JPS6119234B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the production of blue pigments produced by β-glucosidase of gardenia iridoid glycosides, and in particular to an improved method for producing lightened and vivid blue pigments with good quality reproducibility by industrially easy means. . More specifically, the present invention combines a gardenia iridoid glycoside or a substance containing it and β-glucosidase or a substance containing it in the presence of a primary amino group-containing substance under aerobic conditions (molecular oxygen or (in the presence of molecular oxygen-containing gas),
β-glucosidase color of gardenia iridoid glycoside When forming a blue pigment, the glycoside or its containing substance and the β-glucosidase or its containing substance are prepared in advance under microaerobic conditions (lower than oxygen requirement). (oxygen amount conditions), for example, under non-stirring conditions for a sufficient amount of time, preferably about 10 hours or more, and then under aerobic conditions (oxygen amount conditions exceeding the required oxygen amount), for example, under stirring conditions (active or forced oxygen conditions). The present invention relates to a method for producing a lightened natural blue pigment, preferably having a maximum absorption wavelength in the visible region of 594 mμ or more, which is further allowed to react for about 5 hours or more under (aerobic conditions). It is well known that gardenia fruits contain iridoid glycosides, and when certain iridoid glycosides are treated with β-glucosidase, they produce red to
It has been known for a long time that it produces colors ranging from purple to blue (Tetrahedron Letters, 2347-2350
Page, 1969). Furthermore, iridoid glycosides are β-
Genipin, which is formed by enzymatic decomposition with enzymes such as glucosidase, easily reacts with amino acids such as glycine, leucine, and glutamic acid, which contain primary amino groups, giving it a blue-purple color, and when it comes into contact with the skin, it turns the skin dark purple. It has been well known for a long time that organic chemistry changes (Journal of Organic Chemistry, Vol. 25, 2174-2177)
Page, 1960). Furthermore, JP-A No. 52-53934 discloses a follow-up test of the reaction between genipin and a primary amino group, in which genipin obtained by enzymatic decomposition of an iridoid glycoside is collected, and this genipin and monomethylamine hydrochloride are combined. When the reaction with salt is carried out under a nitrogen stream with the air completely removed, the color tone changes from yellow to yellow-orange to brown, and the known reddish-purple color tone obtained under aerobic conditions cannot be obtained. It has been shown that It has been shown that when oxygen is introduced into this and the reaction is carried out under aerobic conditions, a reddish-purple color tone is obtained.
In addition, this Japanese Patent Application Laid-Open No. 52-53934 discloses that geniposide is dissolved in 2% hydrochloric acid water and decomposed by heating, and this solution is
Adjust the pH to 8 and further heat reaction with glycine.
It is shown that a dark purple dye with a visible absorption maximum of 593 nm was formed. In this way, gardenia iridoid glycosides or their containing substances are allowed to react with β-glucosidase or its containing substances in the presence of a primary amino group-containing substance under aerobic conditions to produce gardenia iridoid glycosides. It has been known for a long time that β-glucosidase in the body causes the formation of color pigments. This coloration occurs when genipins, which are formed by chemically, microbially, or enzymatically decomposing iridoid glycosides, react with substances containing primary amino groups, and this reaction product polymerizes in the presence of oxygen. The color tone can vary over a wide range, such as yellow-green, green, green-blue, blue, red-purple, and dark purple. However, it is possible to obtain a bright and clear blue gardenia iridoid glycoside β-glucosidase coloring pigment by an industrially easy means and with good quality reproducibility without blending with other natural or synthetic pigments. was difficult. The present inventors have conducted research to develop a method for producing a blue color pigment produced by β-glucosidase of gardenia iridoid glycoside, which exhibits a bright and clear blue color, by an industrially easy means and with good quality reproducibility. It came. As a result, β-
The glucosidase coloring pigment forming reaction is carried out under controlled aerobic conditions, that is, the reaction is carried out under conditions that satisfy the binding conditions of the reaction under microaerobic conditions and the reaction under aerobic conditions. In particular, it has been discovered that a brightened and vivid blue colorant can be formed easily and with good reproducibility of color tone quality. Furthermore, such controlled aerobic conditions require that gardenia iridoid glycosides or substances containing them be reacted with β-glucosidase or substances containing them in advance, for example, without stirring. This can be carried out by allowing the reaction system to react sufficiently under the conditions, and then further acting under the stirring conditions, for example, by stirring to bring the reaction system into active contact with molecular oxygen. Absorption wavelength is 594 mμ or more, e.g. 594 to 601
