JP3057369B2 - Method for producing lightened natural blue pigment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial applications>   The present invention relates to a method for producing a gardenia-derived natural blue pigment.
Law related, especially safe food and drinks, luxury goods
Products, health pharmaceuticals, cosmetics, etc.
During the manufacturing process, complicated reaction operations and
Does not require strict management of conditions and is special and complicated
Without the need for a simple purification step
Blue-based pigments with clearer color tones
(Maximum absorption wavelength λ_max= 600nm ~ 625nm) for easy mass production
The present invention relates to a method for producing a natural blue dye. <Conventional technology>   In recent years, while the safety of synthetic dyes has been questioned,
Colors of foods and drinks, luxury goods, health pharmaceuticals, cosmetics, etc.
There is an increasing demand for harmless natural pigments,
Has little blue pigment. A typical natural blue pigment
Pigments from gardenia and spirulina
Are practically used. Gardenia fruit
It has been known for a long time that skin dyes the skin blue to violet.
Have been. And dye this skin blue to blue-purple
Containing iridoid glycosides thought to be involved in
Genipi from ripe fruits of Sano tree (Genipa americana L.)
The crystalline solids of the genipin are separated
Insight into physical and chemical properties and chemical structure,
Means that the genipin is a primary amino group-containing compound (amino
Acids such as glycine, leucine, glutamic acid)
It has been reported that a blue pigment is produced in response.
(J.Org.Chem., 25,2175,1960)   Gardenia fruits often contain iridoid glycosides
It is known, and certain iridoid glycosides have β-
When glucosidase is applied, it turns reddish purple or blue
It has long been known to produce color. (Tetrahe
Delon Letters 2347-2350,1969)   Furthermore, the coloring mechanism of the blue pigment derived from gardenia
Is an iridoid glycoside found in gardenia nuts
Geniposide and the like produce enzymes such as β-glucosidase
Or bacteria belonging to the genus Bacillus or Aspergillus
Action of a microorganism having the ability to produce β-glucosidase,
Or hydrolyzed by chemical action such as hydrochloric acid
To generate genipin, and the genipin is converted to a primary amino group.
Contained substances (amino acids such as glycine, leucine, glue
Tannic acid) to form a blue pigment
It has been reported. The mechanism of color development of this blue pigment
Utilizing genipin and various primary amino group-containing substances
After reacting, various dyes (blue-purple-purple-red-purple-green-yellow-yellow-orange
-Colors having various color tones ranging from orange to brown to brown)
Has been advocated. (Japanese Patent Publication No. 54-13451)   Furthermore, in the production of blue pigments from gardenia,
Thus, instead of allowing the β-glucosidase to act,
Microorganisms of the genus Bacillus (JP-B No. 52-13971) and Monasca
Maximum absorption using microorganisms of the genus Azot (Special Publication No. 59-16751)
Collection wavelength (λ_max) Produces a dark blue dye of about 590nm
There are examples that have been successful.   However, the color development of these blue pigments from gardenia
Blue dyes obtained by various reactions based on canism
Is a mixture of many compounds as a result of a complex reaction,
As a result, a dark blue-violet color (maximum absorption wavelength: λ_max= 593nm or less
Lower, half width = 100-140 nm)
Supply of bright blue pigments
I have.   Conventionally, the lightening of the blue dye obtained by this reaction
Improvements were attempted. ((A) Japanese Patent Publication No. 61-19234,
(B) JP-A-61-47167)   The former “(a) JP-B-61-19234 (JP-A-56-92792)
No.) ”, the gardenia iridoid glycoside
Body-containing substance and β-glucosidase are preliminarily subjected to microaerobic conditions.
After sufficient action under the
To obtain a blue dye with a narrow polymerization distribution.
Complex reaction procedures and particularly strict control of the reaction conditions
A blue pigment is produced through the process. But
Et al., The method of “(a) Japanese Patent Publication No. 61-19234”
Β-glucosidase used in the reaction system is expensive
Not reusable, maximum absorption of generated blue dye
Collection wavelength (λ_max) Is 594-601nm and is fully lightened
Not only that, but also complicated reaction operation process and
It requires strict control and is very
It is difficult to control various conditions such as air conditions and stirring conditions.
Difficult and generated when these conditions fluctuate
The maximum absorption wavelength (λ_maxIs also affected
And the maximum absorption wavelength (λ_max) Moves to shorter wavelength side
There are various problems that the effect of coloring is significantly reduced.
You.   On the other hand, the method of the latter "(b) JP-A-61-47167"
If a mixture of many dyes is
It is obtained by purification by chromatography.
It is complicated and the yield of blue dye is extremely low.
There is. <Problems to be solved by the invention>   Method for producing lightened blue pigment according to the present invention
Is β-glucosidase or β-glucosidase
In the presence of viable bacteria, iridoid glycosides of Gardenia plants
Or the iridoid glycoside-containing substance (hereinafter referred to as “iridoid
Glycoside or iridoid glycoside-containing substance ''
And simply referred to as “iridoid glycosides (containing substances)”
Primary amino group-containing compound or primary amino group-containing
Substances (hereinafter referred to as “primary amino group-containing compounds or primary
The term “amino group-containing substance” is collectively referred to simply as “primary amino group-containing substance”.
Compound (substance) ")
In the method for producing, an electrolyte is added to the reaction system.
Under conditions that reduce the water solubility of the resulting blue dye
By reacting under the below, the light blue color pigment
Completed based on the discovery that it was created
It is.   And, similarly, the related art involves the reaction
Of primary amino group-containing compounds (substances)
By using a material with a high molecular weight
It was discovered that a lighter blue pigment was produced.
