CN112251430A - Method for extracting gardenia blue pigment - Google Patents
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Abstract
The invention relates to the field of food, and aims to solve the problems of gardenia blue pigment in the fixing process. Then the obtained immobilized beta-glucosidase is used for catalyzing geniposide. Under the catalytic condition of beta-glucosidase, geniposide is hydrolyzed to generate glucose and genipin, and the genipin reacts with amino acid to generate the gardenia blue pigment. The prepared gardenia blue pigment has the characteristics of good human body compatibility and high safety, and the immobilized beta-glucosidase has good characteristics.
Description
Technical Field
The invention relates to the field of food, and in particular relates to a method for extracting gardenia blue pigment.
Background
Cape jasmine fruit, another name: gardenia jasminoides Ellis, Gardenia jasminoides Ellis and Cinobufagin are fruits of Gardenia jasminoides Ellis of Rubiaceae. The fruit of gardenia is a traditional Chinese medicine, belongs to the 1 st batch of medical and edible dual-purpose resources issued by the ministry of health, and has the functions of protecting liver, benefiting gallbladder, reducing blood pressure, calming, stopping bleeding, reducing swelling and the like. Is used for treating icteric hepatitis, sprain, contusion, hypertension, diabetes and other diseases in traditional Chinese medicine clinic. Contains crocetin glycoside group, and can be used as yellow dye. The geniposide is extracted and refined from dried mature fruit of Gardenia jasminoides Ellis of Rubiaceae by high-tech production process. The geniposide is iridoid glycoside compound, namely geniposide. The geniposide has multiple purposes, can be fermented under different conditions, can be made into natural edible colorants gardenia blue and gardenia red, and is also a raw material medicine for treating cardiovascular and cerebrovascular diseases, liver and gall diseases and diabetes.
The immobilized enzyme technology can not only ensure the recycling of the enzyme, but also improve the stability of the enzyme in the catalytic process, thereby leading the industrial application of the enzyme to be more economical. At present, glutaraldehyde is generally used as a cross-linking agent in the process of extracting gardenia blue pigment for immobilization, however, research shows that: the use amount of glutaraldehyde is strictly controlled, the activity of the immobilized enzyme is inversely proportional to the use amount of glutaraldehyde, excessive glutaraldehyde can cause unnecessary intermolecular crosslinking of the enzyme, and excessive crosslinking can distort the structure of the enzyme, so that the activity of the enzyme is reduced. In addition, the biological toxicity of glutaraldehyde limits the application range, so that a simple, non-toxic and mild method for high-efficiency green immobilization of the enzyme is urgently needed to be found. The novel immobilized enzyme prepared by immobilizing beta-glucosidase on a carrier through dopamine autopolymerization reaction by using magnetic silica as the carrier is a green immobilization method.
Beta-glucosidase (EC 3.2.1.21), also known as beta-D-glucoside hydrolase, is capable of hydrolyzing the beta-D-glucoside bond bound to the non-reducing end, and releasing beta-D-glucose and corresponding aglycone; can also be used for synthesizing glucoside compounds by catalytic reverse hydrolysis reaction in a micro-water environment. The catalytic property determines that the beta-glucosidase has important application prospects in various aspects of improving fruit juice flavor, increasing fruit wine flavor, increasing tea flavor, producing soybean isoflavone active aglycone, degrading cellulose and the like.
Disclosure of Invention
In order to solve the problems of the gardenia blue pigment in the fixing process, the invention provides the method for extracting the gardenia blue pigment, and the prepared gardenia blue pigment has the characteristics of good human body compatibility and high safety, and the immobilized beta-glucosidase has good characteristics.
The invention is realized by the following technical scheme: the extraction method of gardenia blue pigment comprises the following steps:
(1) preparing immobilized beta-glucosidase; the preparation method of the immobilized beta-glucosidase comprises the following steps:
(1.1)Fe3O4preparing nano particles;
preparation of Fe by solvothermal method3O4Nanoparticle: firstly, ferric trichloride, sodium citrate and sodium acetate are dissolved in ethylene glycol in turn under stirring, the stirring is continued until a yellow homogeneous solution is formed, and then the solution is transferred to a high-pressure reaction kettle and reacted for 8 to 12 hours at the temperature of 220 ℃. After cooling, the black solid was washed several times with water and ethanol in sequence and dried under vacuum.
