TW201735798A

TW201735798A - A new gardenia blue pigment, preparation and use thereof

Info

Publication number: TW201735798A
Application number: TW106106138A
Authority: TW
Inventors: 安德列迪斯特洛; 王曉燕; 黃小平; 鄧惠文; 林冰東; 賀慶; 安潔亞布巴瑞洛
Original assignee: 帝斯曼知識產權資產管理有限公司
Priority date: 2016-03-17
Filing date: 2017-02-23
Publication date: 2017-10-16
Also published as: WO2017156744A1

Abstract

The present application is related to a new gardenia blue pigment derived from a reaction of genipin with an amino acid which is brighter than commercial gardenia blue pigment and suitable for industrial application. The present application is also related to a process for producing the new gardenia blue pigment and the use thereof in food and beverage industries.

Description

新穎的梔子藍色素及其製備與用途Novel gardenia blue pigment and its preparation and use

發明領域本申請案有關一種從京尼平(genipin)與胺基酸之反應衍生而得之新穎的梔子藍色素、一種製備之方法以及於食品以及飲料產業上之用途。FIELD OF THE INVENTION This application relates to a novel gardenia blue pigment derived from the reaction of genipin with an amino acid, a method of preparation, and use in the food and beverage industries.

發明背景梔子藍色素是一種廣泛應用於食品、藥學以及化妝品產業之水溶性天然色素。BACKGROUND OF THE INVENTION Gardenia Blue is a water soluble natural pigment widely used in the food, pharmaceutical and cosmetic industries.

現今，梔子藍色素通常是茜草科山黃梔(Gardenia Jasminoides Ellis )所含之梔子苷原料，經過β-葡萄糖苷酶處理梔子苷產生京尼平，其再與胺基酸反應獲得梔子藍色素之製程處理產生的。(見Shijing WU,et al., National Food Additive Communications, 1992 (3): 90-93)。然而，由此製程獲得之梔子藍色素暗沈、具低色值、低品質以及在低pH飲料中易沈澱。因此其不適合一些如食品以及飲料之應用。(見如CN103509368A以及CN102021210A)。Nowadays, gardenia blue pigment is usually the raw material of gardenia Jasminoides Ellis , which is produced by β-glucosidase treatment of geniposide, which is then reacted with amino acid to obtain hydrazine. Produced by the processing of sub-blue pigment. (See Shijing WU, et al., National Food Additive Communications, 1992 (3): 90-93). However, the gardenia blue pigment obtained by this process is dull, has a low color value, low quality, and is easily precipitated in a low pH beverage. Therefore it is not suitable for applications such as food and beverages. (See, for example, CN103509368A and CN102021210A).

本申請案提供一種從京尼平與胺基酸之反應衍生而得之新穎的梔子藍色素，其比業界已知的梔子藍色素明亮。The present application provides a novel gardenia blue pigment derived from the reaction of genipin with an amino acid, which is brighter than the gardenia blue pigment known in the art.

本申請案亦提供一種製備以上梔子藍色素之方法，其包含下列步驟： a)用葡萄糖苷酶處理梔子苷，以獲得一水解產物；以及 b)用一溶劑萃取於步驟a)中所獲得之該水解產物，以及在該萃取後移除該溶劑以獲得一含京尼平的產物；以及 c)使於步驟b)中所獲得之該含京尼平的產物與胺基酸和/或其鹽之一水溶液反應，以產生該梔子藍色素，以及較佳地將該含京尼平的產物溶於一水溶性溶劑中，產生一溶液，且就這樣使用該溶液。The present application also provides a method for preparing the above gardenia blue pigment, comprising the steps of: a) treating geniposide with glucosidase to obtain a hydrolyzate; and b) extracting with a solvent in step a) Obtaining the hydrolyzed product, and removing the solvent after the extracting to obtain a product containing genipin; and c) bringing the genipin-containing product obtained in step b) with an amino acid and/or An aqueous solution of one of its salts is reacted to produce the gardenia blue pigment, and preferably the genipin-containing product is dissolved in a water-soluble solvent to produce a solution, and the solution is used as it is.

本申請案另外提供以上梔子藍色素於食品以及飲料產業上之用途。This application additionally provides the use of the above scorpion blue pigment in the food and beverage industry.

本申請案另外又提供含如本申請案之梔子藍色素之飲料以及食品。Further, the present application further provides a beverage containing scorpion blue pigment as in the present application and a food.

本發明之詳細說明在第一態樣中，本申請案提供一種從京尼平與胺基酸之反應衍生而得之新穎的梔子藍色素，其比市面上商購的梔子藍色素明亮。DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present application provides a novel gardenia blue pigment derived from the reaction of genipin with an amino acid, which is brighter than commercially available gardenia blue pigment. .

特別是，本申請案提供一種新穎的梔子藍色素，其在CIELAB色標(color scale)上具有從220至245，較佳地從230至240之範圍內的色調(color shade)h，以及在CIELAB色標上具有從-12至-7，較佳地從-11至-9之範圍內的色值“a*”。較佳地，如本申請案之梔子藍色素含有一或多種具式(I)-(IV)之化合物、其幾何異構物、其互變異構體、其鹽或其等之組合：，In particular, the present application provides a novel gardenia blue pigment having a color shade h ranging from 220 to 245, preferably from 230 to 240, on a CIELAB color scale, and The color value "a*" in the range of from -12 to -7, preferably from -11 to -9, is present on the CIELAB color scale. Preferably, the gardenia blue pigment as in the present application contains one or more compounds of the formula (I)-(IV), geometric isomers thereof, tautomers thereof, salts thereof or combinations thereof: ,

其中：R彼此獨立地為H或C_1-7 烷基，較佳地甲基；以及R’彼此獨立地選自於由下列所構成之群組：C_1-7 烷基，較佳地乙基、丙基、丁基以及戊基，任擇地經羧基、苯基、咪唑基或胍基基團取代。Wherein: R is independently H or a C _1-7 alkyl group, preferably a methyl group; and R' is independently selected from the group consisting of C _1-7 alkyl, preferably B. The group, propyl, butyl and pentyl are optionally substituted by a carboxy, phenyl, imidazolyl or fluorenyl group.

