CN116253708A - Method for extracting anthocyanin from black rice - Google Patents
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Abstract
The invention relates to a method for extracting anthocyanin from black rice, which aims to solve the technical problems of high extraction cost, complex process, long period and the like of the conventional acylated anthocyanin. The method comprises the steps of 1, taking black rice raw materials, and taking the black rice raw materials according to the mass of the black rice raw materials: extraction solvent volume = 1g: (3-8) adding extraction solvent into ml, extracting for multiple times to obtain black rice extract; the extraction solvent comprises an organic solvent and a compound acid with the volume ratio of 3 to 6 per mill with the extraction solvent; 2. ultrafiltering, and collecting ultrafiltrate; nanofiltration and collection of nanofiltration membrane retentate to obtain anthocyanin extract; 3. adding ethanol solution, pyridine, methyl benzoate and lipase into the anthocyanin extract to carry out acylation to obtain acylated anthocyanin concentrated solution; 4. adding 3-8 mill hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.02-1.07, adding the mixture into a macroporous adsorption resin chromatographic column, collecting eluent, concentrating and drying to obtain the acylated anthocyanin.
Description
Technical Field
The invention relates to an extraction method of anthocyanin, in particular to a method for extracting anthocyanin with high stability and high bioavailability from black rice.
Background
Black rice is a medical food in traditional Chinese medicine, can be used as edible rice, is a precious variety in rice, contains abundant proteins, amino acids, unsaturated fatty acids, vitamin C, mineral nutrient elements such as calcium, iron, zinc and the like, and also contains anthocyanin with medicinal value, wherein the anthocyanin contains 8.5-12.5% of crude protein, 2.7-3.8% of crude fat, 75-84% of carbohydrate and 1.7-2% of crude ash. When the black rice is used for making porridge, the cooked black rice porridge is fragrant and sticky, has a smooth and greasy taste, is suitable for people with weak constitution, is also an excellent tonic for healthy people, and is especially suitable for pregnant women and patients with excessive blood loss.
The black rice grains are flat, and are eaten in most places in China and are used as cash crops, wherein long-term black rice planting history exists in Hunan, guizhou and Shaanxi provinces. The ancient books record that the black rice can protect the liver, the kidney, the stomach and the lung of a human body, has special efficacy on eyesight, can be eaten as food, can prevent dim eyesight, lumbar pain, ear hearing loss and the like after being eaten for a long time, and can improve physical quality and immunity after being eaten frequently.
Anthocyanin belongs to flavonoid substances in plant polyphenol, is a water-soluble natural pigment widely existing in nature, has the functions of resisting oxidation, preventing cardiovascular and cerebrovascular diseases and cancers, protecting liver, protecting eyesight and the like, has the functions of absorbing harmful radioactive substances and protecting organs from radiation injury, and has important application in the aspects of foods, medicines, cosmetics and the like. Since anthocyanin has a cationic structure and is unstable in a free state, it is difficult to exist alone in nature, so 97% of anthocyanin exists in plants, and further fatty acid and aromatic acid groups are combined on anthocyanin in an acylated form to form various acylated anthocyanin.
In recent years, more and more researchers find that anthocyanin has the functions of protecting and improving eyesight, treating cardiovascular diseases, preventing obesity, preventing cerebral nerve aging, preventing cancers and the like through continuous exploration. But because it has these three disadvantages: (1) The water-soluble property of anthocyanin polyhydroxy greatly restricts the application in oil food; (2) The anthocyanin has poor stability in the environment outside the original plant cells and is easy to degrade and lose physiological activity, so that the effective concentration is reduced in application, and the expected effect is difficult to play; (3) The inherent characteristic of anthocyanin leads to the fact that the anthocyanin is not easy to permeate phospholipid bilayer biological membrane, and the bioavailability of the anthocyanin is also directly low, so that the anthocyanin becomes a bottleneck of application of the anthocyanin in the food and drug industries.
Anthocyanin is unstable as a natural pigment and is easily influenced by a plurality of external factors. In order to improve the stability of anthocyanin and expand the application field of the pigment, the stability of anthocyanin is improved by adopting a method of adding auxiliary pigment, antioxidant, chelating agent microcapsule and chemical modification.