It was completed based on the above.   The present invention relates to a gardenia-derived iridoid glycoside (containing
Substance) and a primary amino group-containing compound (substance) by β
The presence of glucosidase or β-glucosidase producing bacteria
When producing a blue dye by reacting under
Strict control of reaction conditions and reaction conditions
Maximum absorption without the need for special purification steps
Wavelength (λ_max) At 600-625 nm in ethanol aqueous solution
It is stable for a long time when dissolved, and even in a wide pH range
Stable, bright blue color that withstands fading tests
To provide a method for producing a large amount of
And for the purpose. <Means to solve the problem>   To achieve the above objectives,   Manufactures a lightened natural blue pigment according to the present invention
The way to do   [Β-glucosidase or β-glucosidase production
In the presence of fungi, the iridoid glycosides of Gardenia plants
Is a compound containing a primary amino group or a compound containing a primary amino group.
Reacts with primary amino group-containing substances to produce blue dye
In the method   The concentration of the reaction system is 10% by weight or more and 30% by weight or less.
Blue color produced by adding electrolyte to the range
Reaction under conditions that reduce the aqueous solubility of
A method for producing a lightened natural blue pigment. 』 According to the present invention.   The inventor has proposed a light-colored blue pigment according to the present invention.
The mechanism of the manufacturing method is estimated as follows.
In other words, although the reason is not clear,
In the process for producing a bright blue dye,
Water-soluble in involved primary amino group-containing compounds (substances)
Use primary amino group-containing compounds (substances) with low
Or a primary amino group-containing compound having a large molecular weight
(Substance) to generate blue-based pigments
Reduce water solubility or produce
Reaction in a reaction system with reduced aqueous solubility of
As a result, the blue dye precipitates as soon as it is formed.
Is taken out of the reaction system and moves to the next reaction
Is suppressed as much as possible, and no further polymerization reaction will occur.
And a pigment with a uniform degree of polymerization is produced.
Guessed that a blue dye
I have. Of course, the present invention has no restrictions on such inference principles.
Not promised.   Gardenia iridoi used in the method according to the present invention
Glycosylate or its containing substance
De, methyl deacetylasperloside, genipingen
Examples include thiopicide, gardenoside, and geniposidic acid
Can be shown. Then, a commercially available gardenia dried extract (geni
Poside content = about 9%: manufactured by Nippon Powder Co., Ltd.
You can use it. Extracted from gardenia fruits, etc.
The product (ie, iridoid glycoside or the substance concerned)
To obtain, for example, crush the gardenia fruit crush
Are methanol, ethanol, isopropanol, aceto
Organic solvent, or any combination of water and the organic solvents
Extract with the mixture and evaporate or distill off various solvents
To prepare a concentrate or a concentrated dry product or an evaporated dry product
can do.   In the reaction system of the method for producing a blue dye according to the present invention,
Initial concentration of iridoid glycosides (contained substances) in
Can be selected, usually as geniposide content, 0.01 ~
1 wt% is available, with concentrations of 0.05 to 0.6 wt% being preferred.
(Wt% indicates weight%; the same applies hereinafter)   Next, other reactions that can be used in the method according to the present invention
Substances, ie, primary amino group-containing compounds (substances)
Any compound or substance containing an amino group can be used.
You. For example, amino acids, peptides, proteins, protein components
Amino sugars such as digests and chitosan and the like;
Various salts (for example, metal salts, inorganic acid salts, etc.)
Combination of these with various substances
Single or various compounds (substances) selected from the compound group
Can use a mixture of two or more compounds (substances).   The primary amino which can be used in the method according to the present invention.
Detailed explanation of specific examples of group-containing compounds (substances)
I will tell.   First, the amino acids that can be used in the present invention include
Soluble or high molecular weight amino acids, such as
For example, the molecular weight is preferably about 120 or more. Ma
In addition, the peptides that can be used in the present invention include dipepti
Compounds, tripeptides, tetrapeptides,
Oligopeptide in which the amino acid consists of 2 to 10 amino acids,
A polypeptide consisting of 10 to 100 amino acids or it
The above macropeptides can also be used,
Whether they are linear or cyclic peptides
And can be used in homocyclic peptides or heterocyclic peptides.
Peptides are also available. Furthermore, it can be used in this invention
Protein and various salts of the protein include various proteins
White matter and metal salts of the protein (eg, sodium
Salt, potassium salt, etc.) and inorganic acid salts (for example, hydrochloride
) Can be used, but the microorganisms (lactic acid bacteria)
Assimilable and dusty, skim milk, casein milk and
These various salts are particularly preferred. Also, in this invention
What can be made is casein milk (Hamarstein method)
Protease titration such as (Wako Pure Chemical Industries, Ltd.)
Casein and the like used as a substrate for use can also be used. It
Against casein (acid hydrolyzate) (Sigma Chemical
Company) is the first class A used in the present invention.
Less preferred as a mino group-containing compound (substance)
No. Because, for example, the casein (milk) (acid degradation
It is considered that the decomposition state is advanced.
Zein (acid hydrolyzate) contains water-soluble amino acids and low molecular weight
Amino acids (such as glycine)
It is considered that the lightening of the generated blue pigment is inferior,
λ_maxTend to produce only dark blue-violet pigments with low
Because there is.   Further, the light-colored blue pigment according to the present invention
The characteristic reaction conditions in the production
Adds denaturation to reduce the water solubility of the resulting blue pigment
Reaction under the same conditions "
Add various electrolytes such as inorganic salts into the reaction system
This can be achieved by: By adding to this reaction system
Sodium chloride, potassium chloride, sulfuric acid
Neutral salts such as sodium acid are most preferred. Other
Examples of inorganic salts that can be added include, in principle, the pH of the reaction system.
