CN109706205B - Stable-performance black rice anthocyanin monomer and molecular modification preparation method - Google Patents
Stable-performance black rice anthocyanin monomer and molecular modification preparation method Download PDFInfo
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- CN109706205B CN109706205B CN201910031833.7A CN201910031833A CN109706205B CN 109706205 B CN109706205 B CN 109706205B CN 201910031833 A CN201910031833 A CN 201910031833A CN 109706205 B CN109706205 B CN 109706205B
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Abstract
The invention relates to a molecular modification method for improving stability of a black rice anthocyanin monomer (cyanidin-3-glucoside, C3G). The specific operation steps are as follows: (1) cleaning and removing impurities; (2) crushing and leaching; (3) carrying out centrifugal separation; (4) purifying the resin; (5) nano-filtering and concentrating; (6) vacuum freeze drying; (7) C3G extracting and purifying; (8) vacuum freeze drying; (9) enzymatic modification C3G; (10) separating a C3G modified product; (11) and (5) vacuum freeze drying. The method firstly utilizes mild conditions to separate and purify the black rice anthocyanin monomer C3G from the black rice, so as to ensure the integrity of reaction substrates; and the enzymatic modification of C3G is carried out, the whole process is safe and efficient, the reaction conditions are mild and convenient to control, the selected Novozym 435 lipase has strong specificity and single modification product, the preparation type liquid chromatography can be used for high-efficiency separation and purification, and finally the modification product is dried by the vacuum freeze drying technology, so that the stability of the anthocyanin structure is maintained to the greatest extent.
Description
Technical Field
The invention belongs to the technical field of pigment research, and particularly relates to a stable-performance black rice anthocyanin monomer (cyanidin-3-glucoside, C3G) and a molecular modification preparation method.
Background
Anthocyanins, also known as anthocyanidins, are water-soluble natural pigments widely present in plants in nature, belong to flavonoid compounds, and are also the main coloring substances in plant petals. In recent years, a great deal of research on the physiological functions of anthocyanins has been carried out domestically and abroad, and fruitful research results have been obtained. Researches show that the anthocyanin can obviously improve vision, relieve liver dysfunction, inhibit tumor activity and slow the deposition of senile plaques by being eaten frequently, and has the functions of resisting oxidation and delaying senescence. Meanwhile, the anthocyanin is a relatively safe natural pigment, and is developed in a large amount, so that the anthocyanin can meet the requirements of people on the anthocyanin as a medicine, a cosmetic and a health-care food, and can replace the use of an artificially synthesized pigment harmful to human bodies.
However, the anthocyanin structure determines that the anthocyanin is unstable in a natural state and is easy to degrade; meanwhile, external environmental factors are also important factors influencing the stability of the anthocyanin, such as illumination, pH value, temperature, enzyme, redox agent and the like. Therefore, the improvement of the stability of anthocyanin has a positive promoting effect for widening the utilization of anthocyanin. At present, the application of natural active substances in the food industry is mainly improved by a microcapsule method, but the wall materials of the microcapsules are mostly easily dissolved in the common water environment in food, so the application of the microcapsule technology is limited to a certain extent. In the previous experiments, the main monomer (C3G) of the black rice anthocyanin is subjected to acylation reaction with an acyl donor in a nonaqueous environment by taking a chemical or biological enzyme as a catalyst, so that the stability of the obtained derivative on metal ions, temperature and light is remarkably improved, and the proliferation effect on probiotics is also greatly improved.
Disclosure of Invention
In order to improve the stability of anthocyanin and reduce the generation of byproducts in the reaction process, the invention provides a black rice anthocyanin monomer with stable performance and a molecular modification preparation method.
A black rice anthocyanin monomer with stable performance is acylated cyanidin-3-glucoside, which is prepared from cyanidin-3-glucosidep-coumaric acid, malic acid, lauric acid and p-hydroxybenzoic acid, and the acylated cyanidin-3-glucoside is a tan powder with a purity of not less than 95%.