It has been discovered that a brightened blue colorant of mμ can be formed with good quality reproducibility and with easy operation. The reason for this is not clear, but due to the preliminary action under microaerobic conditions in the first half, the enzymatic sugar separation reaction of gardenia iridoid glycosides and the reaction of the decomposed products with primary amino group-containing substances are facilitated. progresses evenly,
The subsequent polymerization reaction proceeds moderately and uniformly, suppressing its excessive progress and uneven progress, and the latter half of the reaction under aerobic conditions produces a product with a narrow polymerization degree distribution and an appropriate degree of polymerization. However, because it is formed homogeneously, it is speculated that a brightened and vivid blue pigment is selectively formed. Of course, it should be understood that the present invention is not limited in any way by such speculation. Therefore, the object of the present invention is to provide a natural blue pigment that exhibits brightened and vivid blue color and can be produced by β-glucosidase coloring of gardenia iridoid glycosides with good color tone quality reproducibility and by easy means. To provide the manufacturing method. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. Examples of the gardenia iridoid glycoside or its containing substance used in the method of the present invention include crushed gardenia fruit, extracts and concentrates thereof, dried products, and iridoid glycosides separated therefrom. Many gardenia iridoid glycosides are known, such as geniposide, methyl deacetylasperloside,
Examples include genipin gentiopicide, and in the present invention, such gardenia iridoid glycosides or substances containing them can be used. To obtain extracts from gardenia fruits, e.g.
The crushed gardenia fruit can be obtained by extraction with water or organic solvents such as methanol, ethanol, isopropanol, acetone, water or any mixture thereof. Further solvent can be removed to obtain a concentrate. Further, as the β-glucosidase or the substance containing it used in the method of the present invention, any substance having a β-glucosidase action to hydrolyze iridoid glycosides can be used regardless of its origin. Specific examples include acylase (Amano Pharmaceutical Products), biozyme (Amano Pharmaceutical Products), pronase (Kaken Kagaku), Nakaze Bromelain G (Nagase Sangyo), protease "Amano" (Amano Pharmaceutical), morcin (Seishin Pharmaceutical) ), Cellulase P-1500 “Onozuka” (All Nippon Seikagaku), Meicelase P (Meiji Pharmaceutical), Coclase (Sankyo Co., Ltd.), Lipase AP
(Amano Pharmaceutical), A-D-C (Takeda Pharmaceutical), Coclase SS (Sankyo), Coclase (Takadiastase A) (Sankyo), and Driselase N-10-20 (Kyowa Hakko). can be easily obtained at. The amount of β-glucosidase or its containing substance can be selected as appropriate. For example, the amount of β-glucosidase or its containing substance can be selected as appropriate.
About 0.1 to about 30% by weight, more preferably about 0.5 to about
An example of a usage amount is about 10% by weight. Further, examples of primary amino group-containing substances used in the method of the present invention include proteins such as gelatin, casein, albumin, and enzyme proteins; peptides such as peptone and protein hydrolysates; glycine, leucine, and alanine. , valine, aspartic acid, glutamic acid, and the like. The amount of these primary amino group-containing substances to be used can be selected as appropriate, for example, from about 1 to about 80% by weight based on the iridoid glycoside or its containing substance. According to the method of the present invention, a gardenia iridoid glycoside or a substance containing it, as exemplified above;
β-glucosidase or its containing substance,
The reaction is carried out under aerobic conditions in the presence of a substance containing primary amino groups. At this time, the aqueous reaction liquid system containing these is sufficiently worked in advance under microaerobic conditions, for example, under non-stirring conditions. In addition to water, caustic soda, caustic potash, and the like can be added as a PH regulator to form the aqueous reaction solution. According to the method of the present invention, in order to avoid forcible mixing of the reaction liquid system and air as described above, it is preferable to allow the above-mentioned components to act sufficiently in advance, for example, under non-stirring conditions. Most commonly, a method is employed in which the surface of the reaction solution is allowed to stand still under conditions in which air is in contact with the surface of the reaction solution. At this time, the reaction zone may be in an open state or a closed state. The dissolved oxygen concentration in the reaction solution (measured near the center of the reaction solution: dissolved oxygen meter) is preferably less than 0.5 ppm, preferably 0.1 ppm or less, and more preferably around 0 ppm. This pre-action may be carried out for at least about 10 hours, more preferably for about 15 hours or more, such as from about 15 to
A duration of action such as about 60 hours may be exemplified. The working temperature is preferably about 30° to about 70°C, and about 40°C.