Wherein the mass ratio of ferric trichloride, sodium citrate and sodium acetate is 1: 0.25-0.3: 1.5-2.3, and the preferred dosage of ethylene glycol is as follows: the mass and volume ratio of ferric trichloride, sodium citrate and sodium acetate to glycol is 8-12 g: 100 mL.
(1.2)Fe3O4@SiO2Preparing nano particles;
using improvements
Preparation of Fe by hydrolysis3O4@SiO2Nanoparticle: under the action of ultrasonic wave, Fe is mixed3O4Uniformly dispersing the nano particles in a mixed system of water, ethanol and ammonia water, dropwise adding tetraethoxysilane (TEOS, AR), stirring at room temperature for 6-7h, magnetically separating the product, washing off unreacted raw materials with water, and drying to obtain Fe3O4@SiO2Nanoparticles.
Fe3O4The mass-volume ratio of the nano particles to the tetraethoxysilane is as follows: 0.1-0.2 g: 1 mL.
The volume ratio of water, ethanol and ammonia water in the mixed system is 5: 1.
Preferably, the ultrasonic dispersion has a frequency of 80-120Hz and a time of 0.5-1 h.
(1.3) immobilization of β -glucosidase.
Taking Fe3O4@SiO2Dispersing nanoparticles in Tris buffer solution (Tris with purity not less than 99.5%) under ultrasonic action, adding dopamine hydrochloride, stirring at room temperature for 22-26 hr, magnetically separating the product, and washing with water to remove unreacted dopamine to obtain Fe3O4@SiO2@ PDA nanoparticles. Then adding Fe3O4@SiO2The @ PDA nano particles are uniformly dispersed in a sodium citrate buffer solution (the purity is more than or equal to 99 percent), beta-glucosidase is added, and immobilization is carried out for 24 hours at room temperature. Magnetically separating the product, and washing off the non-immobilized beta-glucosidase with water to obtain the immobilized beta-glucosidase.
Fe3O4@SiO2The ratio of the amount of the nano particles to the amount of the dopamine hydrochloride is 1: 8-9. Preferably, Fe3O4@SiO2The mass-volume ratio of the nano particles to the tris buffer solution is 1.5-2.5 mg: 1 mL.
The addition amount of the beta-glucosidase is 50U/g Fe3O4@SiO2@ PDA nanoparticles, preferably Fe3O4@SiO2The mass-volume ratio of the @ PDA nano-particles to the sodium citrate buffer solution is 8-12 mg: 1 mL.
The magnetic carrier material is used as the immobilized material, and the magnetic carrier material has the advantages of no toxicity, unique magnetic responsiveness and easy separation from the substrate solid.
Dopamine is a biological neurotransmitter, can perform autopolymerization reaction in an alkaline aerobic solution, thereby forming a polydopamine layer strongly adhered to the surface of a solid material, and has excellent dispersibility, biocompatibility and mild reaction conditions.
(2) Extracting geniposide from gardenia: the extraction method for extracting geniposide from gardenia comprises the following steps:
(2.1) crushing the dried gardenia raw material, sieving the crushed gardenia raw material by a sieve of 20 to 60 meshes, and collecting coarse powder for later use;
(2.2) enzymolysis: weighing coarse powder, adding ethanol for soaking, adding biological enzyme, and soaking and performing enzymolysis at pH 4-6 and temperature 40-50 deg.C for 1-2 hr to obtain enzymolysis solution;
the biological enzyme is selected from one or more of pectinase, cellulase and hemicellulase. The addition amount of the biological enzyme is 0.1-0.3% of the weight of the coarse powder.
Preferably, the feed-liquid ratio of the coarse powder to the ethanol is 1 g: 3-5mL, wherein the mass concentration of the ethanol is 50% -70%.