In order to improve the lipophilicity, anthocyanin and fatty alcohol, vinyl ester and other weak polar substances are subjected to esterification or acylation reaction, so that the purposes of introducing lipophilic groups and improving the lipophilicity are achieved. Lipophilic molecule modification generally includes two methods: chemical and enzymatic processes. However, the chemical method has complex reaction process, side reaction can occur in the process, the enzyme method is greener and mild, and in the enzyme catalytic reaction process, the operation is easy, and the generated product is single because of the high efficiency and specificity of the enzyme.
The Chinese patent with publication number of CN103181544A adopts novel membrane separation technology and microencapsulation or liposome encapsulation technology to obtain anthocyanin with stable property, wherein the anthocyanin can comprise cyanidin and paeoniflorin of more than 60%.
The Chinese patent with publication number of CN106632204A combines aqueous solution extraction with microbial degradation, and realizes stable structure of anthocyanin acylation through microbial degradation to obtain anthocyanin with good stability.
The Chinese patent with publication number CN110699397B adopts macroporous resin immobilized enzyme technology and column chromatography technology to obtain acylated anthocyanin.
The method for stabilizing anthocyanin mainly comprises the following steps: microencapsulation technology, microbial metabolism technology and column adsorption method modification, but have the problems of (1) high cost and complex process for preparing microcapsule materials; (2) The microorganism growth condition is relatively strict, and the requirements on production equipment and environment are high; (3) The immobilized enzyme-resin enzymatic acylation reaction time is longer, and the production period is prolonged.
Disclosure of Invention
The invention aims to solve the technical problems of high cost, complex process, long period and the like of the conventional anthocyanin acylation extraction, and provides a method for extracting anthocyanin from black rice.
The technical scheme provided by the invention is as follows:
the method for extracting anthocyanin from black rice is characterized by comprising the following steps of:
s1, taking black rice raw materials, wherein the black rice raw materials are prepared according to the mass of the black rice raw materials: extraction solvent volume = 1g: (3-8) adding extraction solvent into the mixture, stirring and extracting the mixture at the temperature of between 30 and 60 ℃ for 2 to 4 times, each time for 1 to 3 hours, filtering the mixture, and combining the filtrates to obtain black rice extract;
the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is ethanol or methanol with the mass fraction of 60% -80%, and the mass of the compound acid is as follows: the volume of the extracting solvent is 3-6 permillage g/ml;
s2, ultrafiltering the black rice extract by adopting a hollow fiber membrane, and collecting ultrafiltrate;
s3, nanofiltration is carried out on the ultrafiltrate by adopting a nanofiltration membrane, and a nanofiltration membrane retentate is collected to obtain anthocyanin extract;
s4, extracting the mass of the paste according to anthocyanin: 40-60% ethanol solution volume = 1mg: adding 1ml of ethanol solution, respectively adding pyridine, methyl benzoate and lipase to form an acylation reaction mixed solution, and carrying out constant-temperature stirring reaction at 50 ℃ for at least 4 hours for acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
s5, adding 3-8%o hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.02-1.07, adding the acylated anthocyanin concentrated solution with the adjusted specific gravity into a macroporous adsorption resin chromatographic column, eluting with 3-8%o hydrochloric acid solution until eluent is colorless, and removing impurities; eluting with eluent, collecting eluate, concentrating, and drying to obtain acylated anthocyanin.
Further, in step S1, the complex acid is sulfuric acid and citric acid, wherein the citric acid: the mass ratio of the sulfuric acid is 1: (1-3).
Sulfuric acid is adopted in the compound acid, but hydrochloric acid and phosphoric acid are very commonly used because the hydrochloric acid is easy to volatilize, the extraction effect is affected, and the ash powder of the final product extracted by the phosphoric acid is large; citric acid has low toxicity and low cost, and can also be selected from vitamin C (L-ascorbic acid).
Further, in step S1, the compound acid mass: the volume of the extraction solvent is 4 per mill g/ml;
in the complex acid, citric acid: the mass ratio of the sulfuric acid is 1:1.5;
the organic solvent is ethanol with the mass fraction of 70%;
the black rice raw material comprises the following components in mass: extraction solvent volume = 1g:5ml.