°C to about 60 °C is more preferred. The pH of the reaction solution system can be adjusted within a fairly wide range, for example, within a pH range of about 4 to about 9. If the above pre-treatment step is omitted or insufficient, it is impossible to form a brightened and clear blue color pigment, especially bright colors with a maximum absorption wavelength in the visible region of 594 mμ or more, preferably 594 to 601 mμ. It is difficult to form a vivid blue pigment. If this step is carried out under stirring conditions that create aerobic conditions,
Brightened clear blue pigment cannot be obtained.580m
It becomes a pigment with a color tone ranging from μ to 598 μm. In the method of the present invention, the reaction system that has undergone preliminary reaction under microaerobic conditions as described above is then further reacted, for example, under stirring conditions, that is, aerobic conditions. The stirring may be mechanical stirring using a stirrer or stirring by blowing molecular oxygen or a molecular oxygen-containing gas such as air. At this time, it is preferable to conduct the reaction so that the dissolved oxygen concentration in the reaction solution exceeds 0.5 ppm. This reaction under aerobic conditions is preferably carried out for about 5 hours or more, and can be exemplified by a reaction time of about 1 to about 400 hours. Good results are usually obtained in about 10 to about 40 hours. The reaction temperature and system PH can be exemplified by the same conditions as described for the preliminary reaction. After the reaction is completed, the reaction solution is heated to about 70 to 120°C to inactivate the enzyme, and then filtered to obtain a clear blue pigment. The lightened blue pigment liquid obtained as described above can be used in a wide range of fields as it is as a solution or concentrated and further dried as a powder by means such as hot air drying, freeze drying, vacuum drying, etc. It can be used as a natural colorant. When used, it can be used alone or in combination with other colorants. The lightened natural blue pigment obtained by the method of the present invention is useful in fields of application such as food and drink, luxury goods, health pharmaceuticals, and cosmetics. For example, natural coloring agents for food and drinks such as drops, candy, chocolate, ice cream, sherbet, milk drinks, yokan, sweet bean paste, porridge, jelly, boiled beans, dried vegetables, seafood, meat processed foods, and pickles; , natural colorants for health and pharmaceutical products such as tablets, liquid oral medicines, powdered oral medicines and poultices; or alternatively, for example,
It is useful as a natural source colorant in cosmetics such as soaps, detergents, shampoos, eye shadows, mascaras, lip balms and lotions. Hereinafter, several embodiments of the production of the blue pigment of the present invention will be illustrated in more detail with reference to Examples. Example 1 8 kg of water was added to 1 kg of coarsely ground dried gardenia fruit, and the mixture was stirred and extracted at 50°C for 3 hours. Insoluble matter was separated to obtain an extract. This was then concentrated under reduced pressure to obtain 500 g of gardenia fruit concentrate (solid content 70%). Add 590g of warm water (50℃) to 10g of this concentrate and
Dissolve in a volume flask and pH with 1N-NaOH.
5.0 Adjusted. Next, while maintaining the temperature at 50° C., 1 g of acylase (manufactured by Amano Pharmaceutical Co., Ltd.) was added and stirred until dissolved, then stirring was stopped and the mixture was allowed to stand for 15 hours (dissolved oxygen concentration 0.1 ppm or less). Stirring (500 rpm) was started 15 hours later, and the reaction was carried out under stirring conditions (dissolved oxygen concentration 1.5 to 5 ppm) for 40 hours. Next, heat the reaction solution at 90℃ for 30 minutes to inactivate the enzyme.
Heating was carried out for a minute to obtain 570 g of a blue pigment-containing solution. The maximum absorption wavelength of this solution was measured using a Hitachi Model 124 spectrophotometer and was found to be 595 mμ, with an absorbance of 40
It was hot. This solution was concentrated under reduced pressure using an evaporator, and alcohol was added to the concentrate to obtain 40 g of a gardenia blue pigment product (liquid product). Example 2 600 g of warm water (50°C) was added to 100 g of the gardenia extract obtained in Example 1, the mixture was dissolved in a 2-volume flask, and the pH was adjusted to 6.0 with 1N-NaOH. Next, while maintaining the temperature at 50°C, 15 g of acylase (manufactured by Amano Pharmaceutical Co., Ltd.) was added and stirred until dissolved, then stirring was stopped and the mixture was allowed to stand still for 48 hours. Stirring (500 rpm) was started 48 hours later, and the reaction was stirred for 20 hours. Next, the reaction solution was heated at 90° C. for 30 minutes to inactivate the enzyme, yielding 668 g of a blue pigment-containing substance. The maximum absorption wavelength of this solution is 598m
The absorbance was 450. Example 3 8 kg of alcohol was added to 1 kg of coarsely ground dried gardenia fruit, and the mixture was stirred and extracted at 70° C. for 5 hours. Insoluble materials were separated to obtain an extract. The alcohol was then recovered under reduced pressure to obtain 40 g of gardenia alcohol extract.