(2.3) eluting the enzymolysis solution, collecting the eluent, and drying to obtain geniposide;
the method comprises the following specific steps: passing the obtained enzymolysis liquid through a macroporous adsorption resin column, eluting with 2-5 times of column volume of distilled water, eluting with 2-5 times of column volume of 60-80% ethanol by mass concentration, and collecting ethanol eluate;
the macroporous adsorption resin is one of X-5, LSA-21 and WLD; the reagent can be recovered after use.
(3) Geniposide is used for extracting genipin by beta-glucoside immobilized enzyme;
the extraction method comprises the following steps: weighing geniposide, adding an immobilized beta-glucosidase aqueous solution, and carrying out hydrolysis reaction at the temperature of 50-60 ℃ and the pH of 4-5 for 6-8h to obtain genipin.
The addition amount of the immobilized beta-glucosidase aqueous solution is 0.1-0.3% of the mass of geniposide, and the mass concentration of the immobilized beta-glucosidase aqueous solution is 7-9%.
(4) Gardenia blue pigment is extracted from genipin.
The extraction method comprises the following steps: adding amino acid into genipin, and reacting at 70-90 deg.C under neutral pH condition for 8-12 to obtain gardenia blue pigment.
The amino acid comprises methionine, glycine, lysine and alanine. The molar ratio of the amino acid to the genipin is 5-7: 1.
According to the invention, gardenia is selected as a base material, magnetic silicon dioxide with a core-shell structure is used as an immobilized carrier, and beta-glucosidase is immobilized on the carrier through dopamine autopolymerization, so that various immobilization conditions are optimized. Then the obtained immobilized beta-glucosidase is used for catalyzing geniposide. Under the catalytic condition of beta-glucosidase, geniposide is hydrolyzed to generate glucose and genipin, and the genipin reacts with amino acid to generate the gardenia blue pigment.
The gardenia blue pigment obtained by the extraction method of the gardenia blue pigment has good effect on food coloring. The invention adopts the biological engineering technology, and the extracted substance has the function of food coloring and has the advantages of good human body compatibility and high safety. Compared with the existing preparation method of the gardenia blue pigment, the magnetic nanoparticle material applied by the process can effectively improve the production efficiency of the gardenia blue pigment, and the activity of the prepared immobilized beta-glucosidase is obviously superior to that of the traditional glutaraldehyde crosslinking immobilized beta-glucosidase. The catalyst has high reaction selectivity and good stability, and can be recycled.
Compared with the prior art, the invention has the beneficial effects that:
(1) the production process does not cause pollution to the environment;
(2) the required materials are simple and easy to obtain, and the production cost is low;
(3) the obtained product has good human body compatibility and high safety;
(4) the catalytic activity of the adopted immobilized enzyme is higher than that of free enzyme;
(5) the recovery rate and the reuse rate of the immobilized enzyme are high.
Drawings
FIG. 1 is an HPLC chart of gardenia blue pigment;
FIG. 2 is a graph showing thermodynamic stability of immobilized β -glucosidase and free enzyme;
FIG. 3 shows the acid and alkali resistance of immobilized beta-glucosidase and free enzyme.
Detailed Description
The present invention will be described in further detail below with reference to examples and the accompanying drawings, in which the starting materials are commercially available or can be prepared by conventional methods.
In the examples, the ultrasonic dispersion was carried out at a frequency of 100Hz for a period of 1 hour.
Example 1
(1) Preparation of immobilized beta-glucosidase:
(1.1)Fe3O4preparing nano particles: preparation of Fe by solvothermal method3O4Nanoparticles. First, 3g of ferric chloride, 0.87g of sodium citrate and 6g of sodium acetate were weighed out and dissolved in 100mL of ethylene glycol in succession with stirring, and stirring was continued until a yellow homogeneous solution was formed. Then, the mixture is transferred to a high-pressure reaction kettle and reacted for 8 hours at 220 ℃, after cooling, the black solid is washed for a plurality of times by water and ethanol in sequence and dried for 24 hours under vacuum.
(1.2)Fe3O4@SiO2Preparing nano particles: using improvements
Preparation of Fe by hydrolysis3O4@SiO2Nanoparticles. Under the action of ultrasonic wave, 0.2g of Fe3O4The nano particles are uniformly dispersed in a mixed system of water, ethanol and ammonia water. Then, 1.8mL of TEOS was added dropwise, stirred at room temperature for 7h, the product was magnetically separated, unreacted starting material was washed with water, and dried under vacuum for 24h.