Relatively little hindrance to enzyme regulation
Relatively low inhibition or microbial action
Inorganic added as long as each condition such as relatively small is satisfied
There is no particular limitation on the salt, for example, sodium acetate
Such as salts of weak acids and strong alkalis, and ammonium sulfate.
Any salt of a weak alkali and a strong acid can be used.
The amount of the inorganic salt added to the reaction system is 0.5
-30 wt% can be used, and 1-20 wt% is preferred. And
This characteristic reaction condition in the present invention
As tested in the examples, one reactant, primary
Formed regardless of the amino group-containing compound (substance)
The maximum absorption wavelength (λ_max) On the long wavelength side
It turned out to be moved to. Table 2 shows the reaction
The salt concentration of the system was 0% (no addition = control), 10wt%, 15wt
%, Water solubility of blue dyes produced as 20 wt% is low
To form a reaction system that contains various amino acids.
And the iridoid glycoside of the gardenia fruit
Blue color produced when reacting in the presence of dase
Maximum absorption wavelength (λ_max) And half width and
λ_max5 shows an example of the variation of the above. (See Table 2)   Table 2 shows that all 17 amino acids tested
Acids and peptides can be added to the reaction system by adding salt.
The blue dye produced tends to lighten (i.e.
Large absorption wavelength (λ_max) Tends to shift to the longer wavelength side]
are doing.   Next, the related art related to the method according to the present invention will be described.
Another reaction condition, “containing primary amino group with low water solubility”
Organic compound (substance) or primary amino group with high molecular weight
Containing compounds (substances) and gardenia iridoid glycosides
Or reacting with the contained substance (compound). "
Under the reaction conditions A. When the primary amino group-containing compound (substance) is an amino acid
If (A) The amino acids having low water solubility include, for example,
For example, leucine, isoleucine, phenylalanine, hiss
Tidine, tyrosine, tryptophan, cystine, etc.
Colored blue pigment (λ_max= 593-600 nm)
It can be exemplified as one that can be particularly effectively used. In addition,
It is a matter of course that the amino acids are not limited to those exemplified. (B) The amino acid having a large molecular weight has a molecular weight of
About 120 or more amino acids such as leucine, isoleuc
Syn, methionine, histidine, tyrosine, tryptophan
Cyan, cysteine, cystine, etc. were lightened to some extent
As an example that can be used effectively for the production of blue dyes
Wear. (See Table 2). (C) Other.   Other suitable amino acids available for this related technology
Thus, valine, threonine and the like can be exemplified. (See Table 2
See) B. A compound (substance) containing a primary amino group that is hardly soluble in water,
Or primary amino group-containing compound (substance) having a high molecular weight
Is other than an amino acid, (A) Peptide, protein or protein degradation product or
These various salts (eg, metal salts, hydrochlorides, etc.)
Can be used.   Examples of peptides include glutathione and others.
Various peptides (from dipeptide to macropeptide)
Various peptides) can be used. The protein and
Examples of the metal salt include casein Na (dairy case).
In-Na) (trade name “Sanlacto S” Taiyo Chemical Co., Ltd.)
Made), casein milk (made by Wako Pure Chemical Industries, Ltd.), skimmy
Luc (Snow Brand Milk Products Co., Ltd.), Qurite No.1 [Kaze
In milk (made by Kewpie Tamago Co., Ltd.)
Zane (importer of Nippon Protein Co., Ltd.
Do Daily Product), Casein Na (Gunze)
Sangyo Co., Ltd.), Casein (Hamarstein method)
(Manufactured by Wako Pure Chemical Industries, Ltd.) can be used. The above
Of course, it is not limited to the example.   As the proteolysates, enzymatic decomposition of the above various proteins
Products, microbial degradation products, etc. can be used.   Utilizing the peptide, protein or protein degradation product
Sometimes, the maximum absorption wavelength (λ_max)
Is lightened to 600 to 610 nm. (Table 2
To Table 4)   In addition, of the protein degradation products, casein (acid degradation product)
(Made by Sigma Chemical Company)
Amino acids such as glycine in the degradation product.
Is considered to be a compound containing a primary amino group (compound)
Quality), the blue pigment produced is sufficient.
Because it may not be lightened in minutes,
When using, the degree of decomposition is relatively low,
For example, it is important to use degradation products up to the peptide level.
is there.   In addition, said peptide, protein or said protein and other
Complexes with compounds can also be used. Furthermore, chitosan
Amino sugars and the like can also be used. In addition, the said protein etc.
In particular, dairy casein was exemplified, but
Is the lactic acid bacterium used in this related technology a lactic acid bacterium?
Using β-glucosidase, or
When using bacteria other than lactic acid bacteria, primary amino groups
Proteins other than dairy proteins as contained compounds (substances)
The decomposed product, the various salts or the complex can be used.
Of course.   And, in the reaction system used in the method of the present invention,
Concentration of primary amino group-containing compounds (substances)
Can be appropriately selected, but amino acids or
Is 0.01 to 10% by weight in the case of a peptide, preferably 0.1 to 10% by weight.
2.0 wt% available, relatively high molecular weight peptides and proteins
Or protein degradation products or various salts thereof
In the case of these various composites, 0.1 to 30% by weight, preferably 1 to 2%
5wt% is available.   Next, the microorganism used in the method of this related art is:
It belongs to lactic acid bacteria and is Streptococcus (Stre
ptococcus), Lactobacillus
Various fungi can be used.   In addition, the reaction of the method according to this related technique
Acid bacteria) in the presence of
Has a temperature of 22-45 ° C (preferably 35-42 ° C) and a pH of about 4-
8. Under standing or shaking conditions (100-150 rpm)
Until the production of blue dyes reaches a steady state.