The molecular modification preparation method of the black rice anthocyanin monomer with stable performance comprises the following specific operation steps:
drying a reaction medium by using a 4A molecular sieve, adding an acyl donor and cyanidin-3-glucoside (C3G) powder into the dried reaction medium according to a molar ratio of 10-50: 1, wherein the solid-to-liquid ratio of a reactant to the reaction medium is (1: 1-2), continuously stirring until the reactant and the reaction medium are completely dissolved, adding Novozym 435 lipase, the concentration of the Novozym 435 lipase in the solution is 5-40 mg/mL, finally adding a 4A molecular sieve with the concentration of 100 mg/mL to absorb water generated in the reaction process, and reacting for 100-168 hours at 30-50 ℃ to obtain a cyanidin-3-glucoside reaction liquid;
the reaction medium is one of tert-amyl alcohol, acetonitrile or n-butyl alcohol;
the acyl donor isp-one of coumaric acid or malic acid or lauric acid or p-hydroxybenzoic acid;
the cyanidin-3-glucoside (C3G) powder has a purity of greater than 99.0%;
separating by preparative liquid chromatography, eluting with acetonitrile to obtain a separation solution containing only cyanidin-3-glucoside modified product;
pre-freezing the separation liquid, and drying in a vacuum freeze dryer at the vacuum degree of 0.05-0.1 Mpa and the freeze drying temperature of-20-60 ℃ until the water content is less than or equal to 5 percent to obtain the brown powdered acylated cyanidin-3-glucoside.
The procedure for the preparation of cyanidin-3-glucoside (C3G) is as follows:
(1) cleaning to remove impurities
Removing macroscopic impurities and mildewed particles in the black rice;
(2) pulverizing and extracting
Cleaning the black rice after impurity removal by using deionized water, crushing, and sieving by using a 50-100-mesh sieve; taking black rice and water according to the mass volume ratio of 1 Kg to 10-20L, adding a citric acid solution with the concentration of 1-3% to adjust the pH value to 2.5-6.0, and leaching for 2-5 hours at the temperature of 20-55 ℃ to obtain black rice anthocyanin leaching liquor;
(3) centrifugal separation
Centrifuging the black rice anthocyanin extract liquid to separate solid from liquid, and taking supernatant liquid to obtain black rice anthocyanin water extract liquid;
(4) resin purification
Purifying the black rice anthocyanin water extract by using AB-8 resin, eluting by using ethanol, and collecting black rice anthocyanin ethanol eluent;
(5) nanofiltration concentration
Concentrating the black anthocyanin ethanol eluate by using a nanofiltration membrane with the molecular weight of 200-400 Da, and collecting trapped fluid to obtain a black anthocyanin concentrated solution;
(6) preparation of crude extract of anthocyanin from black rice
Vacuum freeze drying the black rice anthocyanin concentrated solution until the water content is less than or equal to 5 percent to obtain a powdery black rice anthocyanin crude extract;
(7) extraction and purification of cyanidin-3-glucoside
According to the volume ratio of 1: 3-5, redissolving the crude extract of the black rice anthocyanin by using acid liquor, and eluting by using methanol and acetonitrile through preparative liquid chromatography to obtain a separation solution only containing cyanidin-3-glucoside;
(8) vacuum freeze drying
Vacuum freeze drying the separated solution of cyanidin-3-glucoside until the water content is less than or equal to 5% to obtain cyanidin-3-glucoside (C3G) powder.
The technical scheme for preparing cyanidin-3-glucoside (C3G) is further defined as follows:
and (3) removing impurities such as starch, protein, polysaccharide and the like in the black rice anthocyanin leaching liquor by using a disc type centrifuge at 5000-10000 r/min.
In the step (4), the column is eluted by deionized water with the volume of 3-9 times of the column volume, then the sample is loaded according to 1-4 times of the volume of the AB-8 resin, and finally the ethanol with the concentration of 65-95% is used for elution, wherein the elution volume is 2-5 times of the column volume.
In the step (5), nanofiltration concentration conditions are as follows: freeze drying under the conditions of pressure of 0.4-0.8 MPa and temperature of 20-45 ℃.
In the step (6), the vacuum drying operation is as follows: pre-freezing the black rice anthocyanin concentrated solution for 0.5-1 h at the temperature of 0-minus 2 ℃; in a vacuum freeze dryer, the vacuum degree is 0.05-0.1 Mpa, and the temperature is-20 to-60 ℃.