Further, in the step S2, the pore diameter of the hollow fiber membrane is 0.1-1 μm, and the ultrafiltration pressure is 0.2-0.4 Mpa; hollow fiber membranes are used to remove coarse impurities.
Further, in the step S3, the molecular weight cut-off of the nanofiltration membrane is 600-900 Da, and the nanofiltration pressure is 0.3-0.5 Mpa.
Further, in step S4, the addition amount of pyridine is specifically that the volume of pyridine is: anthocyanin extract quality= (5-15) ml:100mg;
the concentration of the methyl benzoate in the acylation reaction mixed solution is 0.3-0.7 mol/L;
the lipase is Aspergillus oryzae fermented lipase, and the addition amount is 260-350U per gram of anthocyanin extract.
Further, in the step S5, the macroporous adsorption resin is XDA-6, LX-180S, LS-180 and D101;
volume of macroporous adsorption resin added into chromatographic column: black rice raw material mass=1 ml: (2-5) g.
Further, in step S5, the eluent includes 60-90% ethanol and hydrochloric acid;
the addition amount of the hydrochloric acid is 0.4 to 0.7 per mill of the volume of the eluent.
Hydrochloric acid has volatility, and in the subsequent operation, the content of the product is not influenced after the hydrochloric acid volatilizes, and the corresponding impurity removal process is not needed, so that the flow is simplified.
Further, in step S5, the eluting with the eluent specifically includes:
the ratio of the eluent to the macroporous adsorption resin is (700-1000) ml:100g;
the flow rate of the eluent is 1.0mL/min-3.0mL/min.
Further, in step S4, the addition amount of pyridine is the volume of pyridine: anthocyanin extract mass = 10ml:100mg;
the concentration of the methyl benzoate in the acylation reaction mixed solution is 0.5mol/L;
the lipase is Aspergillus oryzae fermented lipase, and the addition amount is 300U per gram of anthocyanin extract;
in the step S5, macroporous resin is LX-180S;
in the eluent, the mass concentration of ethanol is 70%, and the addition amount of hydrochloric acid is 0.4 per mill of the volume of the eluent.
The invention has the beneficial effects that:
1. the invention combines the complex acid, membrane filtration and lipase acylation technology, firstly, the complex acid is utilized to not only enhance the solubility of the black rice anthocyanin, but also prevent the degradation of the active ingredients in the extraction process; secondly, enriching anthocyanin by using a membrane separation technology to prevent the anthocyanin structure from being damaged by high temperature; finally, acylation is carried out on the anthocyanin structure by utilizing enzyme catalysis to introduce a lipophilic group, so that on one hand, the stability of the anthocyanin is enhanced, and on the other hand, the bioavailability of the water-soluble anthocyanin is improved.
2. The method has the advantages of mild whole process condition, simple operation and strong specificity, and is relatively suitable for large-scale production.
Detailed Description
The technical scheme of the invention is further described below by combining specific embodiments.
Example 1
1. Taking 100kg of black rice raw material (anthocyanin content is 0.34%), adding 5 times (500L) of extraction solvent, stirring and extracting at 50deg.C for 3 times each for 2 hr, filtering, and mixing filtrates to obtain black rice extract; the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is ethanol with the mass fraction of 70%, and the mass of the compound acid is as follows: the volume of the extraction solvent is 4 per mill g/ml; the compound acid is sulfuric acid and citric acid, citric acid: the mass ratio of the sulfuric acid is 1:1.5.
2. ultrafiltering fructus Zizaniae Caduciflorae extractive solution with hollow fiber membrane with pore size of 0.45 μm under ultrafiltration pressure of 0.3Mpa, and collecting ultrafiltrate.