(1.3) immobilization of β -glucosidase: self-polymerization with dopamine: taking 300mg of Fe3O4@SiO2The nano particles are uniformly dispersed in 150mL of Tris buffer solution under the action of ultrasound, and dopamine hydrochloride (Fe) is added3O4@SiO2The ratio of the nano particles to the dopamine hydrochloride is 1: 8), and stirring is carried out for 24 hours at room temperature. The product was magnetically separated and unreacted dopamine was washed away with water. 50mg of Fe3O4@SiO2@ PDA nano particles are uniformly dispersed in 5mL sodium citrate buffer solution, beta-glucosidase is added according to 50U/g carrier, and immobilization is carried out for 24h at room temperature. Magnetic separation, washing off the non-immobilized beta-glucosidase with water to obtain the immobilized beta-glucosidase 1.
(2) Extracting geniposide from gardenia:
(2.1) crushing 8g of dried gardenia raw material, sieving by a 60-mesh sieve, and collecting coarse powder for later use;
(2.2) enzymolysis: weighing 3g of coarse powder, adding 12mL of 60 wt% ethanol for soaking, adding 0.2% of hemicellulase by weight of the coarse powder, and soaking and performing enzymolysis for 2 hours at the pH value of 4 and the temperature of 40 ℃;
(2.3) putting the obtained enzymolysis liquid on a macroporous adsorption resin column X-5, eluting by using distilled water with the volume of 3 times that of the column, eluting by using 60 wt% ethanol with the volume of 3 times that of the column, and collecting ethanol eluent; recovering reagent, and vacuum drying the eluate to obtain geniposide, which is also geniposide;
(3) extracting genipin from geniposide: taking 1g of geniposide, adding immobilized beta-glucosidase with the geniposide amount of 0.1 wt% and the mass concentration of 8%, and performing full hydrolysis reaction at the temperature of 50 ℃ and under the condition of pH of 4 for 6 hours to obtain genipin;
(4) extracting gardenia blue pigment from genipin: adding glycine into genipin, wherein the molar ratio of the glycine to the genipin is 5: 1, and reacting at 70 ℃ at the pH of 7.0 for 12 h; after the reaction is fully carried out, vacuum drying is carried out to obtain the gardenia blue pigment 1.
Example 2:
(1) preparation of immobilized beta-glucosidase:
(1.1)Fe3O4preparing nano particles: preparation of Fe by solvothermal method3O4Nanoparticles. First, 3.25g of ferric chloride, 0.87g of sodium citrate and 6g of sodium acetate were weighed out and dissolved in 100mL of ethylene glycol in succession with stirring, and stirring was continued until a yellow homogeneous solution was formed. Then, the mixture was transferred to an autoclave and reacted at 200 ℃ for 10 hours. After cooling, the black solid was washed several times with water and ethanol in sequence and dried under vacuum for 24h.
(1.2)Fe3O4@SiO2Preparing nano particles: using improvements
Preparation of Fe by hydrolysis3O4@SiO2Nanoparticles. Under the action of ultrasonic wave, 0.3g of Fe3O4The nano particles are uniformly dispersed in a mixed system of water, ethanol and ammonia water. Then, 1.8mL of TEOS was added dropwise, stirred at room temperature for 6h, the product was magnetically separated, unreacted starting material was washed with water, and dried under vacuum for 24h.
(1.3) immobilization of β -glucosidase: self-polymerization with dopamine: taking 300mg of Fe3O4@SiO2The nano particles are uniformly dispersed in 150mL of Tris buffer solution under the action of ultrasound, and dopamine hydrochloride (Fe) is added3O4@SiO2The ratio of the nano particles to the dopamine hydrochloride is 1: 8), and stirring is carried out for 24 hours at room temperature. The product was magnetically separated and unreacted dopamine was washed away with water. 50mg of Fe3O4@SiO2@ PDA nano particles are uniformly dispersed in 5mL sodium citrate buffer solution, beta-glucosidase is added according to 50U/g carrier, and immobilization is carried out for 24h at room temperature. Magnetic separation, then washing off the non-immobilized beta-glucosidase with water. Obtaining the required immobilized beta-glucosidase 2.