In the step (7), the acid solution has a concentration of 100%pCoumaric acid or malic acid, lauric acid or p-hydroxybenzene-A at a concentration of 100%And (4) acid.
In the step (8), pre-freezing the cyanidin-3-glucoside separation solution for 0.5-1 h at the temperature of 0-2 ℃; drying in a vacuum freeze dryer under the conditions that the vacuum degree is 0.05-0.1 Mpa and the temperature is-20 to-60 ℃ until the water content is less than or equal to 5 percent to obtain powdered cyanidin-3-glucoside powder.
The beneficial technical effects of the invention are embodied in the following aspects:
(1) according to the method, the stability of the anthocyanin is improved (the stability of the modified anthocyanin monomer C3G is improved by more than 50%) by using the Novozym 435 enzyme modified anthocyanin monomer C3G; the anthocyanin modified product is separated and purified by using preparative high performance liquid chromatography, so that the purification efficiency (the purity is more than 95%) of the anthocyanin modified product is improved.
(2) The modification method adopts enzyme method to modify
The chemical modification reaction conditions are difficult to control, the reaction products are complex, and the later separation and purification are difficult; the invention utilizes Novozym 435 enzyme to modify anthocyanin monomer cyanidin-3-glucoside, the enzyme modification has the advantages of high specificity, safety, high efficiency, mild reaction condition, convenient control and the like, and the enzyme modification product is single, and is combined with preparative liquid chromatography, so that the product is easy to separate and purify. Reacting black rice anthocyanin monomer (C3G) with Novozym 435 enzyme as catalyst in nonaqueous environmentpThe coumaric acid, the malic acid, the lauric acid, the p-hydroxybenzoic acid and the like are subjected to acylation reaction, and the stability of the obtained derivative acylated cyanidin-3-O-glucoside) is obviously improved.
(3) Stability of anthocyanins is improved
In the whole extraction and purification process, the anthocyanin monomer is ensured not to be damaged to the greatest extent by adopting medium-temperature leaching, centrifugal impurity removal and freeze-drying; on the basis, the stability of the anthocyanin is improved by changing the structure of the anthocyanin under the condition of keeping the original activity of the anthocyanin basically unchanged. At present, natural products are mainly embedded by a microcapsule method, so that the influence of external environmental factors on the stability of the natural products is avoided, commonly used wall materials such as Arabic gum, maltodextrin and the like are dissolved in water, most of foods contain a water system, and the wall materials of the microcapsules are damaged, so that the embedding effect of the microcapsules is influenced. The invention starts from the structure of anthocyanin, improves the stability of anthocyanin and delays the degradation of anthocyanin under different conditions by changing the relevant spatial structure, and referring to fig. 2, compared with unmodified cyanidin-3-glucoside, the cyanidin-3-glucoside modified product obtained by the method has the stability improved by more than 50 percent under the conditions of normal temperature, illumination and the like, and the stability of acylated cyanidin-3-O-glucoside is further improved.
Drawings
FIG. 1 is a product liquid chromatogram of cyanidin-3-glucoside.
FIG. 2 is C3G quiltpMass spectra successfully modified with coumaric acid as the acyl donor (as an example).