3. Nanofiltration is carried out on the ultrafiltrate under the pressure of 0.4Mpa nanofiltration by adopting a nanofiltration membrane with the molecular weight cutoff of 800Da, and the interception matters of the nanofiltration membrane are collected to obtain 1.3kg of anthocyanin extract;
4. 1300L of 50% ethanol solution is added into 1.3kg of anthocyanin extract, 130L of pyridine, methyl benzoate and 390000U of lipase Lipozyme RM are respectively added to form an acylation reaction mixed solution, and the concentration of the methyl benzoate is 0.5mol/L; stirring and reacting at a constant temperature of 50 ℃ for at least 4 hours to carry out acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
5. adding 5%o hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.03, adding the acylated anthocyanin concentrated solution with the specific gravity adjusted into a chromatographic column containing 25L LX-180S macroporous adsorption resin, eluting with 4BV 5%o hydrochloric acid solution until the eluent is colorless, and removing impurities; eluting with 8BV 70% ethanol (containing 0.5%hydrochloric acid), collecting eluate, concentrating, drying to obtain acylated anthocyanin 1.1kg, precisely weighing acyl groupDissolving 18.78mg of anthocyanin sample, placing the anthocyanin sample into a 50ml first volumetric flask, adding 40ml of 2% hydrochloric acid-methanol solution, carrying out ultrasonic treatment for 30 minutes, diluting the anthocyanin sample to the 50ml scale of the first volumetric flask by using 2% hydrochloric acid-methanol, transferring the anthocyanin sample into a 2ml to 50ml second volumetric flask, diluting the anthocyanin sample to the 50ml scale of the second volumetric flask by using 2% hydrochloric acid-methanol solution, obtaining a sample solution, measuring the absorbance value of the sample solution at 535nm to 0.4652, and adopting the following content calculation formula:
wherein a is the measured absorbance, w is the sample amount of the acylated anthocyanin sample, Q is the content of the acylated anthocyanin in the acylated anthocyanin sample, and q=30.36% is calculated. Compared with the original content of 0.34 percent of the raw materials, the method improves 88.3 times; the obtained acylated anthocyanin can be used by purifying to a desired content range.
Example 2
1. Taking 10kg of black rice raw material, adding 3 times (30L) of extraction solvent, stirring and extracting at 60 ℃ for 3 times, 1 hour each time, filtering and combining the filtrates to obtain black rice extract; the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is methanol with the mass fraction of 80%, and the mass of the compound acid is as follows: the volume of the extraction solvent is 3 per mill g/ml; the compound acid is sulfuric acid and citric acid, citric acid: the mass ratio of the sulfuric acid is 1:1.
2. ultrafiltering fructus Zizaniae Caduciflorae extractive solution with hollow fiber membrane with pore size of 0.85 μm under ultrafiltration pressure of 0.2Mpa, and collecting ultrafiltrate.
3. Nanofiltration is carried out on the ultrafiltrate under the pressure of 0.35Mpa nanofiltration by adopting a nanofiltration membrane with the molecular weight cutoff of 850Da, and the interception matters of the nanofiltration membrane are collected to obtain 138g of anthocyanin extract;
4. to 138g of the anthocyanin extract was added 172.5L of 55% ethanol solution (anthocyanin extract: ethanol solution=0.8 mg:1 ml), and the volume of pyridine was calculated as follows: anthocyanin extract mass = 8ml: adding 100mg of pyridine, adding 330U of lipase RM per gram of anthocyanin extract, and adding methyl benzoate according to the final concentration of 0.4mol/L of methyl benzoate to form an acylation reaction mixed solution; stirring and reacting at a constant temperature of 50 ℃ for at least 4 hours to carry out acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
5. adding 8%o hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.02, adding the acylated anthocyanin concentrated solution with the adjusted specific gravity into a chromatographic column containing 3.3L XDA-6 macroporous adsorption resin, eluting with 4BV hydrochloric acid solution of 6%o until the eluent is colorless, and removing impurities; eluting with 8BV of 60% ethanol (containing 0.6% hydrochloric acid), collecting eluate, concentrating, drying to obtain acylated anthocyanin 115g, and purifying to obtain acylated anthocyanin with desired content range by measuring method in example 1 to obtain acylated anthocyanin content of 31.28%.
Example 3
1. Taking 10kg of black rice raw material, adding 8 times (80L) of extraction solvent, stirring and extracting at 30 ℃ for 2 times each for 3 hours, filtering and combining the filtrates to obtain black rice extract; the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is ethanol with the mass fraction of 60%, and the mass of the compound acid is as follows: the volume of the extraction solvent is 5 per mill g/ml; the compound acid is sulfuric acid and citric acid, citric acid: the mass ratio of the sulfuric acid is 1:2.