較佳地，R’彼此獨立地選自於由下列所構成之群組： Preferably, R' is independently selected from the group consisting of:

更佳地，該具式(I)-(IV)之化合物是具式(a)-(h)之化合物、其幾何異構物、其互變異構物、其鹽以及其等之組合： More preferably, the compounds of formula (I)-(IV) are compounds of formula (a)-(h), geometric isomers thereof, tautomers thereof, salts thereof, and combinations thereof;

在此使用之術語幾何異構物意指一樣的結構但在雙鍵處具不同的構形之異構物(即，E或Z異構物，或順式/反式異構物)。用於說明，圖例1顯示一對二個幾何異構物之例子，在此該二個異構物之間唯一不同的是在雙鍵處之構形。 The term geometric isomer as used herein refers to an isomer of the same structure but having a different configuration at the double bond (ie, an E or Z isomer, or a cis/trans isomer). For purposes of illustration, Figure 1 shows an example of a pair of two geometric isomers, where the only difference between the two isomers is the configuration at the double bond.

在此使用之術語互變異構物意指可經互變異構作用轉換之化合物。互變異構轉換是業界已知的，通常指圖例2所示之反應。在大部分之情況下，該反應中之基團G是氫；以及X、Y與Z彼此獨立地為碳原子。 The term tautomer as used herein refers to a compound that can be converted by tautomerism. Tautomeric conversion is known in the art and generally refers to the reaction shown in Figure 2. In most cases, the group G in the reaction is hydrogen; and X, Y and Z are each independently a carbon atom.

在此使用之術語"鹽"應理解為包括具有陽離子作為相對離子，諸如鹼金屬離子(如，Na、K等等)、鹼土金屬離子(如，Mg²⁺ 、Ca²⁺ 等等)、銨離子(如，NH₄ 或有機銨離子)等等之鹽類，以及具有陰離子作為相對離子，諸如無機陰離子(如，Cl、SO₄ 、Br、HSO₄ 等等)或有機陰離子(如，羧酸陰離子，諸如甲酸鹽、醋酸鹽等等)之鹽類。The term "salt" as used herein is understood to include having a cation as a relative ion, such as an alkali metal ion (eg, Na, K, etc.), an alkaline earth metal ion (eg, Mg ²⁺ , Ca ^{2+ ,} etc.), ammonium. Salts of ions (eg, NH ₄ or organic ammonium ions), and the like, and having anions as relative ions, such as inorganic anions (eg, Cl, SO ₄ , Br, HSO _{4 ,} etc.) or organic anions (eg, carboxylic acids) Salts of anions such as formates, acetates, and the like.

在根據本申請案之從京尼平與麩胺酸鈉或麩胺酸之反應衍生而得之梔子藍色素之實施例中，該梔子藍色素另外具有下列特徵： GPC之分子量：600-5000Da，較佳地1000-4000 Da，更佳地1200-3000Da；亮藍色部分之含量：18-45重量%，較佳地20-40重量%，更佳地25-35重量%，和/或；等電點：1.5-3.0，較佳地1.8-2.8，以及更佳地1.9-2.2。In the examples of gardenia blue pigment derived from the reaction of genipin with sodium glutamate or glutamic acid according to the present application, the gardenia blue pigment additionally has the following characteristics: GPC molecular weight: 600- 5000Da, preferably 1000-4000 Da, more preferably 1200-3000 Da; content of bright blue portion: 18-45 wt%, preferably 20-40 wt%, more preferably 25-35 wt%, and / Or; isoelectric point: 1.5-3.0, preferably 1.8-2.8, and more preferably 1.9-2.2.

在本申請案中，該亮藍色部分指的是根據以下所提之方法獲得之梔子藍色素中，具有最大吸收波長在600nm以上之部分。In the present application, the bright blue portion refers to a portion of gardenia blue pigment obtained by the method described below having a maximum absorption wavelength of 600 nm or more.

在第二態樣中，本申請案提供一種用於製備以上梔子藍色素之方法，其包含下列步驟： a)用葡萄糖苷酶處理梔子苷，以獲得一水解產物；以及 b)用一溶劑萃取於步驟a)中所獲得之該水解產物，以及在該萃取後移除該溶劑以獲得一含京尼平的產物；以及 c)使於步驟b)中所獲得之該含京尼平的產物與胺基酸和/或其鹽之一水溶液反應，以產生該梔子藍色素，以及較佳地將該含京尼平的產物溶於一水溶性溶劑中，產生一溶液，且就這樣使用該溶液。In a second aspect, the present application provides a method for the preparation of the above gardenia blue pigment, comprising the steps of: a) treating geniposide with glucosidase to obtain a hydrolyzate; and b) using one Solvent extraction of the hydrolyzate obtained in step a), and removal of the solvent after the extraction to obtain a product containing genipin; and c) the genipin obtained in step b) The product is reacted with an aqueous solution of an amino acid and/or a salt thereof to produce the gardenia blue pigment, and preferably the genipin-containing product is dissolved in a water-soluble solvent to produce a solution, and This solution was used in this way.

在該方法中，用作為原料之梔子苷可來自各種來源。其可經由任何已知之方法，例如中國專利公開案CN102732050A中所揭示之方法，萃取山黃梔(Gardenia Jasminoides Ellis )果實而獲得。此外，梔子苷粉，其含有約20重量%至70重量%之梔子苷且可從市場上購得，以及梔子黃生產之廢物流，其含有約40重量%之梔子苷且亦可從市場上購得，可直接用於該方法或在例如超過濾之簡單的精製後使用於該方法(見CN103509368A、CN103525883A等等)。In this method, geniposide used as a raw material can be derived from various sources. It can be obtained by extracting the fruit of Gardenia Jasminoides Ellis by any known method, for example, the method disclosed in Chinese Patent Publication No. CN102732050A. Further, geniposide powder, which contains about 20% to 70% by weight of geniposide and is commercially available, and a waste stream produced from gardenia yellow, which contains about 40% by weight of geniposide and is also It is commercially available and can be used directly in the process or after simple refining such as ultrafiltration (see CN103509368A, CN103525883A, etc.).