And gardenia fruit iridoid glycosides (geniposide)
3-4 days at the initial concentration of about 0.2wt%)
To produce a blue pigment. (See FIGS. 1 and 2)
In addition, the stationary culture referred to herein means a minimum of about 0 to 2 per day.
Degree or about 1 to 2 times per hour
Shake and agitate the bottle to shake the reaction contents and promote the reaction.
And so on. And, the shaking culture and the stationary culture
The difference between the two is that static culture is better than shaking culture.
Is also the maximum absorption wavelength (λ_max)
In this case, the color shifts to the longer wavelength side of
Direction.   Furthermore, the reaction of the method according to the related art
When performed in the presence of glucosidase, β-glucosidase
The amount of the enzyme used can be appropriately selected, for example, 0.01 to 1 m
g / ml, preferably 0.05-0.5 mg / ml
The reaction conditions are 20 to 50 ° C. (preferably 35 to 43
C) at a pH of about 4-9, under standing or shaking conditions.
Until the formation of a blue pigment enters a steady state, that is, 3 to 4
Incubate for days to produce a blue pigment. In addition,
The shaking conditions and the like in the shaking culture depend on the reaction by the microorganism.
Same as in the case.   By the way, the generated blue-based dye is
Heat the nutrient solution as it is (about 95 ° C or more under reflux in a water bath,
After about 15 minutes) or with organic solvent added
Heat (under reflux in a water bath, immediately after warming up to about 15 minutes) and lose enzyme
After performing live or microbial sterilization,
The nutrient solution is separated into solid and liquid by a known method such as centrifugation to obtain a liquid phase.
Separates pigments and solids (cells, precipitated pigments, etc.) dissolved in
And then dilute it as is or dilute as appropriate
Partial absorption spectrum and maximum absorption wavelength (λ_max)
Λ of the resulting dye_maxAnd production
Can be specified. And if necessary, a suitable organic
Extracting the dye from the liquid phase and the solid phase using a solvent,
The λ_maxAnd the amount of production (absorbance) can be measured
Wear. For example, from the liquid phase, such as n-butanol
Extract the pigment using an organic solvent that is not completely soluble in
After that, the maximum absorption wavelength (λ_max) And absorbance
It can also be measured. On the other hand, from the solid phase, ethanol
Water, methanol, isopropanol, acetone, etc.
Solvent, the mixed solution or the organic solvent and the like and water
Arbitrary mixture (preferably 100 to 50 vol% aqueous solution)
The blue dye can be extracted using
Measure the absorption spectrum and determine the maximum absorption wavelength (λ_maxMeasure)
And the λ_maxMeasure the absorbance at the dye
Can be measured. (However, vol% is volume
Indicates the amount%. same as below)   The maximum absorption depends on the method of extracting the generated dye.
Collection wavelength (λ_max) Value and absorbance change.
Revealed. That is, heat sterilization of microorganisms after the reaction
Alternatively, the blue dye produced by heat inactivation of the enzyme
Extraction method only (blue pigment remains in the precipitate
), And after adding an organic solvent, heat
Simultaneous sterilization of substances, inactivation of enzyme and extraction of produced pigment
Method [For example, add the same amount of ethanol (100%)
The solution was made into ethanol solution with a final concentration of 50 vol% and then added under reflux.
Heat to sterilize microorganisms or deactivate enzymes.
The blue dye generated at that time is replaced with the organic solvent (in this case, 50 v
ol% ethanol aqueous solution).
I do. As a result, the above is obtained more than the above
The maximum absorption wavelength (λ_max) Is long wavelength and color
The color tone is lighter and the absorbance, which indicates pigment productivity, is higher.
It's gone. This is because the blue pigment produced is water-soluble
Exists as a precipitate outside the reaction system, and this precipitate
Is a blue dye that dissolves in organic solvents
As a considerable amount and dissolved in organic solvents
Toward the maximum absorption wavelength (λ_max) Is a long wavelength
This suggests that there are many bright blue pigments.
available.   Furthermore, the blue pigment produced is in any case
Light, heat and pH, and is suitable for fading tests
It can withstand enough. <Example>   Next, the present invention will be described based on embodiments.
However, the present invention is not limited to these examples, and
Needless to say, there is no restriction by these examples
Nor. i) Preparation of Gardenia Fruit Iridoid Glycoside (A) Gardenia dried extract:   Commercial gardenia dried extract (Nippon Powder Co., Ltd.)
Use as it is. 2 g of this gardenia dried extract
The gel in the reaction solution dissolved in 98 ml of Lubein buffer (pH 6.8)
Niposide content was 0.18 wt%. (B) Preparation of X% Gardenia Decoction:   "Sanshishi powder (Gardenia crude drug name)" (Nippon Powder Co., Ltd.
Water is added to Xg at a ratio of 100 ml of water, and the water bath (about 95
℃) for 1 hour under reflux, followed by filtration and solid-liquid separation.
After that, the water in the filtrate is evaporated and evaporated to dryness.
Kilbane buffer (pH 6.8) was added to bring the total volume to 100 ml
Use something. The geniposide content of the 4% decoction is 2.02
mg / ml or about 0.2 wt%. (C) Preparation of 2% methanol extract of gardenia fruit:   Add 50 ml of methanol to 2 g of Sanshishi powder and leave in a water bath for 1 hour.
The mixture is heated under reflux, cooled and filtered, and the filtrate is evaporated to dryness.
This was dissolved in McIlvaine buffer (pH 6.8) and 10
0 ml. ii) A method for producing (manufacturing / producing) a blue pigment. (A) A method for producing (manufacturing) a blue pigment by lactic acid bacteria.   20 g of skim milk powder and the dried gardenia extract of i (a) above
(Geniposide content about 9%) 2 g of McKilbain buffer
(PH 6.8) The reaction solution to which 98 ml was added was mixed at about 95 ° C. in a water bath.