2. ultrafiltering fructus Zizaniae Caduciflorae extractive solution with hollow fiber membrane with aperture of 1 μm under ultrafiltration pressure of 0.4Mpa, and collecting ultrafiltrate.
3. Nanofiltration is carried out on the ultrafiltrate under the pressure of 0.5Mpa nanofiltration by adopting a nanofiltration membrane with the molecular weight cutoff of 750Da, and the interception matters of the nanofiltration membrane are collected to obtain 135g of anthocyanin extract;
4. to 135g of the anthocyanin extract was added 112.5L of 40% ethanol solution (anthocyanin extract: ethanol solution=1.2 mg:1 ml), and the volume of pyridine was respectively: anthocyanin extract mass = 12ml: adding 100mg of pyridine, adding 280U of lipase RM per gram of anthocyanin extract, and adding methyl benzoate according to the final concentration of 0.7mol/L of methyl benzoate to form an acylation reaction mixed solution; stirring and reacting at a constant temperature of 50 ℃ for at least 4 hours to carry out acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
5. adding 6%o hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.07, adding the acylated anthocyanin concentrated solution with the adjusted specific gravity into a chromatographic column containing 2.5L XDA-6 macroporous adsorption resin, eluting with 4BV hydrochloric acid solution to 3%o until the eluent is colorless, and removing impurities; eluting with 8BV of 90% ethanol (containing 0.4% hydrochloric acid), collecting eluate, concentrating, drying to obtain 123g of acylated anthocyanin, and purifying to obtain the final product with the content of acylated anthocyanin of 31.05% by the measurement method in example 1.
Example 4
1. Taking 50kg of black rice raw material, adding 6 times (300L) of extraction solvent, stirring and extracting for 4 times at 40 ℃ for 1 hour each time, filtering and combining the filtrates to obtain black rice extract; the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is ethanol with the mass fraction of 75%, and the mass of the compound acid is as follows: the volume of the extraction solvent is 5 per mill g/ml; the compound acid is sulfuric acid and citric acid, citric acid: the mass ratio of the sulfuric acid is 1:1.5.
2. ultrafiltering fructus Zizaniae Caduciflorae extractive solution with hollow fiber membrane with pore size of 0.25 μm under ultrafiltration pressure of 0.25Mpa, and collecting ultrafiltrate.
3. Nanofiltration is carried out on the ultrafiltrate under the pressure of 0.45Mpa nanofiltration by adopting a nanofiltration membrane with the molecular weight cutoff of 600Da, and the interception matters of the nanofiltration membrane are collected to obtain 642g of anthocyanin extract;
4. to 642g of the anthocyanin extract was added 713.3L of 60% ethanol solution (anthocyanin extract: ethanol solution=0.9 mg:1 ml), and the volume of pyridine was calculated as follows: anthocyanin extract mass = 5ml: adding 100mg of pyridine, adding 350U of lipase RM per gram of anthocyanin extract, and adding methyl benzoate according to the final concentration of 0.3mol/L of methyl benzoate to form an acylation reaction mixed solution; stirring and reacting at a constant temperature of 50 ℃ for at least 4 hours to carry out acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
5. adding 3%hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.05, adding the acylated anthocyanin concentrated solution with the specific gravity adjusted into a chromatographic column containing 10L LS-180 macroporous adsorption resin, eluting with 4BV hydrochloric acid solution to 8%of the eluent until the eluent is colorless, and removing impurities; eluting with 8BV of 75% ethanol (containing 0.45% hydrochloric acid), collecting eluate, concentrating, drying to obtain 538g of acylated anthocyanin, and purifying to obtain the desired content range by the measurement method in example 1 to obtain acylated anthocyanin content of 30.66%.
Example 5
1. Taking 50kg of black rice raw material, adding 4 times (200L) of extraction solvent, stirring and extracting at 55 ℃ for 2 times each for 3 hours, filtering and combining the filtrates to obtain black rice extract; the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is 65% of methanol by mass percent, and the compound acid is prepared from the following components by mass percent: the volume of the extraction solvent is 6 per mill g/ml; the compound acid is sulfuric acid and citric acid, citric acid: the mass ratio of the sulfuric acid is 1:3.