根據Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes by the Reactions they Catalyse，葡萄糖苷酶是EC 3.2.1下之一種酵素。較佳地，葡萄糖苷酶是纖維素酶(EC 3.2.1.4)或β-葡萄糖苷酶(EC 3.2.1.21)或其混合物。葡萄糖苷酶之例子包括，但不限於，可從Sunson Biotechnology Co. Ltd. (Guangzhou, China)購得之纖維雙糖酶、可從DSM (China) Ltd. (Shanghai, China)購得之纖維素酶4000以及可從DSM (China) Ltd. (Shanghai, China)購得之Rapidase®。葡萄糖苷酶可於步驟a)中添加，每克梔子苷中添加之數量範圍從10U至1000U，較佳地數量範圍從50U至500U，更佳地數量範圍從100U至400U，最佳地數量範圍從200U至300U。According to the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes by the Reactions they Catalyse, the glucosidase is an enzyme under EC 3.2.1. Preferably, the glucosidase is a cellulase (EC 3.2.1.4) or a beta-glucosidase (EC 3.2.1.21) or a mixture thereof. Examples of glucosidases include, but are not limited to, cellobiase available from Sunson Biotechnology Co. Ltd. (Guangzhou, China), cellulose available from DSM (China) Ltd. (Shanghai, China). Enzyme 4000 and Rapidase® available from DSM (China) Ltd. (Shanghai, China). Glucosidase may be added in step a), the amount added per gram of geniposide ranges from 10 U to 1000 U, preferably in an amount ranging from 50 U to 500 U, more preferably in an amount ranging from 100 U to 400 U, optimally. The range is from 200U to 300U.

該步驟a)中之處理可在pH於3.0至6.5之範圍內，較佳地在pH於3.6至6.0之範圍內以及更佳地在pH於4.0至4.6之範圍內進行。較佳地，該步驟a)中之處理在可提供以上之pH範圍之緩衝溶液中進行。此緩衝溶液是業界已知的，例子包括，但不限於，HCOOH/NaOAc水溶液或檸檬酸/Na₂ HPO₄ 水溶液。每克梔子苷中可添加該緩衝溶液之數量範圍從8mL至80mL，較佳地數量範圍從10mL至50mL，更佳地數量範圍從20mL至40mL。The treatment in this step a) can be carried out at a pH in the range of from 3.0 to 6.5, preferably in the range of from 3.6 to 6.0, and more preferably in the range of from 4.0 to 4.6. Preferably, the treatment in step a) is carried out in a buffer solution which provides the above pH range. This buffer solution is known in the art and examples include, but are not limited to, HCOOH/NaOAc aqueous solution or citric acid/Na ₂ HPO ₄ aqueous solution. The buffer solution may be added in an amount ranging from 8 mL to 80 mL per gram of geniposide, preferably in an amount ranging from 10 mL to 50 mL, more preferably in an amount ranging from 20 mL to 40 mL.

該步驟a)中之處理可在從約20^o C至約60^o C之溫度範圍內，較佳地約50°C之溫度下進行。The step a) of the process may, preferably be carried out at a temperature of about 50 ° C in the range of from about 20 ^o C to a temperature in the range of about 60 ^o C.

該步驟a)中之處理可持續約10個小時至約30個小時。所獲得的水解產物含有作為主要組份之京尼平以及其它組份。該水解產物可直接用於步驟b)中之萃取。The treatment in this step a) can last from about 10 hours to about 30 hours. The obtained hydrolyzate contains genipin as a main component and other components. This hydrolyzate can be used directly in the extraction in step b).

在先前的方法中，從步驟a)所獲得的水解產物正常情況下是直接與胺基酸反應產生梔子藍色素。然而，從先前的方法中獲得之梔子藍色素暗沈，此對於一些諸如食品以及飲料之產業應用並不好。本發明之發明人意外地發現，額外的萃取步驟b)產生天空藍的顏色，一種亮藍色素，其更適合於食品以及飲料之應用。In the previous method, the hydrolyzate obtained from step a) is normally reacted directly with an amino acid to produce gardenia blue pigment. However, the hazelnut blue pigment obtained from the previous method is dull, which is not good for some industrial applications such as food and beverages. The inventors of the present invention have unexpectedly discovered that the additional extraction step b) produces a sky blue color, a bright blue pigment, which is more suitable for food and beverage applications.

任何熟悉此技藝之人士當能理解，步驟b)中之萃取所使用的溶劑很重要，且可為任何一種適合本發明之目的之溶劑，例如包括，但不限於，二***、醋酸乙酯、丁醇、丁醇與石油和/或己烷之混合物，或其等之混合物。在該溶劑是丁醇與石油和/或己烷之混合物之實施例中，丁醇與己烷和/或己烷之比在從1：5至5：1之範圍內，較佳地在從1：3至3：1之範圍內以及更佳地在從1：2至2：1之範圍內。Anyone skilled in the art will understand that the solvent used in the extraction in step b) is important and can be any solvent suitable for the purposes of the present invention, including, but not limited to, diethyl ether, ethyl acetate, Butanol, a mixture of butanol with petroleum and/or hexane, or a mixture thereof. In embodiments where the solvent is a mixture of butanol and petroleum and/or hexane, the ratio of butanol to hexane and/or hexane is in the range of from 1:5 to 5:1, preferably from It is in the range of 1:3 to 3:1 and more preferably in the range of 1:2 to 2:1.

於步驟b)中，每毫升之水解產物所使用之溶劑的數量可從1mL至5mL，較佳地從1mL至3mL。步驟b)之萃取可在從10°C至60°C之溫度範圍內，較佳地在室溫下進行。根據本發明，可重複步驟b)之萃取二至四次。In step b), the amount of solvent used per ml of hydrolyzate may range from 1 mL to 5 mL, preferably from 1 mL to 3 mL. The extraction of step b) can be carried out at a temperature ranging from 10 ° C to 60 ° C, preferably at room temperature. According to the invention, the extraction of step b) can be repeated two to four times.

