesperidin concentration in the reaction solution at the start of the transglycosylation reaction was 1.9 g / L, which was a supersaturated dissolution state significantly higher than the solubility at 25 ° C. (0.02 g / L).
実施例５
加熱温度を１２０℃、圧力を０．３ＭＰａとした以外は実施例３と同様にヘスペリジン製剤を加熱処理し、金属焼結フィルターを通し、加熱処理液を得た。加熱処理の１０分後に、得られた加熱処理液をロータリーエバポレータで濃縮した（液温は４０℃とした）。加熱処理終了時点から１２０分後に、得られた加熱処理液の濃縮液１０ｍＬをガラス瓶に入れ、実施例１と同様に糖転移反応を行い、ヘスペリジンの糖付加物を得た。糖転移反応開始時の反応液中のヘスペリジン濃度は６．０ｇ／Ｌであり、２５℃における水への溶解度よりも顕著に高い過飽和溶解状態であった。
Example 5
The hesperidin preparation was heat-treated in the same manner as in Example 3 except that the heating temperature was 120 ° C. and the pressure was 0.3 MPa, and a heat treatment liquid was obtained through a metal sintered filter. Ten minutes after the heat treatment, the obtained heat treatment liquid was concentrated by a rotary evaporator (the liquid temperature was 40 ° C.). 120 minutes after the end of the heat treatment, 10 mL of the concentrated heat treatment liquid obtained was placed in a glass bottle and subjected to a sugar transfer reaction in the same manner as in Example 1 to obtain a hesperidin sugar adduct. The hesperidin concentration in the reaction solution at the start of the transglycosylation reaction was 6.0 g / L, which was a supersaturated dissolution state significantly higher than the solubility in water at 25 ° C.
実施例６
モノグルコシルヘスペリジン製剤（林原ヘスペリジンＳ（商品名）、（株）林原生物化学研究所製、ヘスペリジン含有量１７％、モノグルコシルヘスペリジン（ｍＧＨＥＳ）含有量７４％）を１．１ｇ／Ｌ溶解した蒸留水にヘスペリジン製剤を１０ｇ／Ｌで分散したスラリーを用いた以外は実施例４と同様に加熱処理し、金属焼結フィルターを通し、加熱処理液を得た。次いで、加熱処理終了時点から３０分後にγ-シクロデキストリンに代えて澱粉（和光純薬工業（株）製、溶性）を用いた以外は実施例１と同様に糖転移反応を行い、ヘスペリジンの糖付加物を得た。糖転移反応開始時の反応液中のヘスペリジン濃度は２．８ｇ／Ｌであり、２５℃における水への溶解度よりも顕著に高い過飽和溶解状態であった。
Example 6
Distilled water in which a monoglucosyl hesperidin preparation (Hayashibara Hesperidin S (trade name), manufactured by Hayashibara Biochemical Laboratory, 17% hesperidin content, 74% monoglucosyl hesperidin (mGHES) content) was dissolved in 1.1 g / L. And heat treatment was performed in the same manner as in Example 4 except that a slurry in which the hesperidin preparation was dispersed at 10 g / L was used, and a heat treatment liquid was obtained through a metal sintered filter. Then, 30 minutes after the end of the heat treatment, a sugar transfer reaction was performed in the same manner as in Example 1 except that starch (manufactured by Wako Pure Chemical Industries, Ltd., soluble) was used instead of γ-cyclodextrin, and hesperidin sugar An adduct was obtained. The hesperidin concentration in the reaction solution at the start of the transglycosylation reaction was 2.8 g / L, which was a supersaturated dissolution state significantly higher than the solubility in water at 25 ° C.