2. ultrafiltering fructus Zizaniae Caduciflorae extractive solution with hollow fiber membrane with pore size of 0.1 μm under ultrafiltration pressure of 0.35Mpa, and collecting ultrafiltrate.
3. Nanofiltration is carried out on the ultrafiltrate by adopting a nanofiltration membrane with the interception molecular weight of 900Da under the pressure of 0.3Mpa nanofiltration, and the interception matters of the nanofiltration membrane are collected to obtain 662g of anthocyanin extract;
4. to 662g of the anthocyanin extract, 601.8L of 45% ethanol solution (anthocyanin extract: ethanol solution=1.1 mg:1 ml) was added, and the volumes of pyridine were respectively: anthocyanin extract mass = 15ml: adding 100mg of pyridine, adding 260U of lipase RM per gram of anthocyanin extract, and adding methyl benzoate according to the final concentration of 0.6mol/L of methyl benzoate to form an acylation reaction mixed solution; stirring and reacting at a constant temperature of 50 ℃ for at least 4 hours to carry out acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
5. adding 4%hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.04, adding the acylated anthocyanin concentrated solution with the adjusted specific gravity into a chromatographic column containing 25L D101 macroporous adsorption resin, eluting with 4BV hydrochloric acid solution to 7%hydrochloric acid until the eluent is colorless, and removing impurities; eluting with 8BV of 85% ethanol (containing 0.7% hydrochloric acid), collecting eluate, concentrating, drying to obtain 565g of acylated anthocyanin, and purifying to obtain acylated anthocyanin with desired content range by measuring method in example 1 to obtain acylated anthocyanin content of 30.36%.
Claims (10)
1. A method for extracting anthocyanin from black rice, which is characterized by comprising the following steps:
s1, taking black rice raw materials, wherein the black rice raw materials are prepared according to the mass of the black rice raw materials: extraction solvent volume = 1g: (3-8) adding extraction solvent into ml, stirring at 30-60deg.C for extracting for multiple times, filtering, and mixing filtrates to obtain fructus Zizaniae Caduciflorae extractive solution;
the extraction solvent comprises an organic solvent and a compound acid, wherein the organic solvent is ethanol or methanol with the mass fraction of 60% -80%, and the mass of the compound acid is as follows: the volume of the extracting solvent is 3-6 permillage g/ml;
s2, ultrafiltering the black rice extract by adopting a hollow fiber membrane, and collecting ultrafiltrate;
s3, nanofiltration is carried out on the ultrafiltrate by adopting a nanofiltration membrane, and a nanofiltration membrane retentate is collected to obtain anthocyanin extract;
s4, extracting the mass of the paste according to anthocyanin: 40-60% ethanol solution volume= (0.8-1.2) mg: adding 1ml of ethanol solution, respectively adding pyridine, methyl benzoate and lipase to form an acylation reaction mixed solution, and carrying out constant-temperature stirring reaction at 40-60 ℃ for at least 4 hours for acylation; after the reaction is finished, decompressing and concentrating to remove pyridine and ethanol, and obtaining an acylated anthocyanin concentrated solution;
s5, adding 3-8%o hydrochloric acid solution into the acylated anthocyanin concentrated solution to adjust the specific gravity to 1.02-1.07, adding the acylated anthocyanin concentrated solution with the adjusted specific gravity into a macroporous adsorption resin chromatographic column, eluting with 3-8%o hydrochloric acid solution until eluent is colorless, and removing impurities; eluting with eluent, collecting eluate, concentrating, and drying to obtain acylated anthocyanin.
2. The method for extracting anthocyanin from black rice of claim 1, wherein:
in the step S1, the compound acid is sulfuric acid and citric acid, wherein the citric acid: the mass ratio of the sulfuric acid is 1: (1-3).
3. The method for extracting anthocyanin from black rice of claim 2, wherein:
in step S1, the mass of the complex acid: the volume of the extraction solution is 4 per mill g/ml;
in the complex acid, citric acid: the mass ratio of the sulfuric acid is 1:1.5;
the organic solvent is ethanol with the mass fraction of 70%;
the black rice raw material comprises the following components in mass: extraction solvent volume = 1g:5ml.