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
The preparation method of cyanidin-3-glucoside (C3G) powder comprises the following steps:
(1) cleaning and impurity removal: removing impurities and mildewed particles which are visible to naked eyes in the black rice;
(2) crushing and leaching: crushing the black rice after impurity removal, and sieving the crushed black rice with a 50-mesh sieve; taking black rice and water according to the mass volume ratio of 1 Kg to 10L, adjusting the pH value to 2.5 by using 1% citric acid solution, and leaching for 2 h at 20 ℃ to obtain black rice anthocyanin leaching liquor;
(3) centrifugal separation: performing solid-liquid separation on black rice anthocyanin leaching liquor by a disc type centrifuge at 5000 r/min, removing impurities such as starch, protein, polysaccharide and the like in the leaching liquor, and taking supernate to obtain black rice anthocyanin water leaching liquor;
(4) resin purification: purifying the black rice anthocyanin water extract by using AB-8 resin, eluting a column by using deionized water with the volume of 3 times of the column volume, then loading according to 1 time of the resin volume, finally eluting by using ethanol with the concentration of 65 percent, eluting the column by using the volume of 2 times of the column volume, and collecting black rice anthocyanin ethanol eluent;
(5) nanofiltration and concentration: concentrating black rice anthocyanin ethanol eluate with nanofiltration membrane with molecular weight of 200 Da at operating pressure of 0.4 MPa and temperature of 20 deg.C, and collecting trapped fluid to obtain black rice anthocyanin concentrated solution;
(6) vacuum freeze drying: pre-freezing the black rice anthocyanin concentrated solution for 0.5h at the temperature of 0 ℃; placing into a vacuum freeze drier, and drying at-20 deg.C under 0.05Mpa of vacuum degree until the water content is less than or equal to 5% to obtain powdered crude extract of black rice anthocyanin, i.e. black rice anthocyanin powder;
(7) extraction and purification: according to the volume ratio of 1: 3 redissolving the crude extract of black rice anthocyanin with 100% acid solutionpCoumaric acid, eluted by preparative liquid chromatography with methanol and acetonitrile, obtaining a separation containing only C3G;
(8) vacuum freeze drying: pre-freezing cyanidin-3-glucoside separation liquid for 0.5h at the temperature of 0 ℃, putting the separation liquid into a vacuum freeze dryer, and drying the separation liquid until the water content is less than or equal to 5% under the conditions that the vacuum degree is 0.05Mpa and the freeze drying temperature is-20 ℃ to obtain powder, namely C3G powder; the purity of the cyanidin-3-glucoside (C3G) powder was 99.15%. The product liquid chromatogram of cyanidin-3-glucoside is shown in FIG. 1.
Example 2
The preparation method of cyanidin-3-glucoside (C3G) powder comprises the following steps:
(1) cleaning and impurity removal: removing macroscopic impurities and mildewed particles in the black rice;
(2) crushing and leaching: crushing the black rice after impurity removal, and sieving the crushed black rice with a 100-mesh sieve; taking black rice and water according to a mass-volume ratio of 1:20 (Kg/L), adjusting the pH value to 6.0 by using a 3% citric acid solution, and leaching for 5 hours at 55 ℃ to obtain black rice anthocyanin leaching liquor;
(3) centrifugal separation: performing solid-liquid separation on black rice anthocyanin leaching liquor by a disc centrifuge at 10000 r/min, removing impurities such as starch, protein, polysaccharide and the like in the leaching liquor, and taking supernatant liquid to obtain black rice anthocyanin water leaching liquor;
(4) resin purification: purifying the black rice anthocyanin water extract by using AB-8 resin, eluting a column by using deionized water with the volume of 9 times of the column volume, then loading according to 4 times of the resin volume, finally eluting by using ethanol with the concentration of 95%, wherein the elution volume is 5 times of the column volume, and collecting black rice anthocyanin ethanol eluent;
(5) nanofiltration and concentration: concentrating black rice anthocyanin ethanol eluate with nanofiltration membrane with molecular weight of 400 Da at operating pressure of 0.8 MPa and temperature of 45 deg.C, and collecting trapped fluid to obtain black rice anthocyanin concentrated solution;
(6) vacuum freeze drying: pre-freezing the black rice anthocyanin concentrated solution for 1h at the temperature of-2 ℃, putting the pre-frozen black rice anthocyanin concentrated solution into a vacuum freeze dryer, and drying the pre-frozen black rice anthocyanin concentrated solution until the water content is less than or equal to 5% under the conditions of the vacuum degree of 0.1Mpa and the freeze drying temperature of-60 ℃ to obtain powder, namely the black rice anthocyanin crude extract;
(7) C3G extraction and purification: according to the volume ratio of 1: 5 redissolving the anthocyanin crude extract with acid liquor, wherein the acid liquor is malic acid with the concentration of 100%, and eluting with methanol and acetonitrile through preparative liquid chromatography to obtain a separation solution only containing C3G;
(8) vacuum freeze drying: pre-freezing cyanidin-3-glucoside separation liquid for 1h at the temperature of-2 ℃, putting the separation liquid into a vacuum freeze dryer, and drying the separation liquid until the water content is less than or equal to 5% under the conditions that the vacuum degree is 0.1Mpa and the freeze drying temperature is-60 ℃, thus obtaining powder, namely C3G powder; the purity of the cyanidin-3-glucoside (C3G) powder was 99.15%.