在步驟b)中收集有機相並移除溶劑後，獲得該含京尼平的產物。該溶劑亦可回收。該萃取程序以及萃取期間用於收集溶劑與移除/回收溶劑之程序，是熟悉此技藝之人士已知的。因此，在此不再詳細的討論。After the organic phase is collected in step b) and the solvent is removed, the genipin-containing product is obtained. The solvent can also be recovered. This extraction procedure, as well as procedures for collecting solvents and removing/recovering solvents during extraction, are known to those skilled in the art. Therefore, it will not be discussed in detail here.

如業界所知的，該梔子藍色素是京尼平與胺基酸或其鹽之產物。據此，該方法之步驟c)為使於步驟b)中所獲得之該含京尼平的產物與胺基酸或其鹽反應，產生該梔子藍色素。在本發明中，適合該反應之胺基酸可選自於由下列所構成之群組：麩胺酸、***酸、組胺酸、白胺酸、異白胺酸、精胺酸以及其等之任一混合物。該鹽可為任一鹼金屬鹽，諸如鈉鹽。較佳地，該鹽是麩胺酸鈉。本發明之發明人發現，步驟c)中所使用之胺基酸以及鹽亦很重要，因為其等可提供如本發明所揭示之天空藍顏色。As is known in the art, the gardenia blue pigment is a product of genipin and an amino acid or a salt thereof. Accordingly, step c) of the process produces the scorpion blue pigment by reacting the genipin-containing product obtained in step b) with an amino acid or a salt thereof. In the present invention, the amino acid suitable for the reaction may be selected from the group consisting of glutamic acid, phenylalanine, histidine, leucine, isoleucine, arginine, and the like. Any mixture. The salt can be any alkali metal salt such as a sodium salt. Preferably, the salt is sodium glutamate. The inventors of the present invention have found that the amino acids and salts used in step c) are also important because they provide a sky blue color as disclosed herein.

在本發明中，使用該胺基酸或其鹽之水溶液與該含京尼平的產物反應產生該梔子藍色素，以及於步驟c)之反應混合物中，該水溶液所添加之數量為可提供京尼平與該胺基酸間之莫耳比為1：0.5-10，較佳地1：1-8，更佳地1：2-6以及最佳地1：3-4之數量。In the present invention, an aqueous solution of the amino acid or a salt thereof is reacted with the genipin-containing product to produce the scorpion blue pigment, and in the reaction mixture of the step c), the amount of the aqueous solution added is available. The molar ratio between genipin and the amino acid is from 1:0.5 to 10, preferably from 1:1 to 8, more preferably from 1:2 to 6 and most preferably from 1:3-4.

此外，本發明之發明人發現，將該含京尼平的產物溶於水溶性溶劑中形成一均質反應系統，有利於步驟c)中之反應。據此，根據本發明，該步驟b)中所獲得之該含京尼平的產物，較佳地是溶於水溶性溶劑中。該水溶性溶劑可為任何業界已知可溶解該含京尼平的產物者，例子包括，但不限於，C_1-10 烷醇類，諸如甲醇以及乙醇、C_3-10 酮類，諸如丙酮。較佳地，該水溶性溶劑是甲醇、乙醇或丙酮或其等之任一混合物。Further, the inventors of the present invention have found that the homonipine-containing product is dissolved in a water-soluble solvent to form a homogeneous reaction system, which is advantageous for the reaction in the step c). Accordingly, according to the present invention, the genipin-containing product obtained in the step b) is preferably dissolved in a water-soluble solvent. The water-soluble solvent may be any one known in the art to dissolve the product containing genipin, and examples include, but are not limited to, C _1-10 alkanols such as methanol and ethanol, C _3-10 ketones such as acetone. . Preferably, the water soluble solvent is any mixture of methanol, ethanol or acetone or the like.

步驟c)之反應可在約40^o C至90^o C，較佳地約60^o C至80^o C，諸如65^o C、70^o C以及75^o C下進行。可利用任何已知之方法，諸如HPLC以及TLC監控該反應之進程。Step c) may be reacted at about 40 ^o C to 90 ^o C, preferably about 60 ^o C to 80 ^o C, carried out at 65 ^{^o} C, 70 ^o C and 75 ^o C, such as. The progress of the reaction can be monitored by any known method, such as HPLC and TLC.

較佳地，該步驟c)是在pH值從7.0-11，更佳地8-9.5之範圍內進行。在一些實施例中，添加選自於，但不限於，NaOH、KOH、NaCO₃ 以及NaHCO₃ 之鹼，以便調整該步驟c)之反應混合物至適當的pH值。Preferably, step c) is carried out at a pH in the range of from 7.0 to 11, more preferably from 8 to 9.5. In some embodiments, a base selected from, but not limited to, NaOH, KOH, NaCO _{3 ,} and NaHCO ₃ is added to adjust the reaction mixture of step c) to an appropriate pH.

該反應完成後，移除該反應混合物中之有機溶劑以及水，可獲得呈固態之梔子藍色素。據此，本發明之方法任擇地包含通過例如凍乾法或噴霧乾燥法移除該溶劑以及水，以提供梔子藍色素固體之步驟。After completion of the reaction, the organic solvent in the reaction mixture and water are removed to obtain a solid blue pigment. Accordingly, the method of the present invention optionally comprises the step of removing the solvent and water by, for example, lyophilization or spray drying to provide a scorpion blue pigment solid.

任擇地，可通過任何業界已知之程序，諸如超過濾進一步純化該獲得的梔子藍色素，以便獲得甚至更純的梔子藍色素。本發明之方法產生天空藍之梔子藍色素，其比已知方法所產生如群青之藍色更明亮，因此更受一些如飲料產業應用之歡迎。Optionally, the obtained gardenia blue pigment can be further purified by any procedure known in the art, such as ultrafiltration, to obtain even more pure gardenia blue pigment. The method of the present invention produces sky blue scorpion blue pigment which is brighter than the ultramarine blue produced by known methods and is therefore more popular with applications such as the beverage industry.

此外，大部分所獲得的梔子藍色素具有色值＞100，意指在應用時可使用低劑量。此外，通過額外的萃取步驟，意外地可在無複雜的操作下，從該反應混合物中分開以及純化所獲得的梔子藍色素。In addition, most of the obtained gardenia blue pigments have a color value > 100, meaning that a low dose can be used when applied. Furthermore, by an additional extraction step, the obtained gardenia blue pigment can be accidentally separated and purified from the reaction mixture without complicated operations.