(2) Extracting geniposide from gardenia:
(2.1) crushing 8g of dried gardenia raw material, sieving with a 40-mesh sieve, and collecting coarse powder for later use;
(2.2) enzymolysis: weighing 3g of coarse powder, adding 9mL of 50 wt% ethanol for soaking, adding 0.1 wt% of cellulase in the coarse powder, and soaking and performing enzymolysis for 1.5h at the pH value of 5 and the temperature of 45 ℃;
(2.3) putting the enzymolysis liquid on a macroporous adsorption resin column LSA-21, eluting by using distilled water with the volume of 3 times of the column volume, then eluting by using ethanol with the volume of 3 times and 70 wt%, and collecting ethanol eluent; recovering the reagent, and drying in vacuum to obtain geniposide;
(3) extracting genipin from geniposide: taking 1g of geniposide, adding immobilized beta-glucosidase with the mass concentration of 8 percent and 0.2wt percent of the dosage of the geniposide, and carrying out full hydrolysis reaction at the temperature of 55 ℃ and the pH value of 4 for 7 hours to obtain genipin;
(4) extracting gardenia blue pigment from genipin: adding methionine into the product 2, reacting at pH 7.0 and 80 deg.C for 10 hr at a molar ratio of methionine to geniposide of 6: 1, and vacuum drying to obtain gardenia blue pigment 2.
Example 3:
(1) preparation of immobilized beta-glucosidase:
(1.1)Fe3O4preparing nano particles: preparation of Fe by solvothermal method3O4Nanoparticles. First, 3.4g of ferric chloride, 0.87g of sodium citrate and 6g of sodium acetate were weighed out and dissolved in 100mL of ethylene glycol in succession with stirring, and stirring was continued until a yellow homogeneous solution was formed. Then, the mixture was transferred to an autoclave and reacted at 180 ℃ for 12 hours. After cooling, the black solid was washed several times with water and ethanol in sequence and dried under vacuum for 24h.
(1.2)Fe3O4@SiO2Preparing nano particles: using improvements
Preparation of Fe by hydrolysis3O4@SiO2Nanoparticles. Under the action of ultrasonic wave, 0.35g of Fe3O4The nano particles are uniformly dispersed in a mixed system of water, ethanol and ammonia water. Then, 1.8mL of TEOS was added dropwise, stirred at room temperature for 6h, the product was magnetically separated, unreacted starting material was washed with water, and dried under vacuum for 24h.
(1.3) immobilization of β -glucosidase: self-polymerization with dopamine: taking 300mg of Fe3O4@SiO2The nano particles are uniformly dispersed in 150mL of Tris buffer solution under the action of ultrasound, and dopamine hydrochloride (Fe) is added3O4@SiO2The ratio of the nano particles to the dopamine hydrochloride is 1: 8), and stirring is carried out for 24 hours at room temperature. The product was magnetically separated and unreacted dopamine was washed away with water. 50mg of Fe3O4@SiO2@ PDA nano particles are uniformly dispersed in 5mL sodium citrate buffer solution, beta-glucosidase is added according to 50U/g carrier, and immobilization is carried out for 24h at room temperature. Magnetic separation, then washing off the non-immobilized beta-glucosidase with water. Obtaining the required immobilized beta-glucosidase 3.