Example 3
The preparation method of cyanidin-3-glucoside (C3G) powder comprises the following steps:
(1) cleaning and impurity removal: removing macroscopic impurities and mildewed particles in the black rice;
(2) crushing and leaching: crushing the black rice after impurity removal, and sieving the crushed black rice with a 100-mesh sieve; taking black rice and water according to a mass-volume ratio of 1:20 (Kg/L), adjusting the pH value to 6.0 by using a 3% citric acid solution, and leaching for 5 hours at 55 ℃ to obtain black rice anthocyanin leaching liquor;
(3) centrifugal separation: performing solid-liquid separation on black rice anthocyanin leaching liquor by a disc centrifuge at 10000 r/min, removing impurities such as starch, protein, polysaccharide and the like in the leaching liquor, and taking supernatant liquid to obtain black rice anthocyanin water leaching liquor;
(4) resin purification: purifying the black rice anthocyanin water extract by using AB-8 resin, eluting a column by using deionized water with the volume of 9 times of the column volume, then loading according to 4 times of the resin volume, finally eluting by using ethanol with the concentration of 95%, wherein the elution volume is 5 times of the column volume, and collecting black rice anthocyanin ethanol eluent;
(5) nanofiltration and concentration: concentrating black rice anthocyanin ethanol eluate with nanofiltration membrane with molecular weight of 400 Da at operating pressure of 0.8 MPa and temperature of 45 deg.C, and collecting trapped fluid to obtain black rice anthocyanin concentrated solution;
(6) vacuum freeze drying: pre-freezing the black rice anthocyanin concentrated solution for 0.5h at the temperature of 0 ℃, putting the pre-frozen black rice anthocyanin concentrated solution into a vacuum freeze dryer, and drying the pre-frozen black rice anthocyanin concentrated solution until the water content is less than or equal to 5% under the conditions of the vacuum degree of 0.1Mpa and the freeze drying temperature of-60 ℃ to obtain powder, namely the black rice anthocyanin crude extract;
(7) C3G extraction and purification: according to the volume ratio of 1: 5 redissolving the anthocyanin crude extract with an acid solution, wherein the acid solution is 100% lauric acid solution, and performing preparative liquid chromatography by using methanol and acetonitrile to obtain a separation solution only containing C3G;
(8) vacuum freeze drying: pre-freezing cyanidin-3-glucoside separation liquid for 0.5h at the temperature of 0 ℃, putting the separation liquid into a vacuum freeze dryer, and drying the separation liquid until the water content is less than or equal to 5% under the conditions of the vacuum degree of 0.1Mpa and the freeze drying temperature of-60 ℃ to obtain powder, namely C3G powder; the purity of the cyanidin-3-glucoside (C3G) powder was 99.15%.