Immediately after reaching the warming temperature, sterilize by heating for about 30 minutes.
× 10⁷~ 2.0 × 10⁷Inoculate pcs / ml. Incubate still at 38 ° C (however,
And a minimum of about 0 to 2 times / day to a maximum of about 1 to 2 times / hour
The reaction solution was shaken and agitated at an appropriate ratio to culture. Hereinafter the same
Ji). After culturing for 72 to 96 hours, lactic acid bacteria are
Sterilized by heating at 95 ℃ for about 10 minutes, blue pigment by lactic acid bacteria
Ends the manufacturing process.   Thereafter, blue pigment is extracted from this reaction solution (culture solution).
Moved to the process. After sterilization, centrifuge the reaction solution.
Measure the visible spectrum using the liquid phase of (culture solution) as the dye solution.
The maximum absorption wavelength (λ_max) And
The color tone and production of the dye
The amount is measured and the degree of lightening of the produced dye is compared and examined.
(Note that the results in Tables 1 to 4 are all
Of the aqueous solution (aqueous phase) as it is after solid-liquid separation of
Absorption wavelength (λ_max) Are measured and compared. )   In addition, without using the gardenia dried extract
When using shi decoction, add 2 g of the gardenia dry extract
The cuticle was replaced with 98 ml of McKilbain buffer (pH 6.8).
Add 100ml of pear decoction as a reaction solution (culture solution)
Will be used. (B) Production of a blue pigment by β-glucosidase (manufactured by
Manufacturing and production) methods.   20 g of skim milk powder (5 for protein or protein degradation products)
g, 0.05 g to 0.2 g or 0.005 M to 0 for amino acids.
02 M (M = mol /), Gardenia Dried Extract 2g and Mackill
98 ml of Bain buffer (pH 6.8) was added, and the mixture was heated to about 95 ° C in a water bath.
Immediately after reaching the temperature, heat sterilize for about 30 minutes, then cool,
Add cosidase at a rate of 0.1 mg / ml and allow to stand
Approximately 0 to 2 times / day to a maximum of 1 to 2 times / hour
Includes agitation culture in which the solution (culture solution) is shaken and agitated. Less than
The reaction is performed for 48 hours to (72 hours) to 96 hours. Anti
After completion of the reaction, heat the mixture to about 95 ° C in a water bath for about 10 minutes to
Inactivate lucosidase and add blue to β-glucosidase
The color pigment production process is completed.   Thereafter, blue pigment is extracted from this reaction solution (culture solution).
Moved to the process. After inactivation of the enzyme,
The liquid phase (aqueous phase) of the reaction solution (culture solution) is
Maximum absorption wave of blue dye generated by measuring yield spectrum
Length (λ_max) And by measuring the absorbance, the dye
The color tone and the production of are measured.   In addition, without using the gardenia dried extract
When using the decoction, the bluish color in the presence of the microorganism
2 g of gardenia dried extract
And 98 ml of McKilbain buffer (pH 6.8)
The reaction solution (culture solution) was prepared by adding 100 ml of the cinnamon decoction.
Will be used. (C) a method for reducing the water solubility of the produced blue dye,
The maximum absorption wavelength (λ_maxRelationship with
Person in charge.   Reduce the water solubility of the blue dye produced
Ii) (a) and ii) (b)
In each reaction system of the pigment production process, food
By adding 10 wt%, 15 wt% and 20 wt% of salt (NaCl)
Decrease the water solubility of the blue dyes produced
On the other hand, a control without salt (0%) was set as a control.
And the blue color produced in each of the four reaction systems
Maximum absorption wavelength of dye (λ_max) And half width,
Compare and study.   Table 1 shows various lactic acid bacteria and β-glucosidase.
From the visible absorption spectrum of the blue dye
The maximum absorption wavelength (λ_max) And the production volume (absorption)
3 shows an example of a test result of (luminous intensity). The blue
The absorbance of the dye stock solution, which indicates the production amount of the color dye, is necessary.
The stock solution is diluted to an appropriate magnification according to_maxTo
After measuring the absorbance of the dye,
The figure shows the value obtained by calculating the absorbance of the stock solution.  From Table 1, it can be seen that Streptococcus (Strepto-coccu
s) Milk of the genus and Lacto-bacillus
Mycobacteria and β-glucosidase are the methods of this related art.
It turns out that it is effective for blue pigmentation.   FIG. 1 shows lactic acid bacteria (Streptococcus faecalis IFO 1296).
FIG. 5 is a diagram showing the state of the formation of a blue pigment over time according to 5).
The figure shows the generation of blue pigments over time by β-glucosidase
It is a figure showing a state. According to FIGS. 1 and 2,
The production of a blue pigment according to the present invention is carried out for 72 hours to 96 hours.
In between (3-4 days)
I have.   Next, Tables 2 to 4 show that an electrolyte [neutral salt
0% (no addition: control), 10 wt.
%, 15wt%, and 20wt%, respectively.
Reduce the water solubility of the blue dye produced
In the presence of microorganisms or β-glucosidase
Gardenia iridoid glycoside and containing the primary amino group
Formation of blue dye when reacted with compound (substance)
State (λ_max, Half-width value, etc.).   Primary amino group-containing compounds in Tables 2 to 4
(Substance) and gardenia iridoid glycoside (geniposide)
The primary amino group-containing
When the compound (substance) is an amino acid or peptide
(Table 2) or non-dairy proteins or
In the case of white matter decomposition products or amino sugars (Table 3)
In the above and cases, β-glucosida
2 shows an example of the result of a reaction in the presence of sucrose. one
On the other hand, the primary amino group-containing compound (substance) is made of skim milk, etc.