Put 10g of this extract and 1000g of water into a 2-volume flask, heat and dissolve, then adjust the pH to 5.5 with 1N-NaOH, add 4g of gelatin, and adjust the dissolution temperature to 40℃.
After adding 1.2 g of lipase AP and stirring until it was uniformly dispersed in the reaction solution, the stirring was stopped. After the reaction was allowed to stand at 40°C for 20 hours, stirring (1000 rpm) was started, and the reaction was carried out with stirring at 40°C for 20 hours. The reaction solution was heated to 90℃ to inactivate the enzyme.
After heating for 1 hour, 955 g of a solution containing a blue pigment was obtained.
The maximum absorption wavelength of this solution is 596 mμ and the absorbance is 85
It was hot. After the solution was filtered, dextrin was added and spray-dried to obtain 150 g of gardenia blue pigment powder. Comparative Example 1 Gardenia alcohol extract 10 used in Example 3
g and 1000 g of water in a 2-volume flask, heat and dissolve, then adjust the pH to 5.5 with 1N NaOH, add 4 g of gelatin, and after dissolving, maintain the temperature at 40℃.
1.2 g of Lipase AP (manufactured by Amano Pharmaceutical Co., Ltd.) was added, and the reaction was carried out with stirring (1000 rpm) at 40° C. for 40 hours (oxygen concentration 3 ppm). The mixture was heated at 90° C. for 1 hour to inactivate the enzyme, and 950 g of a solution containing a blue pigment was obtained. The maximum absorption wavelength of this solution was 583 mμ, and the absorbance was 98. The reason why the maximum absorption wavelength changed to 583 mμ compared to Example 3 is because the reaction was not carried out under microaerobic conditions during the first half of the reaction.

[Table] Example 4 Gardenia alcohol extract 30 obtained in Example 3
Put g and 1000 g of water into a 2 volume mini jar, heat and dissolve, adjust the pH to 7.0 with 2N-NaOH, and add peptone.
After dissolving, add 3 g of Morsin (manufactured by Seishin Pharmaceutical) and maintain the temperature at 40°C.
A standing reaction was performed for a period of time. After 20 hours, air was blown from the spargeer for 20 hours. The ventilation amount at this time was 1.0vvm. Next, 90 for enzyme deactivation.
C. for 10 minutes to obtain 975 g of a blue dye-containing solution. The maximum absorption wavelength of this solution is 596 mμ, and the absorbance
It was 250. Example 5 20g of the gardenia extract obtained in Example 1 was added with warm water (50g
℃）Add 500g and dissolve in a 2-volume flask to 1N
- Adjusted the pH to 5.0 with NaOH. Then increase the temperature to 50℃
2.5 g of acylase was added and stirred at 50°C for 1.5 hours (50 rpm). The dissolved oxygen concentration at this time is
It was below 0.1ppm. Continue to increase the stirring speed.
The reaction was stirred at 500 rpm for 20 hours at 50°C (dissolved oxygen concentration 1.0-4 ppm). Due to enzyme inactivation
The mixture was heated at 95° C. for 10 minutes to obtain 575 g of a blue dye-containing solution. The maximum absorption wavelength of this solution was 598 mμ, and the absorbance was 82.

Claims (1)

[Scope of Claims] 1 Gardenia iridoid glycoside or a substance containing it and β-glucosidase or a substance containing it are allowed to interact under aerobic conditions in the presence of a substance containing a primary amino group, β-glucosidase coloring of iridoid glycoside When forming a blue pigment, the glycoside or its containing substance and the β-glucosidase or its containing substance are allowed to interact sufficiently in advance under microaerobic conditions. A method for producing a lightened natural blue pigment, which is then further reacted under stirring conditions. 2. A method according to claim 1, characterized in that the preliminary action under microaerobic conditions is carried out for at least about 10 hours. 3. The method according to claim 1, wherein the lightened natural blue pigment has a maximum absorption wavelength in the visible region of 594 to 601 mμ.