Example 4
The preparation method of cyanidin-3-glucoside (C3G) powder comprises the following steps:
(1) cleaning and impurity removal: removing macroscopic impurities and mildewed particles in the black rice;
(2) crushing and leaching: crushing the black rice after impurity removal, and sieving the crushed black rice with a 100-mesh sieve; taking black rice and water according to a mass-volume ratio of 1:20 (Kg/L), adjusting the pH value to 6.0 by using a 3% citric acid solution, and leaching for 5 hours at 55 ℃ to obtain black rice anthocyanin leaching liquor;
(3) centrifugal separation: performing solid-liquid separation on black rice anthocyanin leaching liquor by a disc centrifuge at 10000 r/min, removing impurities such as starch, protein, polysaccharide and the like in the leaching liquor, and taking supernatant liquid to obtain black rice anthocyanin water leaching liquor;
(4) resin purification: purifying the black rice anthocyanin water extract by using AB-8 resin, eluting a column by using deionized water with the volume of 9 times of the column volume, then loading according to 4 times of the resin volume, finally eluting by using ethanol with the concentration of 95%, wherein the elution volume is 5 times of the column volume, and collecting black rice anthocyanin ethanol eluent;
(5) nanofiltration and concentration: concentrating black rice anthocyanin ethanol eluate with nanofiltration membrane with molecular weight of 400 Da at operating pressure of 0.8 MPa and temperature of 45 deg.C, and collecting trapped fluid to obtain black rice anthocyanin concentrated solution;
(6) vacuum freeze drying: pre-freezing the black rice anthocyanin concentrated solution for 1h at the temperature of-2 ℃, putting the pre-frozen black rice anthocyanin concentrated solution into a vacuum freeze dryer, and drying the pre-frozen black rice anthocyanin concentrated solution until the water content is less than or equal to 5% under the conditions of the vacuum degree of 0.1Mpa and the freeze drying temperature of-60 ℃ to obtain powder, namely the black rice anthocyanin crude extract;
(7) C3G extraction and purification: according to the volume ratio of 1: 5 redissolving the crude anthocyanin extract with acid liquor, wherein the acid liquor is p-hydroxybenzoic acid with the concentration of 100%, and eluting with methanol and acetonitrile through preparative liquid chromatography to obtain a separation solution only containing C3G;
(8) vacuum freeze drying: pre-freezing cyanidin-3-glucoside separation liquid for 1h at the temperature of-2 ℃, putting the separation liquid into a vacuum freeze dryer, and drying the separation liquid until the water content is less than or equal to 5% under the conditions that the vacuum degree is 0.1Mpa and the freeze drying temperature is-60 ℃, thus obtaining powder, namely C3G powder; the purity of the cyanidin-3-glucoside (C3G) powder was 99.15%.
Example 5
A molecular modification preparation operation of a stable black rice anthocyanin monomer (C3G) comprises the following steps:
(1) enzymatic modification C3G: the reaction is carried out by utilizing a 4A molecular sieveDrying with tert-amyl alcohol as medium, and adding acyl donorpAdding coumaric acid and cyanidin-3-glucoside (C3G) powder into a dried reaction medium according to a molar ratio of 10:1, continuously stirring until the solid-to-liquid ratio of reactants to the reaction medium is 1:1 and the reactants and the reaction medium are completely dissolved, adding Novozym 435 lipase to ensure that the concentration of the Novozym 435 lipase in the solution is 5 mg/mL, finally adding a 4A molecular sieve with the concentration of 100 mg/mL to absorb water generated in the reaction process, and reacting for 100 hours at 30 ℃ to obtain a cyanidin-3-glucoside reaction liquid;
(2) C3G modified product purification:
separating by preparative liquid chromatography with acetonitrile to obtain a separation solution containing only the modified product of C3G;
(3) vacuum freeze drying:
pre-freezing the modified product separation liquid, placing the pre-frozen modified product separation liquid into a vacuum freeze dryer, and drying the modified product separation liquid under the conditions of vacuum degree of 0.05Mpa and freeze drying temperature of-20 ℃ until the water content is less than or equal to 5 percent to obtain powder, namely the C3G modified product (the purity of the modified product is more than or equal to 95 percent). C3G quiltpThe mass spectrum of the successful modification of coumaric acid as acyl donor (as an example) is shown in figure 2.
Example 6
A molecular modification preparation operation of a stable black rice anthocyanin monomer (C3G) comprises the following steps:
(1) enzymatic modification C3G: drying a reaction medium acetonitrile by using a 4A molecular sieve, adding acyl donor malic acid and cyanidin-3-glucoside (C3G) powder into the dried reaction medium according to a molar ratio of 50:1, wherein the solid-to-liquid ratio of a reactant to the reaction medium is (1: 2), continuously stirring until the reactant and the reaction medium are completely dissolved, adding Novozym 435 lipase to ensure that the concentration of the Novozym 435 lipase in the solution is 40 mg/mL, finally adding the 4A molecular sieve with the concentration of 100 mg/mL to absorb water generated in the reaction process, and reacting for 168 h at 50 ℃ to obtain a cyanidin-3-glucoside reaction liquid;
(2) C3G modified product purification: separating by preparative liquid chromatography with acetonitrile to obtain a separation solution containing only the modified product of C3G;
(3) vacuum freeze drying: pre-freezing the modified product separation liquid, placing the pre-frozen modified product separation liquid into a vacuum freeze dryer, and drying the modified product separation liquid under the conditions of vacuum degree of 0.1Mpa and freeze drying temperature of-60 ℃ until the water content is less than or equal to 5 percent to obtain powder, namely the C3G modified product (the purity of the modified product is more than or equal to 95 percent).