Dairy-derived protein or its degradation products or
These metal salts and gardenia iridoid glycosides (genipo
(Table 4), the β-glu
Cosidase or the lactic acid bacterium (Streptococcus sp.
Or Lactobacillus sp.)
Examples of the results are shown.   From the results in Table 2 above, in the presence of β-glucosidase
In addition, the gardenia iridoid glycoside (containing substance) and
Produced by reaction with primary amino acids or peptides
The blue dye is produced by adding an electrolyte such as a neutral salt to the reaction system.
By lowering the solubility of the formed blue pigment,
The maximum absorption wavelength (λ_max) Is 2 to 1
It shifts to the long wavelength side of about 5 nm,
Suggests. In particular, the primary amino acids involved in the reaction are
The less water-soluble, or the molecular weight is about 120 or more
Tends to produce brighter blue pigments
For example, leucine as a primary amino acid,
Isoleucine, methionine, histidine, phenylala
Nin, tyrosine, tryptophan, cysteine, etc.
When glutathione or the like is used as a peptide,
Maximum absorption wavelength (λ_max) Is about 598nm or more
(About 598-605 nm)
You. Table 2 shows the maximum absorption of the blue dye produced.
Collection wavelength (λ_max) Is a lightened dye of about 600nm or more.
The reason for this is that, as described above, the color
Even without the method of reducing the water solubility of the element,
The primary amino group-containing compound (substance) itself has low water solubility.
Use a material with a relatively high molecular weight (about 120 or less).
What can be achieved by using
Suggests at the same time.   Reducing the water solubility of the formed dye in the reaction system
As a result, the generated blue dye shifts to the longer wavelength side.
The mechanism of lightening is still unclear.
No, but reduces the water solubility of the blue dye produced
As a result, the produced blue pigment is
It is taken out of the reaction system as a precipitate, and further polymerization occurs.
A light-colored blue pigment with a uniform degree of polymerization is produced
Can be guessed. Of course, this invention
Nothing is constrained by such assumptions.   Also, from the results in Table 3 above, it can be seen that β-glucosidase is present.
In the presence, gardenia iridoid glycosides (contents
) And the primary amino group-containing compound (substance)
The blue dye produced is the primary amino group-containing
The compound (substance) is a non-dairy primary amino acid with a high molecular weight
Group-containing compounds (substances) such as peptides, proteins,
The protein degradation product or amino sugar can be produced.
Suggests that You have to be careful here
This means that when a protein digest is used, the
Amino acids with relatively small or highly soluble amino acids
If acids are mixed, the maximum absorption of the blue dye
Collection wavelength (λ_max) Shifts to the short wavelength side, dark blue purple
Side to obtain the desired lightened blue pigment
It is a tendency to hinder.   Similarly, "yeast extract" used in Table 3,
“Peptone”, “Promois W-32 [(trade name) (product)
Name = collagen degradation product) (manufactured by Seiwa Chemical Co., Ltd.)]]
"Promois A (same as above)", "Promois E-118D [same as above]
Previous) ”, etc.
Because it contained a large amount of primary amino acids,
λ_maxWith a low value, that is, a blue pigment that is not lightened
Can be assumed to have been generated or mixed.   Next, from the results in Table 4 above, β-glucosidase
Or gardenia iridoid in the presence of lactic acid bacteria
Glycosides (substances) and primary amino group-containing compounds (substances)
Blue) dye produced by the reaction with
Secondary amino group-containing substance is poorly soluble in water or high in molecular weight
When it is a primary amino group-containing compound (substance), even more
Suggests easy formation of lighter blue dyes
are doing. In particular, for example, primary amino group-containing compounds
Degreasing with low water solubility and high molecular weight as (substance)
Milk or the dairy protein degradation product or the various salts (metal
Salt) can be used effectively, and the blue dye produced
Absorption wavelength (λ_max) Could not be obtained in the past 610
nm that can produce a very bright blue dye.
And suggests. This is defatting for the growth of lactic acid bacteria
It is also dusty with the assimilation of milk, especially for mass production of blue pigments.
And economical production. That is, β-glucosi
When using an enzyme such as dase, the enzyme itself is expensive.
In addition, collect the used enzyme once and use it several times.
Is extremely complicated and difficult to manage,
And blue dyes can be industrially produced in large quantities, and lactic acid bacteria
Has the advantage that it is much easier to manage
You.   By the way, Table 4 also includes the following:
Suggests. That is, a primary amino acid having a large molecular weight
When the group-containing compound (substance) is a dairy protein, for example,
In the case of using Kimmirum, dairy casein, etc., λ_max= 6
Even when a bright blue color pigment with a wavelength of 00 nm or more was produced
Regardless, the same milk and casein
(Acid decomposition product) ”(λ_max= 590 nm) and
Zein (Hamarstein method) ”(λ
_max= 607nm) means λ of the blue pigment_maxTo
Of the blue dye produced, such as a difference of about 17 nm
(Lightening) may be inferior.   By the way, the inventor used skim milk or dairy casein.
And iridoid glycosides (substances) in the presence of lactic acid bacteria
When a blue pigment is produced by the reaction,
More water-soluble, such as glycine, alanine or leucine
Is formed when an amino acid with a relatively small molecular weight is added
Maximum absorption wavelength (λ_max)
2 to 17 nm shorter wavelength side than when no acid is added
We have gained the knowledge that there is a case where it may shift to.   And some of the results in Tables 2 and 4 are grade 1
Amino acids and pepti as amino group-containing compounds (substances)
And proteins, their degradation products and their
When using metal salts, etc., add salt to the reaction system
Of reduced water solubility of blue dyes
Below these are gardenia iridoid glycosides (containing substances)
Λ of the blue dye produced when_maxYes
Even in the case of misalignment, the blue dye that has shifted to the longer wavelength side and has become brighter
It also suggests that there is a tendency to produce. (D) Blue color from reaction product (reaction solution or culture solution)
The extraction method of the pigment is λ_maxAnd dye collection
Test example of effect on amount. A) Extraction conditions for blue dyes from the reaction solution (culture solution)
Solvent) is λ of the resulting blue dye._maxAnd color
Test example of effects on elementary yield.   The sample for extracting the blue pigment is described in ii) above.
