CN104725341A - Efficient extraction method for blueberry anthocyanin - Google Patents
Efficient extraction method for blueberry anthocyanin Download PDFInfo
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- CN104725341A CN104725341A CN201510072863.4A CN201510072863A CN104725341A CN 104725341 A CN104725341 A CN 104725341A CN 201510072863 A CN201510072863 A CN 201510072863A CN 104725341 A CN104725341 A CN 104725341A
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
The invention discloses an efficient extraction method for blueberry anthocyanin. According to the method, blueberry fruits or blueberry leaves are taken as raw materials for extracting the blueberry anthocyanin and the method comprises extraction and purification of a blueberry anthocyanin crude extract and specifically comprises the following steps of carrying out enzymolysis and alcohol extraction in turn to obtain the blueberry anthocyanin crude extract, then purifying the blueberry anthocyanin crude extract, degreasing by extraction and removing impurities with macroporous resin to obtain the refined blueberry anthocyanin. According to the method disclosed by the invention, solvent extraction and enzyme extraction are simultaneously combined, enzymolysis is firstly used for improving the juice yield of the blueberry anthocyanin and a solvent is further used for extracting the blueberry anthocyanin. By adopting the extraction method, the extraction time can be greatly shortened, the active ingredients can be extracted to the greatest extent, and the extraction rate can be significantly improved; furthermore, the operation is simple and easy to operate, the parameters of the production process are stable, the product quality is stable, and the extraction method is suitable for industrial production; and in addition, the highest content of an extracted and purified blueberry anthocyanin fine product can achieve 49.6%, and the application value is very high.
Description
Technical field
The present invention relates to material extractive technique field, be specifically related to a kind of method of high efficiency extraction vaccinium oxycoccus pigment, to realize high efficiency extraction and the purification process of vaccinium oxycoccus pigment.
Background technology
Vaccinium oxycoccus pigment is a kind of main active ingredient of blueberry, there is very strong oxidation-resistance, and have and promote erythrolabe resynthesis, anti-inflammatory, raising immunizing power, anti-cardiovascular disease, the multiple physiological active functions such as anti-ageing, anticancer, be subject to showing great attention to of domestic and international expert, become one of focus of current blueberry research.
The extraction of vaccinium oxycoccus pigment is the prerequisite of blueberry researchdevelopment, is also the key link that anthocyanidin is produced and come into operation.In the extraction research of vaccinium oxycoccus pigment, prior art mainly contains with extraction, also useful Enzymatic Extraction, but the shortcoming such as these extracting method all exist, and extraction time is long, extraction yield is low, effective constituent loss large, extraction cost is high.
Chinese patent literature application number is the extracting method disclosing a kind of vaccinium oxycoccus pigment in the data of CN201310312112.6, and the method adopts combination organic solvent method to carry out lixiviate, to improve the extraction yield of pigment to fruit juice.Constant for ensureing original character of pigment, extracting in whole process the stability adopting low temperature control acid system maintenance pigment, the problem that concentration is low, the loss of leaching process anthocyanidin is larger of vaccinium oxycoccus pigment extract can be solved.
Chinese patent literature application number is the extracting method disclosing a kind of vaccinium oxycoccus pigment in the data of CN201010246735.4, the method uses the ethanol of organic acid acidifying as extraction agent, under the prerequisite ensureing high extraction, avoid strong acid to the corrosion of equipment, be simultaneously that raw material extracts vaccinium oxycoccus pigment with blue berry, solve the problem that blue berry Seasonal Harvest is not easily stored by force.
Above extracting method all respectively has advantage, but in order to high efficiency extraction vaccinium oxycoccus pigment more, is still necessary to be further improved prior art.
Summary of the invention
Object of the present invention be exactly in order to overcome prior art exist defect and a kind of vaccinium oxycoccus pigment Isolation and purification method that technique is simple, cost is low, efficiency is high is provided.