Example 7
A molecular modification preparation operation of a stable black rice anthocyanin monomer (C3G) comprises the following steps:
(1) enzymatic modification C3G: drying reaction medium n-butanol by using a 4A molecular sieve, adding acyl donor lauric acid and cyanidin-3-glucoside (C3G) powder into the dried reaction medium according to a molar ratio of 10:1, wherein the solid-to-liquid ratio of a reactant to the reaction medium is (1: 1), continuously stirring until the reactant and the reaction medium are completely dissolved, adding Novozym 435 lipase to ensure that the concentration of the Novozym 435 lipase in the solution is 5 mg/mL, finally adding the 4A molecular sieve with the concentration of 100 mg/mL to absorb water generated in the reaction process, and reacting for 100 hours at 30 ℃ to obtain a cyanidin-3-glucoside reaction liquid;
(2) C3G modified product purification: separating by preparative liquid chromatography with acetonitrile to obtain a separation solution containing only the modified product of C3G;
(3) vacuum freeze drying: pre-freezing the modified product separation liquid, placing the pre-frozen modified product separation liquid into a vacuum freeze dryer, and drying the modified product separation liquid under the conditions of vacuum degree of 0.1Mpa and freeze drying temperature of-60 ℃ until the water content is less than or equal to 5 percent to obtain powder, namely the C3G modified product (the purity of the modified product is more than or equal to 95 percent).
Example 8
A molecular modification preparation operation of a stable black rice anthocyanin monomer (C3G) comprises the following steps:
(1) enzymatic modification C3G: drying a reaction medium tert-amyl alcohol by using a 4A molecular sieve, adding acyl donor p-hydroxybenzoic acid and cyanidin-3-glucoside (C3G) powder into the dried reaction medium according to a molar ratio of 50:1, continuously stirring until the solid-to-liquid ratio of reactants to the reaction medium is (1: 2) until the reactants are completely dissolved, adding Novozym 435 lipase to ensure that the concentration of the Novozym 435 lipase in the solution is 40 mg/mL, finally adding a 4A molecular sieve with the concentration of 100 mg/mL to absorb water generated in the reaction process, and reacting for 168 h at 50 ℃ to obtain a cyanidin-3-glucoside reaction solution;
(2) C3G modified product purification: separating by preparative liquid chromatography with acetonitrile to obtain a separation solution containing only the modified product of C3G;
(3) vacuum freeze drying: pre-freezing the modified product separation liquid, placing the pre-frozen modified product separation liquid into a vacuum freeze dryer, and drying the modified product separation liquid under the conditions of vacuum degree of 0.1Mpa and freeze drying temperature of-60 ℃ until the water content is less than or equal to 5 percent to obtain powder, namely the C3G modified product (the purity of the modified product is more than or equal to 95 percent).