據此，本發明之第三態樣提供如本發明之梔子藍色素於食品以及飲料產業上之用途。Accordingly, a third aspect of the present invention provides the use of the garden blue pigment of the present invention in the food and beverage industries.

在第四態樣方面，本發明提供含如本發明之梔子藍色素之飲料以及食品。In a fourth aspect, the present invention provides a beverage comprising the gardenia blue pigment of the present invention and a food product.

通過下列範例進一步說明本發明。此等範例不以任何式限制本發明。範例 The invention is further illustrated by the following examples. These examples do not limit the invention in any way. example

在下列範例中，最大吸收波長以及色值是依照中國之國家標準GB 28311-2012測量。範例1 In the following examples, the maximum absorption wavelength and color value are measured in accordance with China's national standard GB 28311-2012. Example 1

將從Jiatian Biotechnology Co., Ltd. (西安，中國)購得之6.7g的梔子苷粉(35.5重量%)加至47ml之檸檬酸/Na₂ HPO₄ 緩衝水溶液(pH4.0)中。再於反應中加入從Sunson Biotechnology Co. Ltd. (廣州，中國)購得之685.7mg (1000U/g)的纖維雙糖酶，在50^o C下歷時19個小時。在使該反應混合物冷卻至室溫後，用90ml的醋酸乙酯萃取該反應混合物二次。在真空下濃縮醋酸乙酯相，獲得粗製京尼平。6.7 g of geniposide powder (35.5 wt%) commercially available from Jiatian Biotechnology Co., Ltd. (Xi'an, China) was added to 47 ml of a citric acid/Na ₂ HPO ₄ buffered aqueous solution (pH 4.0). And then added to the reaction from Sunson Biotechnology Co. Ltd. (Guangzhou, China) The commercially 685.7mg (1000U / g) of cellobiose enzymes, over at 50 ^o C 19 hours. After the reaction mixture was allowed to cool to room temperature, the reaction mixture was extracted twice with 90 ml of ethyl acetate. The ethyl acetate phase was concentrated under vacuum to give crude genipin.

將所獲得的粗製京尼平溶於16ml的絕對乙醇中，獲得19.4g的溶液(4.79重量%之京尼平，HPLC)。於該溶液中加入配製於10ml的去離子水中之970mg的麩胺酸鈉，在70^o C下反應25個小時。TLC指出京尼平已經完全轉換。The obtained crude genipin was dissolved in 16 ml of absolute ethanol to obtain 19.4 g of a solution (4.79 wt% of genipin, HPLC). Was added 970mg formulated in deionized water of 10ml of sodium bran amine in the solution, reacted at 70 ^o C 25 hours. TLC pointed out that Kinipin has been completely converted.

將該反應液體凍乾，獲得2.1g呈固態粉末狀的梔子藍色素，具最大吸收波長為601nm以及色值為155。範例2 The reaction liquid was lyophilized to obtain 2.1 g of gardenia blue pigment in a solid powder having a maximum absorption wavelength of 601 nm and a color value of 155. Example 2

將從Zhongda HengYuan Biotechnology Co., Ltd. (海南，中國)購得之140g的液態梔子苷(40重量%)與980ml的檸檬酸/Na₂ HPO₄ 緩衝水溶液(pH4.0)混合。將該混合物加熱至50^o C，然後加入從Sunson Biotechnoloty Co. Ltd. (寧夏，中國)購得之42g (400U/g)的纖維二糖酶。在50^o C下攪拌約12個小時後，將該混合物過濾，移除未溶解的固體，同時用約1L的醋酸乙酯萃取二次。利用蒸發移除醋酸乙酯，獲得粗製京尼平，經過HPLC後，純度為92.7%。140 g of liquid geniposide (40% by weight) commercially available from Zhongda HengYuan Biotechnology Co., Ltd. (Hainan, China) was mixed with 980 ml of a citric acid/Na ₂ HPO ₄ buffered aqueous solution (pH 4.0). The mixture was heated to 50 ^o C, followed by addition of the enzyme from the cellobiose Sunson Biotechnoloty Co. Ltd. (Ningxia, China) The commercially 42g (400U / g) in the. After stirring at 50 ^o C for about 12 hours, the mixture was filtered to remove undissolved solids, and extracted twice with ethyl acetate to about 1L. The ethyl acetate was removed by evaporation to obtain crude genipin. After HPLC, the purity was 92.7%.

將6g所獲得的粗製京尼平溶於90ml的絕對乙醇中。使配製於90ml去離子水中之18.5g的麩胺酸鈉與該粗製京尼平乙醇溶液混合。在75^o C下攪拌該混合物約6個小時。6 g of the obtained crude genipin was dissolved in 90 ml of absolute ethanol. 18.5 g of sodium glutamate formulated in 90 ml of deionized water was mixed with the crude genipin ethanol solution. The mixture was stirred at 75 ^o C for about 6 hours.

使該反應溶液蒸發，移除乙醇，之後凍乾獲得約19g呈固態粉末狀之梔子藍產物，具有最大吸收波長為601nm以及色值為約80。範例3 The reaction solution was evaporated, ethanol was removed, and then lyophilized to obtain about 19 g of a gardenia blue product in a solid powder having a maximum absorption wavelength of 601 nm and a color value of about 80. Example 3

本申請案之梔子藍色素之顏色(明度、彩度以及色相)是用HunterLab Ultra Scan Pro分光色度計(Hunter Associates Laboratory, Reston, VA,USA)測定，然後根據CIELAB色標表示。所使用的模型是RSIN，其代表包括鏡面反射。選擇直徑為4.826mm (0.190吋)之小區域視圖(SAV)。顏色之測量依照CIE指南(Commission International d’Eclairage)進行。值表示為平面座標L*，a*，b*，L*為明度之測量值、a*為紅綠軸上之值以及b*為黃藍軸上之值。The color (lightness, chroma, and hue) of the scorpion blue pigment of this application was measured using a HunterLab Ultra Scan Pro spectrophotometer (Hunter Associates Laboratory, Reston, VA, USA) and then expressed according to the CIELAB color scale. The model used is RSIN, the representation of which includes specular reflection. Select a small area view (SAV) with a diameter of 4.826mm (0.190吋). Color measurements were made in accordance with the CIE Guidelines (Commission International d’Eclairage). Values are expressed as plane coordinates L*, a*, b*, L* are measured values of lightness, a* is the value on the red-green axis, and b* is the value on the yellow-blue axis.