Vaccinium oxycoccus pigment Isolation and purification method of the present invention is for raw material with blue berry or Blue berry leaf, adopt first enzymolysis, then the method for alcohol extracting, farthest vaccinium oxycoccus pigment is extracted, through extraction grease removal, macroporous resin removal of impurities, obtain the vaccinium oxycoccus pigment refined.The present invention's blue berry used can be the new blueberry fresh fruit plucked, or low temperature storage the blue berry thawed; Blue berry leaf used is Blue berry leaf dried powder.
Technical scheme of the present invention is as follows:
The present invention is a kind of like this method of high efficiency extraction vaccinium oxycoccus pigment, and it is with blue berry or Blue berry leaf for raw material extracts vaccinium oxycoccus pigment, and it comprises the step extracting vaccinium oxycoccus pigment crude extract and purifying; It adopts first enzymolysis, then the method for alcohol extracting obtains vaccinium oxycoccus pigment crude extract, then carries out purifying to vaccinium oxycoccus pigment crude extract, by the vaccinium oxycoccus pigment obtaining refining after extraction grease removal, macroporous resin removal of impurities.
Preferably, blue berry of the present invention is the blueberry fresh fruit or low temperature storage the blue berry thawed newly plucked; The Blue berry leaf adopted is Blue berry leaf dried powder.
Further, in the method for this high efficiency extraction vaccinium oxycoccus pigment of the present invention, aforesaid extraction vaccinium oxycoccus pigment crude extract comprises the steps:
1) raw materials pretreatment: get the new blueberry fresh fruit plucked, or low temperature storage the blue berry thawed, cleaning removal of impurities is for subsequent use; Or get the Blue berry leaf after cleaning-drying, pulverize for subsequent use with pulverizer;
2) enzymolysis: add the biological enzyme of 0.005% ~ 0.5% in the blue berry or Blue berry leaf of step 1) process, at room temperature lucifuge leaves standstill 1 ~ 5 hour;
3) alcohol extracting: to step 2) in add acidity alcohol solution, at room temperature lucifuge stir 3 ~ 8 hours;
4) concentrated: by the vat liquor filter-cloth filtering of step 3), namely decompression filtrate recycling ethanol obtains vaccinium oxycoccus pigment crude extract.
Wherein, step 2) described in biological enzyme be the combination of one or two or more kinds in cellulase, polygalacturonase, fruit juice enzyme, compound pulp enzyme, dextranase etc.
Wherein, in the acidity alcohol solution described in step 3), acid comprises the one of hydrochloric acid, sulfuric acid, trifluoroacetic acid, citric acid, tartrate, formic acid, acetic acid or propionic acid, and alcohol comprises the one in methyl alcohol, ethanol, propyl alcohol or butanols etc.; PH=2 ~ 4 of acidity alcohol solution, alcohol content is 40% ~ 95%, and solid-liquid ratio is 1:2 ~ 20.
Further, in the method for this high efficiency extraction vaccinium oxycoccus pigment of the present invention, aforesaid purifying comprises the steps:
5) degreasing: be dissolved in appropriate acidic aqueous solution by vaccinium oxycoccus pigment crude extract, adds appropriate ester and extracts 3 times, removing lipid layer;
6) macroporous resin removal of impurities: the non-lipid layer in step 5) is joined in macroporous resin column, presses gradient elution with ethanol water, collect suitable elutriant, decompression recycling ethanol;
7) dry: product step 6) obtained carries out lyophilize, obtains the vaccinium oxycoccus pigment refined.
Wherein, the ester described in step 5) comprises the one in methyl acetate, ethyl acetate, propyl acetate or butylacetate etc.; PH=1 ~ 4 of described acidic aqueous solution, acid wherein comprises the one in hydrochloric acid, sulfuric acid, trifluoroacetic acid, citric acid, tartrate, formic acid, acetic acid or propionic acid etc.
Wherein, the model of the macroporous resin described in step 5) comprises the one in D101, D3520, D4020, DA201, HPD-100, XAD-7, X-5, AB-8, H103, NKA-9, S-8 etc.
Wherein, in the ethanol water described in step 6), the gradient concentration scope of ethanol is 0% ~ 80%.
Wherein, the suitable elutriant described in step 6) refers to that ethanol content is the elutriant of 25% ~ 75%.