Claims (1)
1. A preparation method of a black rice anthocyanin monomer with stable performance is characterized by comprising the following steps: the black rice anthocyanin monomer is acylated cyanidin-3-glucoside, and is prepared by passing cyanidin-3-glucosidepThe-coumaric acid is modified to obtain acylated cyanidin-3-glucoside which is tan powder and has the purity more than or equal to 95 percent;
the preparation method comprises the following specific preparation operation steps:
drying a reaction medium by using a 4A molecular sieve, adding an acyl donor and cyanidin-3-glucoside (C3G) powder into the dried reaction medium according to a molar ratio of 10:1, wherein the solid-to-liquid ratio of a reactant to the reaction medium is 1:1, continuously stirring until the reactant and the reaction medium are completely dissolved, adding Novozym 435 lipase to ensure that the concentration of the Novozym 435 lipase in the solution is 5 mg/mL, finally adding a 4A molecular sieve with the concentration of 100 mg/mL to absorb water generated in the reaction process, and reacting for 100 hours at 30 ℃ to obtain a cyanidin-3-glucoside reaction liquid;
the reaction medium is one of tert-amyl alcohol, acetonitrile or n-butyl alcohol;
the acyl donor isp-coumaric acid;
the cyanidin-3-glucoside (C3G) powder has a purity of greater than 99.0%;
separating by preparative liquid chromatography, eluting with acetonitrile to obtain a separation solution containing only cyanidin-3-glucoside modified product;
pre-freezing the separation liquid, and drying in a vacuum freeze dryer at the vacuum degree of 0.05-0.1 Mpa and the freeze drying temperature of-20-60 ℃ until the water content is less than or equal to 5 percent to obtain brown powdered acylated cyanidin-3-glucoside;
the specific operation steps for preparing cyanidin-3-glucoside are as follows:
(1) cleaning to remove impurities
Removing macroscopic impurities and mildewed particles in the black rice;
(2) pulverizing and extracting
Cleaning the black rice after impurity removal by using deionized water, crushing, and sieving by using a 50-100-mesh sieve; taking black rice and water according to the mass volume ratio of 1 Kg to 10-20L, adding a citric acid solution with the concentration of 1-3% to adjust the pH value to 2.5-6.0, and leaching for 2-5 hours at the temperature of 20-55 ℃ to obtain black rice anthocyanin leaching liquor;
(3) centrifugal separation
Centrifuging the black rice anthocyanin extract liquid to separate solid from liquid, and taking supernatant liquid to obtain black rice anthocyanin water extract liquid;
the specific operation is as follows: removing impurities such as starch, protein, polysaccharide and the like in the black rice anthocyanin leaching liquor by using a disc type centrifuge at 5000-10000 r/min;
(4) resin purification
Purifying the black rice anthocyanin water extract by using AB-8 resin, eluting by using ethanol, and collecting black rice anthocyanin ethanol eluent;
the specific operation is as follows: eluting the column by using deionized water with the volume of 3-9 times of the column volume, then loading according to 1-4 times of the volume of the AB-8 resin, and finally eluting by using ethanol with the concentration of 65-95%, wherein the elution volume is 2-5 times of the column volume;
(5) nanofiltration concentration
Concentrating the black anthocyanin ethanol eluate by using a nanofiltration membrane with the molecular weight of 200-400 Da, and collecting trapped fluid to obtain a black anthocyanin concentrated solution;
and (3) nanofiltration concentration conditions: freeze-drying under the conditions of pressure of 0.4-0.8 MPa and temperature of 20-45 ℃;
(6) preparation of crude extract of anthocyanin from black rice
Vacuum freeze drying the black rice anthocyanin concentrated solution until the water content is less than or equal to 5 percent to obtain a powdery black rice anthocyanin crude extract;
the vacuum drying operation was as follows: pre-freezing the black rice anthocyanin concentrated solution for 0.5-1 h at the temperature of 0-minus 2 ℃; in a vacuum freeze dryer, the vacuum degree is 0.05-0.1 Mpa, and the temperature is-20 to-60 ℃;
(7) extraction and purification of cyanidin-3-glucoside
According to the volume ratio of 1: 3-5, redissolving the crude extract of the black rice anthocyanin by using acid liquor, and eluting by using methanol and acetonitrile through preparative liquid chromatography to obtain a separation solution only containing cyanidin-3-glucoside;
the acid liquor is 100 percentp-coumaric acid;
(8) vacuum freeze drying
Vacuum freeze drying the separated solution of cyanidin-3-glucoside until the water content is less than or equal to 5% to obtain cyanidin-3-glucoside (C3G) powder;
the specific operation is as follows: pre-freezing the cyanidin-3-glucoside separation solution for 0.5-1 h at the temperature of 0-2 ℃; drying in a vacuum freeze dryer under the conditions that the vacuum degree is 0.05-0.1 Mpa and the temperature is-20 to-60 ℃ until the water content is less than or equal to 5 percent to obtain powdered cyanidin-3-glucoside.
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