(2) Extracting geniposide from gardenia:
(2.1) crushing 8g of dried gardenia raw material, sieving with a 20-mesh sieve, and collecting coarse powder for later use;
(2.2) enzymolysis: weighing 3g of coarse powder, adding 15mL of 70 wt% ethanol for soaking, adding 0.3% of pectinase in weight of the coarse powder, and soaking and performing enzymolysis for 1h at the pH value of 6 and the temperature of 50 ℃;
(2.3) putting the obtained enzymolysis liquid on a macroporous adsorption resin column WLD, eluting by using distilled water with the volume of 3 times of the column volume, eluting by using ethanol with the volume of 3 times and 80 wt%, and collecting ethanol eluent; recovering the reagent, and drying in vacuum to obtain geniposide;
(3) extracting genipin from geniposide: taking 1g of geniposide, adding immobilized beta-glucosidase with the mass concentration of 9 percent and 0.3wt percent of the dosage of the geniposide, and carrying out full hydrolysis reaction at the temperature of 60 ℃ and the pH value of 5 for 6 hours to obtain genipin;
(4) extracting gardenia blue pigment from genipin: adding methionine into genipin, wherein the mol ratio of the methionine to geniposide is 7: 1, and the reaction time is 10h at the temperature of 80 ℃ and the pH value of 7.0; after full reaction, vacuum drying to obtain the gardenia blue pigment 3.
Comparative example 1
(1) Extracting geniposide from gardenia:
(1.1) crushing 8g of dried gardenia raw material, sieving the crushed gardenia raw material by a 20-60-mesh sieve, and collecting coarse powder for later use;
(1.2) enzymolysis: weighing 3g of coarse powder, adding 9mL of 50% ethanol for soaking, adding pectinase accounting for 0.1% of the weight of the coarse powder, and soaking and performing enzymolysis for 1.5h under the conditions that the pH value is 5 and the temperature is 45 ℃;
(1.3) passing the obtained concentrated solution through a macroporous adsorption resin column, eluting by using distilled water with the volume of 3 times of the column volume, eluting by using ethanol with the volume of 3 times and 70 percent, and collecting ethanol eluent; recovering the reagent, and drying in vacuum to obtain geniposide;
(2) extracting genipin from geniposide: taking 1g of geniposide, adding 0.2 wt% of the geniposide, and 8% of free beta-glucosidase by mass concentration, and performing full hydrolysis reaction at 55 ℃ and pH of 4 for 7h to obtain genipin;
(3) extracting gardenia blue pigment from genipin: adding methionine into genipin, wherein the molar ratio of the methionine to geniposide is 6: 1, and reacting for 10h at the temperature of 80 ℃ and the pH of 7.0; after full reaction, vacuum drying to obtain the gardenia blue pigment.
Test example 1:
HPLC is used as a detection means, and aims to provide an economical, simple and efficient production process of gardenia blue pigment. The obtained substance has the characteristics of good human body compatibility and high safety. Has the significance of saving cost, being harmless to the environment and the like in the treatment process
The HPLC chart of the gardenia blue pigment obtained in examples 1 to 3 is shown in FIG. 1, and it is understood from the HPLC chart that the gardenia blue pigment can be obtained by the present experimental method.
Test example 2:
(1) the immobilization rates of β -glucosidase prepared in examples 1 to 3 are shown in table 1:
table 1: immobilization rate of beta-glucosidase
| Examples | 1 | 2 | 3 |
| Immobilization rate of enzyme | 67％ | 72％ | 69％ |
(2) The enzyme activity assays of the immobilized β -glucosidase of examples 1-3 and the β -glucosidase of comparative example are shown in table 2.
Table 2: enzymatic activity of beta-glucosidase
| Examples | Example 1 | Example 2 | Example 3 | Example 4 |
| Enzyme activity/(U.g)-1) | 24.09 | 24.43 | 24.24 | 10.08 |
Therefore, the enzyme activity of the immobilized beta-glucosidase is higher than that of the common beta-glucosidase.
(3) The thermodynamic stability curves of the beta-glucosidase 2 prepared in example 2 and the beta-glucosidase 4 prepared in example 4 at different temperatures are shown in fig. 2, and the relative enzyme activities of the beta-glucosidase and the free enzyme are continuously reduced along with the increase of the temperature, and the relative enzyme activity of the immobilized enzyme is always higher than that of the free enzyme. The immobilized enzyme and free enzyme activities at different temperatures are shown in Table 3.