彩度(C*)有時稱作飽和度，描述一顏色之鮮艷或暗淡，其可由下式計算得： C*=√(a*² +b*² )Chroma (C*), sometimes called saturation, describes the vivid or dim color of a color, which can be calculated from: C*=√(a* ² +b* ² )

稱作色相(h)之角度描述如何看待物體的顏色，且可由下式計算得： h=tan(b/a)^(-1) The angle called hue (h) describes how the color of the object is viewed and can be calculated from: h=tan(b/a) ^(-1)

儀器設定： • 色標是CIE L*a*b*/L*C*h • 光源定義：D65日光等效 • 幾何：漫射/8° • 波長：掃描350-1050nm •樣本測量面積直徑：4.826mm • 校正模式：包括鏡面反射Instrument settings: • Color code is CIE L*a*b*/L*C*h • Light source definition: D65 daylight equivalent • Geometry: Diffuse / 8° • Wavelength: Scan 350-1050nm • Sample measurement area diameter: 4.826 Mm • Correction mode: including specular reflection

將範例1與2獲得的梔子藍溶於去離子水中，提供具有最大吸收度為0.337之測試溶液供顏色之測試。與二種商購梔子藍色素(從Qiangjiang Green Sea Treasure Biotechnology Co. Ltd購得之GB1以及從Zhuhai Golden Land Natural Colors Co., Ltd.購得之GB2)比較之測試結果總結於以下表1中。表1 範例4 包含在如本申請案之梔子藍中之化合物的化學式識別如下：The gardenia blue obtained in Examples 1 and 2 was dissolved in deionized water to provide a test for the color of the test solution having a maximum absorbance of 0.337. The test results compared with two commercially available gardenia blue pigments (GB1 purchased from Qiangjiang Green Sea Treasure Biotechnology Co. Ltd and GB2 purchased from Zhuhai Golden Land Natural Colors Co., Ltd.) are summarized in Table 1 below. . Table 1 Example 4 The chemical formulas of the compounds contained in Gardenia Blue as in the present application are as follows:

將15.5mg範例1或2中所獲得的梔子藍產物溶於乙腈/水(50/50，V/V)之混合物中，並在10ml定量瓶中補充體積，得到一測試溶液。注入3ml的該測試溶液，進行HPLC-DAD-MS分析。15.5 mg of the gardenia blue product obtained in Example 1 or 2 was dissolved in a mixture of acetonitrile/water (50/50, V/V), and the volume was replenished in a 10 ml dosing bottle to obtain a test solution. 3 ml of this test solution was injected and subjected to HPLC-DAD-MS analysis.

該HPLC系統由1260四元泵、1290自動取樣器、1290恆慍管柱室以及1290二極體陣列檢測器構成(Agilent Technologies, Waldbronn, Germany)。所使用的質譜儀是裝設有Apollo II電噴霧離子源之Bruker Maxis 4G UHR-QTOF-MS，以及轉向軟體是Compass QTOF (四極柱飛行時間質譜儀)控制版本3.2. (all Bruker Daltonik GmbH, Bremen, Germany)。The HPLC system consisted of a 1260 quaternary pump, a 1290 autosampler, a 1290 constant pressure column chamber, and a 1290 diode array detector (Agilent Technologies, Waldbronn, Germany). The mass spectrometer used was a Bruker Maxis 4G UHR-QTOF-MS equipped with an Apollo II electrospray ion source, and the steering software was a Compass QTOF (Quadrupole Time-of-Flight Mass Spectrometer) control version 3.2. (all Bruker Daltonik GmbH, Bremen , Germany).

在YMC Pro C4 3.0x150mm管柱上施用三元移動相梯度，以0.5ml/min之恆定速度分開該測試樣本。移動相是(A)水、(B)含0.1%甲磺酸的水以及(C)乙腈。梯度排程之時間表如下(表2)：表2 A ternary mobile phase gradient was applied to a YMC Pro C4 3.0 x 150 mm column and the test samples were separated at a constant rate of 0.5 ml/min. The mobile phase is (A) water, (B) water containing 0.1% methanesulfonic acid, and (C) acetonitrile. The schedule for the gradient schedule is as follows (Table 2): Table 2

所獲得的UV-VIS光譜是在5Hz下記錄從200-600nm。質譜儀之離子源參數如下：端板偏移500V、毛細管電壓3500V、噴霧器壓力3.0巴、乾燥氣體9.0 l/min、乾燥溫度250°C。該質譜儀是以正MS模式掃描，掃描範圍為60-2000m/z以及光譜率為1Hz。質量軸之校正是使用醋酸鈉簇之參考質量，其是在各個分析開始時輸注20ml的10mM醋酸鈉溶液後之來源中產生的。此外，使用六(2,2-二氟乙氧基)磷腈進行m/z 622.0290之鎖定質量校正。為此目的，在分析前，將20ml的1000mg/ml之磷腈溶液加至鎖定質量容器中。使用數據分析軟體版本4.3 (Bruker Daltonik GmbH, Bremen, Germany)進行反褶積以及峰檢測(見圖2)以及使用Bruker智能公式演算法從MS數據計算離子分子式。The UV-VIS spectrum obtained was recorded from 200-600 nm at 5 Hz. The ion source parameters of the mass spectrometer were as follows: end plate offset 500V, capillary voltage 3500V, nebulizer pressure 3.0 bar, dry gas 9.0 l/min, drying temperature 250 °C. The mass spectrometer was scanned in a positive MS mode with a scan range of 60-2000 m/z and a spectral rate of 1 Hz. The mass axis calibration was based on the reference mass of sodium acetate clusters generated at the source after infusion of 20 ml of 10 mM sodium acetate solution at the beginning of each analysis. In addition, the lock mass correction of m/z 622.0290 was performed using hexa(2,2-difluoroethoxy)phosphazene. For this purpose, 20 ml of a 1000 mg/ml phosphazene solution was added to the lock mass vessel prior to analysis. Deconvolution and peak detection were performed using data analysis software version 4.3 (Bruker Daltonik GmbH, Bremen, Germany) (see Figure 2) and ion molecular formulas were calculated from MS data using the Bruker smart formula algorithm.