Reaction solution (culture) obtained by the method (a) or ii) (b)
Liquid). That is, 20 g of skim milk powder and gardenia dry
Add 98 ml of McIlvaine buffer (pH 6.8) to 2 g of the dried extract.
Then, apply this to the specified conditions (about 95 ° C in a water bath,
Min) heat-sterilized and cooled, then inoculated with lactic acid bacteria, or
Add β-glucosidase and let stand for 72 to 96 hours
The reaction solution (culture solution) that has been cultured and reacted is used as a blue dye.
Use as a sample for extraction. Extraction condition:   Heat the reaction solution (culture solution) of the sample as it is in a water bath
(About 95 ° C, temperature about 10 minutes) by the enzyme (β-glucose)
Inactivate Sidase or kill the lactic acid bacteria and centrifuge
Of the blue dye in the liquid phase (aqueous phase)
Large absorption wavelength (λ_max) And the amount of dye formation (λ_maxIn
Absorbance). Extraction condition:   An appropriate amount of an organic solvent is added to the reaction solution (culture solution) of the sample.
(Single type of organic solvent or mixture of multiple types of organic solvent
Liquid) to a final organic solvent content of about 20-90 vol%
After heating to reflux in a water bath (delivered temperature about 10 minutes),
Inactivation of the enzyme or sterilization of lactic acid bacteria
The extracted blue dye was extracted into each of the above liquid phases, and then centrifuged.
In the case of a homogeneous liquid phase, the entire liquid phase and the liquid phase become two phases
When separating, maximum absorption wavelength of each organic solvent phase
(Λ_max) And the amount of dye formation (λ_maxAbsorbance at
Method to measure. (Note that when the liquid phase separates into two phases,
Liquid phase, or aqueous phase, is λ_maxAnd absorbance
Not. ) The two are compared and examined.   In this example, as the organic solvent, for example,
If the following organic solvent is added to the reaction solution (culture solution),
The test was performed by adding only the amount. (A) 100% ethanol (final concentration after adding organic solvent is 5
0 vol% ethanol aqueous solution), (B) 100% isopropanol (final after addition of organic solvent)
The concentration is 50 vol% isopropanol aqueous solution), (C) Isobutanol / ethanol (50/50 v / v) mixture
liquid, (D) isobutanol / isopropanol (50/50 v /
v) mixture, (E) 100% n-butanol,   Table 5 shows the extraction conditions and the blue color according to the extraction conditions.
Maximum absorption wavelength (λ_max) And the corresponding λ_maxSucking in
This is a comparison table of luminous intensity (dye production amount), and shows the reaction solution of the sample.
Results when the same amount of the organic solvent was added to (culture solution)
This is an example.   Comparing the two, the extracted blue pigment
Maximum absorption wavelength (λ_max) (603-625 nm) and (λ
_maxDifferences in absorbance)
Was. That is, from Table 5, the above extraction conditions
Dye λ_max, And its production (λ_maxAbsorbance at
Degree) better than the extraction conditions, and
The extraction rate of the blue dye varies depending on the type of organic solvent
Respectively. Furthermore, the conclusion of Table 5
From the result, the light blue color was added to the residue of the extraction conditions.
Contains a considerable amount of base pigments, that is,
The blue dye is poorly soluble in water and often appears on the precipitate side of the reaction product.
This suggests that the amount has shifted. In addition, separately
When extracting with an aqueous ethanol solution, use ethanol
The longer the tool concentration, the longer the wavelength λ_maxTurned out to show
Was. Further, FIG. 3 shows that n-
Visible absorption spectrum of blue dye
Indicate the coutle.B) Blue color that can obtain the pigment extraction conditions (extraction operation procedure, etc.)
Dye λ_maxComparative example of the effect on air. [Preparation of sample (reaction solution)]   The test sample (reaction solution / culture solution) is as described in ii) (d) a) above.
Use the reaction solution (culture solution) prepared by the same method as described.
You. [Example of first extraction method] (See FIG. 4)   The reaction solution (culture solution) is sterilized by heating in a water bath (approx.
Use for about 10 minutes. Heat sterilized anti
The reaction solution is separated into solid and liquid by centrifugation, and the first supernatant (aqueous layer)
(L-1) and the first residue (R-1). The said
1 λ of supernatant (L-1)_maxWas 603 nm. The first
Add 50 vol% ethanol aqueous solution to the residue (R-1) and dye
, And then separated into a solid and a liquid to obtain a second supernatant (L-2).
It is separated from the second residue (R-2). The second supernatant (L
-2) λ of 50% ethanol extract_maxIs 610nm
Was. Further, 2N caustic acid was added to the second residue (R-2).
Extract by adding Lucari (caustic soda), then solid-liquid separation
To obtain a third supernatant (L-3) and a third residue (R-3).
You. Λ of the third supernatant (L-3)_maxWas 600 nm. What
No blue pigment was observed in the third residue.