Table 3: enzyme-immobilized and free enzyme activities at different temperatures
(4) The immobilized beta-glucosidase and the free beta-glucosidase are respectively treated in a buffer solution with the pH value of 2.0-8.0 for 8 hours at the temperature of 30 ℃, the residual enzyme activity is measured, the respective highest enzyme activity is 100%, the relative enzyme activity is calculated, the result is shown in figure 3, the acid and alkali resistance of the figure 3 shows that the pH stability of the immobilized beta-glucosidase is obviously higher than that of the free enzyme, the pH stable range of the immobilized beta-glucosidase is widened after immobilization, and the application range of the immobilized beta-glucosidase is favorably expanded. The relative enzyme activities of immobilized β -glucosidase and free enzyme at the same temperature and different pH are shown in Table 4.
TABLE 4 relative enzyme activities of immobilized and free enzymes at different pH conditions
(5) The yields of gardenia blue pigment obtained in examples 1 to 3 and comparative example are shown in Table 5.
Table 5: (yield/%)
| Examples | Example 1 | Example 2 | Example 3 | Comparative example |
| Yield/%) | 63 | 71 | 69 | 43 |
It can be seen that the yield of gardenia blue pigment using the immobilized enzyme-catalyzed reaction is higher than that using the free enzyme-catalyzed reaction.
(6) The gardenia blue pigment obtained in example 2 was subjected to national standard detection for food safety.
Sensory detection: 0.1g of gardenia blue pigment obtained in example 2 was put in a clean, dry white porcelain dish, and the color and state thereof were observed under natural light, and the smell thereof was smelled. The product is a powdery solid with slightly special aromatic odor and light purple blue after inspection, and meets the sensory requirement.
Physicochemical index detection: the physical and chemical indexes of gardenia blue pigment obtained in example 2 were measured, and the results are shown in table 6. Therefore, the gardenia blue pigment prepared by the process is an excellent product which meets the national standard of food safety.
Table 6: comparison of national food safety standards
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (10)
1. The extraction method of the gardenia blue pigment is characterized by comprising the following steps:
(1) preparing immobilized beta-glucosidase;
(2) extraction of geniposide from gardenia:
(3) geniposide is used for extracting genipin by immobilized beta-glucosidase;
(4) gardenia blue pigment is extracted from genipin.
2. The method of claim 1, wherein the preparation of the immobilized β -glucosidase in step (1) comprises the steps of:
（1.1）Fe3O4preparing nano particles;
（1.2）Fe3O4@SiO2preparing nano particles;
(1.3) immobilization of β -glucosidase.
3. The method for extracting gardenia blue pigment according to claim 1, wherein the method for extracting geniposide from gardenia in the step (2) comprises the following steps:
(2.1) crushing the dried gardenia raw material, sieving the crushed gardenia raw material by a sieve of 20 to 60 meshes, and collecting coarse powder for later use;
(2.2) enzymolysis: weighing coarse powder, adding ethanol for soaking, adding biological enzyme, and soaking and performing enzymolysis at pH 4-6 and temperature 40-50 deg.C for 1-2 hr to obtain enzymolysis solution;
and (2.3) eluting the enzymolysis solution, collecting the eluent, and drying to obtain the geniposide.
4. The method of claim 3, wherein the biological enzyme in step (2.2) is selected from one or more of pectinase, cellulase and hemicellulase.
5. The method according to claim 3 or 4, wherein the amount of the bio-enzyme added is 0.1-0.3% of the weight of the coarse powder.
6. The method of claim 1, wherein the step (3) comprises the steps of: weighing geniposide, adding an immobilized beta-glucosidase aqueous solution, and carrying out hydrolysis reaction at the temperature of 50-60 ℃ and the pH of 4-5 for 6-8h to obtain genipin.
7. The method according to claim 6, wherein the amount of the immobilized β -glucosidase aqueous solution is 0.1-0.3% of the geniposide by mass, and the concentration of the immobilized β -glucosidase aqueous solution is 7-9% by mass.
8. The method of claim 1, wherein the step (4) comprises the steps of: adding amino acid into genipin, and reacting at 70-90 deg.C for 8-12h under neutral pH.
9. The method of claim 8, wherein the amino acids comprise methionine, glycine, lysine, and alanine.
10. The method according to claim 8 or 9, wherein the molar ratio of amino acid to genipin is 5-7: 1.
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