於圖2中，提取峰65與80獲得離子分子式C₃₂ H₃₁ N₂ O₁₂ ，具m/z 635.4872，提取峰55、69、71獲得離子分子式C₃₃ H₃₃ N₂ O₁₂ ，具m/z 649.2029以及提取峰61、76獲得離子分子式C₃₄ H₃₅ N₂ O₁₂ ，具m/z 663.2183。範例5 利用如下之GPC測定本申請案之梔子藍之分子量：使用從180至47100Da之Pullulan/Dextran標準品校正該GPC系統(Polymer Standards Service GmbH, Germany)。將在範例1或2中獲得的梔子藍產物溶於GPC洗提液(見表3)中，並通過1µm的PTFE濾器過濾。以表3所示之條件進行該GPC：表3 In Fig. 2, peaks 65 and 80 are extracted to obtain an ion molecular formula C ₃₂ H ₃₁ N ₂ O ₁₂ with m/z 635.4872, and peaks 55, 69, and 71 are obtained to obtain an ion molecular formula C ₃₃ H ₃₃ N ₂ O ₁₂ with m/ z 649.2029 and the extracted peaks 61, 76 obtained the ionic molecular formula C ₃₄ H ₃₅ N ₂ O ₁₂ with m/z 663.2183. Example 5 The molecular weight of Gardenia Blue of the present application was determined by GPC as follows: The GPC system (Polymer Standards Service GmbH, Germany) was calibrated using a Pullulan/Dextran standard from 180 to 47100 Da. The gardenia blue product obtained in Example 1 or 2 was dissolved in a GPC eluate (see Table 3) and filtered through a 1 μm PTFE filter. The GPC was carried out under the conditions shown in Table 3: Table 3

使用PSS-WinGPC UniChrom Version 8.2計算分子量分佈，結果示於圖3中。範例6 The molecular weight distribution was calculated using PSS-WinGPC UniChrom Version 8.2, and the results are shown in Fig. 3. Example 6

本申請案之梔子藍色素之亮藍部分之含量測定如下： HPLC分析在具有DAD檢測器系統(Agilent, USA)以及6130 Quadrupole MS系統(Agilent, USA)之Agilent HPLC 1260中進行。The content of the bright blue portion of the garden blue pigment of the present application was determined as follows: HPLC analysis was carried out in an Agilent HPLC 1260 with a DAD detector system (Agilent, USA) and a 6130 Quadrupole MS system (Agilent, USA).

將5mg範例1或2中所獲得的梔子藍溶於1mL水中作為測試溶液。在Supelco C18, 75 mm x 4.6 mm x 5 um管柱中，以表4中所述的1.0ml/min相梯度的恆定流速分開該測試溶液樣本。移動相是(A) 0.01% MSA配製於水中，(B) 0.01% MSA配製於CAN中。梯度排程之時間表如下(表4)：表4 5 mg of gardenia blue obtained in Example 1 or 2 was dissolved in 1 mL of water as a test solution. The test solution samples were separated in a Supelco C18, 75 mm x 4.6 mm x 5 um column at a constant flow rate of 1.0 ml/min phase gradient as described in Table 4. The mobile phase was (A) 0.01% MSA in water, and (B) 0.01% MSA in CAN. The schedule for the gradient schedule is as follows (Table 4): Table 4

所獲得的UV-VIS色譜圖是記錄600nm處，如圖4A以及圖4B。The obtained UV-VIS chromatogram was recorded at 600 nm as shown in Fig. 4A and Fig. 4B.

將該梔子藍之波峰分成每部分0.5min進行積分。平均每部分之UV-Vis吸收光譜，以及識別λmax＞ 600nm之部分。λmax＞ 600nm部分之含量計算如下：。The peak of the scorpion blue was divided into 0.5 min for each part for integration. The average UV-Vis absorption spectrum for each part, as well as the portion identifying λmax > 600 nm. The content of the λmax > 600nm portion is calculated as follows: .

如圖4A以及4B所示，在範例1以及2中所獲得的梔子藍產物中，具最大吸收波長大於600nm之部分的含量分別為23%以及41% (面積%)。範例7 As shown in FIGS. 4A and 4B, in the gardenia blue product obtained in Examples 1 and 2, the content of the portion having the maximum absorption wavelength of more than 600 nm was 23% and 41% (area%), respectively. Example 7

在攪拌、室溫下，將10mg之在範例1或2中所獲得的梔子藍產物溶於100mL的軟化水中。之後，將此溶液進一步稀釋直至最後溶液幾乎沒有顯示出任何顏色。用Zetasizer Nano ZS (Malvern, England)，依照製造商提供的使用手冊測定該溶液之等電點。每一個樣本測試二次，數據平均如下(表5)。表5 10 mg of the gardenia blue product obtained in Example 1 or 2 was dissolved in 100 mL of demineralized water under stirring at room temperature. Thereafter, the solution was further diluted until the final solution showed almost no color. The isoelectric point of the solution was determined using a Zetasizer Nano ZS (Malvern, England) according to the manufacturer's instruction manual. Each sample was tested twice and the data was averaged as follows (Table 5). table 5

（無）(no)

圖1 顯示具式(a)-(h)之化合物之化學式。 Figure 1 shows the chemical formula of a compound of formula (a)-(h).

圖2 顯示如本申請案之梔子藍產物之HPLC-DAD-MS量變曲線。 Figure 2 shows the HPLC-DAD-MS quantitative curve of the gardenia blue product as in the present application.

圖3 顯示如本申請案之梔子藍產物之GPC的分子量分佈量變曲線。 Figure 3 shows the molecular weight distribution variation curve of GPC of the gardenia blue product as in the present application.