比較例３
実施例４で得られた加熱処理液１０ｍＬをガラス瓶に入れて静置した。加熱処理終了時点から３６０分後に実施例１と同様に糖転移反応を行い、ヘスペリジンの糖付加物を得た。糖転移反応開始時の反応液中のヘスペリジン濃度は１．９ｇ／Ｌであるが懸濁した状態であった。
Comparative Example 3
10 mL of the heat treatment liquid obtained in Example 4 was placed in a glass bottle and allowed to stand. After 360 minutes from the end of the heat treatment, a sugar transfer reaction was carried out in the same manner as in Example 1 to obtain a sugar adduct of hesperidin. The hesperidin concentration in the reaction solution at the start of the transglycosylation reaction was 1.9 g / L, but was in a suspended state.
比較例４
ヘスペリジン製剤０．０６７ｇを用いた以外は比較例２と同様に糖転移反応を行い、ヘスペリジンの糖付加物を得た。糖転移反応開始時の反応液中のヘスペリジン濃度は６．０ｇ／Ｌであるが懸濁状態であった。
Comparative Example 4
A sugar transfer reaction was performed in the same manner as in Comparative Example 2 except that 0.067 g of the hesperidin preparation was used, and a sugar adduct of hesperidin was obtained. The concentration of hesperidin in the reaction solution at the start of the transglycosylation reaction was 6.0 g / L, but it was in a suspended state.
表１及び２から明らかなように、本発明方法によれば、難水溶性ポリフェノール類の溶解量を顕著に増大させることができ、高い転化率で難水溶性ポリフェノール類の糖付加物を得ることができた。
他方、比較例２及び４では、糖転移反応開始時のルチンやヘスペリジンの溶解量が低く懸濁状態であるため、転化率は低かった。比較例１及び３では、加熱処理終了直後はルチンやヘスペリジンが溶解していたが、加熱処理から１４４０分或いは３６０分経過した後は懸濁状態であり、転化率は低かった。
As is clear from Tables 1 and 2, according to the method of the present invention, the amount of the slightly water-soluble polyphenols dissolved can be remarkably increased, and a sugar adduct of the hardly water-soluble polyphenols can be obtained at a high conversion rate. I was able to.
On the other hand, in Comparative Examples 2 and 4, since the dissolved amount of rutin and hesperidin at the start of the transglycosylation reaction was low and in a suspended state, the conversion rate was low. In Comparative Examples 1 and 3, rutin and hesperidin were dissolved immediately after the end of the heat treatment, but after 1440 minutes or 360 minutes from the heat treatment, the suspension was in a suspended state and the conversion rate was low.
Claims (8)
（１）水性媒体の存在下、難水溶性ポリフェノール類（Ａ）を１００〜１８０℃で加熱処理し、加熱処理液を得、次いで加熱処理温度から９０℃まで低下するのに要した時間から算出される冷却速度が３℃／ｓ以上１００℃／ｓ以下の条件で、加熱処理液を加熱処理温度から９０℃以下まで冷却する工程、
（２）得られた冷却後の加熱処理液に糖供与体（Ｂ）及び糖転移酵素を添加し、加熱処理後３００分以内に糖転移反応を開始し、難水溶性ポリフェノール類（Ａ）の糖付加物を得る工程、
を含む難水溶性ポリフェノール類の糖付加物の製造方法。 Next steps (1) and (2):
(1) In the presence of an aqueous medium, the poorly water-soluble polyphenols (A) are heat-treated at 100 to 180 ° C. to obtain a heat-treated liquid, and then calculated from the time required to decrease from the heat treatment temperature to 90 ° C. step cooling rate under the following conditions 3 ° C. / s or higher 100 ° C. / s, you cool the heat-treated solution from the heat treatment temperature to 90 ° C. or less to be,
(2) The sugar donor (B) and the glycosyltransferase are added to the obtained heat-treated liquid after cooling , and the sugar-transfer reaction is started within 300 minutes after the heat treatment, and the poorly water-soluble polyphenols (A) Obtaining a sugar adduct,
A method for producing a sugar adduct of poorly water-soluble polyphenols, comprising:
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