Was.   Extraction conditions for blue dye from reaction solution (culture solution) (extraction
) Of the dye to be extracted_max
Is found to be affected. Fig. 4 shows the first extraction method
(Example) shows the extraction operation procedure and the like. Λ in the figure_max
Extracted the dye from the sample (reaction solution / culture solution)
Λ of each extract_maxIt shows a comparative example of
Depending on the type of organic solvent used,_max
This suggests that a difference occurs.   From the result of the first example of the extraction method, the light blue
Regarding the order of the dyes, the first place was 50% of the first residue (R-1).
The extraction part of the aqueous solution of tanol, the second place is the first supernatant (L-
1) The third place is 2N caustic saw in the second residue (R-2).
Extract, and each extract has its λ_maxValue is 600
nm or more. And the polarity of the solvent for extraction is small
The lighter (ie, λ_maxBlue color with large value)
This suggests that elements tend to be extracted. iii) the lightened natural blue color produced by this invention
Stability test results of dyes. (A) Light and heat stability of the generated blue dye
Sex test results. 〔Test method〕   The blue dye produced according to the present invention is
Matography (filler: silica gel, eluent: ethanol)
And ethanol aqueous solution).
After eluting the contaminant dye (yellow dye) with 100%, 70 vol%
Eluted ethanol and 50 vol% ethanol
You. 70 vol% ethanol elution part (70% EtOH elution part)
(Λ_max= 602 nm) and 50 vol% ethanol elution part (50
% EtOH elution part) (λ_max= 601 nm)
Dilute with each aqueous ethanol solution of the same concentration and adjust the absorbance value.
Adjust to 0.2-0.5 each, and use the fading tester (Mitsubishi
(Modem MH-1, Xenon lamp, manufactured by Kiki Co., Ltd.)
Irradiate for up to 4 hours to fade. Each measurement setting at this time
After hours (0 hours, 1 hour, 2 hours, 4 hours)
Λ of each sample_maxAfter measuring the absorbance at
The rate is determined by calculation. The fading rate is determined based on each set measurement.
The absorbance value at the fixed time
It is calculated as a value (%) divided by each absorbance value. In addition,
As a comparative control, Blue No. 1 (dye) (λ_max= 625 nm)
Used. Table 6 shows examples of the results of this fading test.   From the results in Table 6, it can be seen that the blue color produced by the present invention
Pigments are used for foods and drinks, luxury goods, health pharmaceuticals and
Representative of a wide range of applications such as cosmetics
What does the fading test compare to the stable Blue No. 1?
It indicates that it is excellent in fading without being inferior
You. Therefore, the light blue color system according to the present invention
Dyes are used for foods and drinks, luxury goods, health pharmaceuticals and fragrances.
Widely applicable to various uses such as cosmetics
Extremely useful. During the fading test,
No color change of each test sample was observed by visual observation
Was. (B) Test results of resistance of the produced blue dye to pH
Fruit. (Sample preparation method)   Skim milk and dried gardenia extract are combined with lactic acid bacteria (Streptoc
occus faecalis IFO 12965) in the presence of [ii)
(A) The blue dye (λ_max= 603nm)
Is used as a sample. 〔Test method〕   McKilbain buffer and 40% ethanol
Each pH in Kilbane buffer (each set pH = 4.03, 5.0
7, 5.97, 6.80 (standard), 7.95, 9.07)
Collection wavelength (λ_max) And absorbance are measured,
Of resistance test result of pH of blue dyes produced
Are shown in Table 7.   From the results in Table 7, generated by the method according to the present invention
Blue dyes are converted from alcoholic acidic to alkaline
Suggests that it is stable over a wide range of
Widely used in foods, luxury goods, health pharmaceuticals, cosmetics, etc.
It suggests that the compound can be used. Each of these settings
Visual observation of this sample in the resistance test to constant pH
No color change was observed. <Effect of the Invention>   The present invention relates to iridoid glycosides of Gardenia plants (including
Β) and a primary amino group-containing compound (substance)
Presence of glucosidase or β-glucosidase producing bacteria
In the method of producing a blue dye by reacting in the presence,
For example, adding an electrolyte such as neutral salt such as salt to the reaction system
By reacting, the reaction system is
Reaction under the conditions of reduced system
Bright coloration that was previously not available just by producing pigments
Blue dye (λ_max= 600-625 nm) for easy mass production
it can. In addition, the method of the present invention can
Reaction conditions such as temperature, reaction time and stirring conditions of the reaction solution
Strict control and complicated separation and purification steps of the produced dye
Not required at all, lighter blue dyes are cheaper and easier
Excellent to achieve the object of invention, such as mass production
It works.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the state of the generation of a blue pigment by lactic acid bacteria over time, and FIG. 2 is a diagram showing the state of the generation of a blue pigment by β-glucosidase over time. FIG. 3 is a diagram showing a visible absorption spectrum of a blue pigment extracted from a reaction solution (culture solution) with n-butanol. FIG. 4 is a diagram showing an extraction operation procedure of an embodiment (first extraction method) relating to a method of extracting a blue pigment from a reaction solution (culture solution).

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 1/00 A23L 1/275 C09B 61/00

Claims (1)

(57) [Claims] In the presence of β-glucosidase or a β-glucosidase-producing bacterium, an iridoid glycoside of a Gardenia plant or a substance containing the same is reacted with a primary amino group-containing compound or a primary amino group-containing substance to form a blue dye. In the production method, an electrolyte is added to the reaction system so that the concentration is in the range of 10% by weight or more and 30% by weight or less, and the reaction is carried out under conditions in which the water solubility of the resulting blue dye is reduced. A method for producing a lightened natural blue-based pigment, characterized in that: 2. The method according to claim 1, wherein the electrolyte is an inorganic salt. 3. 2. The method for producing a lightened natural blue dye according to claim 1, wherein the light-emitting natural blue dye has a visible maximum absorption wavelength of 600 to 625 nm.