圖4A-4B 顯示如本申請案之梔子藍產物之UV-VIS色譜圖。 4A-4B show UV-VIS chromatograms of the gardenia blue product as in the present application.

（無）(no)

Claims

一種梔子藍色素，其在CIELAB色標(color scale)上具有從220至245，較佳地從230至240之範圍內的色調(color shade)h，以及在CIELAB色標上具有從-12至-7，較佳地從-11至-9之範圍內的色值“a*”。A gardenia blue pigment having a color shade h ranging from 220 to 245, preferably from 230 to 240, on a CIELAB color scale, and having from -12 on a CIELAB color scale To -7, preferably the color value "a*" in the range from -11 to -9.

如請求項1之梔子藍色素，其從京尼平(genipin)與胺基酸之反應衍生而得。Such as the scorpion blue pigment of claim 1, which is derived from the reaction of genipin with an amino acid.

如請求項1或2之梔子藍色素，其含有一或多種具式(I)-(IV)之化合物、其幾何異構物、其互變異構體、其鹽或其等之組合：其中：R彼此獨立地為H或C_1-7 烷基，較佳地為甲基；以及R’彼此獨立地選自於由下列所構成之群組：C_1-7 烷基，較佳地為乙基、丙基、丁基以及戊基，任擇地經羧基、苯基、咪唑基或胍基基團取代。A gardening blue pigment of claim 1 or 2 which comprises one or more compounds of formula (I)-(IV), geometric isomers thereof, tautomers thereof, salts thereof or combinations thereof: Wherein: R is independently of each other H or a C _1-7 alkyl group, preferably a methyl group; and R' is independently selected from the group consisting of C _1-7 alkyl groups, preferably It is an ethyl group, a propyl group, a butyl group and a pentyl group, optionally substituted by a carboxyl group, a phenyl group, an imidazolyl group or a fluorenyl group.

如請求項1至3中任一或多項之梔子藍色素，其中該具式(I)-(IV)之化合物是具式(a)-(h)之化合物、其幾何異構物、其互變異構物、其鹽以及其等之組合： A gardening blue pigment according to any one or more of claims 1 to 3, wherein the compound of the formula (I)-(IV) is a compound of the formula (a)-(h), a geometric isomer thereof, Tautomers, their salts, and combinations of these:

如請求項2至3中任一或多項之梔子藍色素，其中該胺基酸是麩胺酸鈉。A gardening blue pigment according to any one or more of items 2 to 3, wherein the amino acid is sodium glutamate.

如請求項5之梔子藍色素，其另外具有下列特徵： GPC之分子量：600-5000Da，較佳地為1000-4000 Da，更佳地為1200-3000 Da；亮藍色部分之含量：18-45重量%，較佳地為20-40重量%，更佳地為25-35重量%，和/或；等電點：1.5-3.0，較佳地為1.8-2.8，以及更佳地為1.9-2.2。The gardening blue pigment of claim 5 additionally has the following characteristics: GPC molecular weight: 600-5000 Da, preferably 1000-4000 Da, more preferably 1200-3000 Da; bright blue portion content: 18 -45% by weight, preferably 20-40% by weight, more preferably 25-35% by weight, and/or; isoelectric point: 1.5-3.0, preferably 1.8-2.8, and more preferably 1.9-2.2.

一種製備如請求項1-6中任一或多項之梔子藍色素之方法，其包含下列步驟： a)用葡萄糖苷酶處理梔子苷，以獲得一水解產物；以及 b)用一溶劑萃取於步驟a)中所獲得之該水解產物，以及在該萃取後移除該溶劑以獲得一含京尼平的產物；以及 c)使於步驟b)中所獲得之該含京尼平的產物與胺基酸和/或其鹽之一水溶液反應，以產生該梔子藍色素，以及較佳地將該含京尼平的產物溶於一水溶性溶劑中，產生一溶液，且就這樣使用該溶液。A method of preparing gardenia blue pigment according to any one or more of claims 1 to 6, comprising the steps of: a) treating geniposide with glucosidase to obtain a hydrolyzate; and b) extracting with a solvent The hydrolyzate obtained in step a), and removing the solvent after the extraction to obtain a product containing genipin; and c) the genipin-containing product obtained in step b) Reacting with an aqueous solution of an amino acid and/or a salt thereof to produce the gardenia blue pigment, and preferably dissolving the product containing genipin in a water-soluble solvent to produce a solution, and thus using The solution.

如請求項7之方法，其中該葡萄糖苷酶是纖維素酶(EC 3.2.1.4)或β-葡萄糖苷酶(EC 3.2.1.21)或其混合物。The method of claim 7, wherein the glucosidase is cellulase (EC 3.2.1.4) or β-glucosidase (EC 3.2.1.21) or a mixture thereof.

如請求項7或8之方法，其中在該步驟b)中所使用之該溶劑為二***、醋酸乙酯、丁醇、丁醇與石油和/或己烷之混合物，或其等之混合物。The method of claim 7 or 8, wherein the solvent used in the step b) is diethyl ether, ethyl acetate, butanol, a mixture of butanol and petroleum and/or hexane, or a mixture thereof.

如請求項7至9中任一或多項之方法，其中在該步驟c)中所使用的胺基酸是選自於由下列所構成之群組：麩胺酸、***酸、組胺酸、白胺酸、異白胺酸、精胺酸以及其等之混合物。The method of any one or more of clauses 7 to 9, wherein the amino acid used in the step c) is selected from the group consisting of glutamic acid, phenylalanine, histidine, A mixture of leucine, isoleucine, arginine, and the like.

一種如請求項1-6中任一或多項之梔子藍色素於食品以及飲料產業上之用途。A use of gardenia blue as claimed in any one or more of claims 1-6 in the food and beverage industry.

一種含有如請求項1-6中任一或多項之梔子藍色素之飲料。A beverage containing gardenia blue pigment as claimed in any one or more of claims 1-6.

一種含有如請求項1-6中任一或多項之梔子藍色素之食品。A food product comprising gardenia blue pigment according to any one or more of claims 1-6.