4. A method for extracting anthocyanin from black rice as claimed in claim 3, wherein:
in the step S2, the pore diameter of the hollow fiber membrane is 0.1-1 mu m, and the ultrafiltration pressure is 0.2-0.4 Mpa.
5. The method for extracting anthocyanin from black rice of claim 4, wherein the step of extracting anthocyanin comprises the steps of:
in the step S3, the molecular weight cut-off of the nanofiltration membrane is 600-900 Da, and the nanofiltration pressure is 0.3-0.5 Mpa.
6. The method for extracting anthocyanin from black rice as claimed in any one of claims 1 to 5, wherein:
in step S4, the addition amount of pyridine is specifically that the volume of pyridine is: anthocyanin extract quality= (5-15) ml:100mg;
the concentration of the methyl benzoate in the acylation reaction mixed solution is 0.3-0.7 mol/L;
the lipase is Aspergillus oryzae fermented lipase, and the addition amount is 260-350U per gram of anthocyanin extract.
7. The method for extracting anthocyanin from black rice of claim 6, wherein the method comprises the steps of:
in the step S5, the macroporous adsorption resin is XDA-6, LX-180S, LS-180 and D101;
volume of macroporous adsorption resin added into chromatographic column: black rice raw material mass=1 ml: (2-5) g.
8. The method for extracting anthocyanin from black rice of claim 7, wherein:
in the step S5, the eluent comprises ethanol and hydrochloric acid with the mass concentration of 60-90%;
the added mass of the hydrochloric acid is 0.4 to 0.7 per mill of the volume of the eluent.
9. The method for extracting anthocyanin from black rice of claim 8, wherein:
in the step S5, macroporous resin is LX-180S;
in the eluent, the mass concentration of ethanol is 70%, and the addition amount of hydrochloric acid is 0.4 per mill of the volume of the eluent.
10. The method for extracting anthocyanin from black rice of claim 9, wherein:
in step S4, the addition amount of pyridine is the volume of pyridine: anthocyanin extract mass = 10ml:100mg;
the concentration of the methyl benzoate in the acylation reaction mixed solution is 0.5mol/L;
the lipase is Aspergillus oryzae fermented lipase, and the addition amount is 300U per gram of anthocyanin extract.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659871A (en) * | 2012-05-08 | 2012-09-12 | 魏有良 | Method for extracting and refining anthocyanin in black rice |
| CN104630315A (en) * | 2013-11-06 | 2015-05-20 | 江苏省农业科学院 | Reduced pressure enzyme process acylation method of black rice pigment |
| CN111206063A (en) * | 2020-01-21 | 2020-05-29 | 合肥工业大学 | Preparation method of high-stability acylated black rice anthocyanin |
| CN112707881A (en) * | 2021-01-22 | 2021-04-27 | 陕西科技大学 | Preparation method of blueberry anthocyanin |
| CN113980485A (en) * | 2021-11-16 | 2022-01-28 | 江苏时代铭阳生物新技术研究院有限公司 | Biological enzyme compound and method for applying biological enzyme compound to plant extraction process |
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- 2023-03-07 CN CN202310209957.6A patent/CN116253708A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659871A (en) * | 2012-05-08 | 2012-09-12 | 魏有良 | Method for extracting and refining anthocyanin in black rice |
| CN104630315A (en) * | 2013-11-06 | 2015-05-20 | 江苏省农业科学院 | Reduced pressure enzyme process acylation method of black rice pigment |
| CN111206063A (en) * | 2020-01-21 | 2020-05-29 | 合肥工业大学 | Preparation method of high-stability acylated black rice anthocyanin |
| CN112707881A (en) * | 2021-01-22 | 2021-04-27 | 陕西科技大学 | Preparation method of blueberry anthocyanin |
| CN113980485A (en) * | 2021-11-16 | 2022-01-28 | 江苏时代铭阳生物新技术研究院有限公司 | Biological enzyme compound and method for applying biological enzyme compound to plant extraction process |
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