Solvent is carried carrying with enzyme simultaneously and is combined by the present invention, first improves the crushing juice rate of vaccinium oxycoccus pigment with enzymolysis, then is extracted by vaccinium oxycoccus pigment with solvent.This extracting method not only can shorten extraction time greatly, farthest by extracts active ingredients out, significantly improves extraction yield, and operation is simple, processing parameter stable, constant product quality, is applicable to suitability for industrialized production; In addition, the most high-content of vaccinium oxycoccus pigment fine work of extraction purification of the present invention can reach 49.6%, has high using value.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of high efficiency extraction vaccinium oxycoccus pigment of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Below provide the specific embodiment of the present invention; the present invention may be better understood; but it should be noted that; enforcement of the present invention is not limited to following embodiment; for the those of ordinary skill of technical field; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Embodiment 1:
By the blueberry fresh fruit newly plucked, first at room temperature spray the cellulase of 0.02%, lucifuge is after 3.5 hours, citric acid-60% ethanol of pH=3.0 is added by solid-liquid ratio 1:10, continue stirring 5 hours, suction filtration, filtrate under reduced pressure rotates recovery ethanol, obtaining vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the citric acid solution of appropriate pH=3.0, be extracted with ethyl acetate 3 times, removing lipid layer, non-lipid layer joins in DA201 macroporous resin column, first uses the ultrapure water wash-out of 4 times of volumes, then uses the ethanol elution of 10%, 30% successively, collect the ethanol eluate of 30%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 48.5%.
Embodiment 2:
-40 DEG C are preserved and the blue berry spent the night that thaws at 0 DEG C, first at room temperature spray 0.04% fruit juice enzyme, lucifuge is after 4 hours, add in citric acid-50% ethanol of pH=3.5 by solid-liquid ratio 1:15, continue stirring 4 hours, suction filtration, filtrate under reduced pressure rotates recovery ethanol, obtaining vaccinium oxycoccus pigment crude extract, is red-purple medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the hydrochloric acid soln of appropriate pH=1.0, be extracted with ethyl acetate 3 times, removing lipid layer, non-lipid layer joins in AB-8 macroporous resin column, first uses the ultrapure water wash-out of 3 times of volumes, then uses the ethanol elution of 10%, 25%, 45% successively, collect the ethanol eluate of 45%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 38.5%.
Embodiment 3:
By the blueberry fresh fruit newly plucked, first at room temperature spray the cellulase-polygalacturonase of 0.02%, lucifuge is after 3.5 hours, citric acid-70% ethanol of pH=3.0 is added by solid-liquid ratio 1:8, continue stirring 6 hours, suction filtration, filtrate under reduced pressure rotates recovery ethanol, obtaining vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the citric acid solution of appropriate pH=3.5, be extracted with ethyl acetate 3 times, removing lipid layer, non-lipid layer joins in D101 macroporous resin column, first uses the ultrapure water wash-out of 4 times of volumes, then uses the ethanol elution of 10%, 25%, 50% successively, collect the ethanol eluate of 50%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 49.6%.
Embodiment 4:
-20 DEG C are preserved and the blue berry spent the night that thaws at 0 DEG C, first at room temperature spray the compound pulp enzyme of 0.008%, lucifuge is after 2 hours, citric acid-80% ethanol of pH=3.0 is added by solid-liquid ratio 1:12, continue stirring 5 hours, suction filtration, filtrate under reduced pressure rotates recovery ethanol, obtaining vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the acetic acid solution of appropriate pH=3.5, with n-butyl acetate extraction 3 times, removing lipid layer, non-lipid layer joins in HPD-100 macroporous resin column, first uses the ultrapure water wash-out of 4 times of volumes, then uses the ethanol elution of 10%, 30%, 60% successively, collect the ethanol eluate of 60%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 49.2 %.
Embodiment 5:
Spray the polygalacturonase of 0.04% to the Blue berry leaf pulverized, at room temperature lucifuge is after 2 hours, adds tartrate-80% methyl alcohol of pH=3.5 by solid-liquid ratio 1:3, continue stirring 3 hours, suction filtration, filtrate under reduced pressure rotates recovery methyl alcohol, obtaining vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the sulphuric acid soln of appropriate pH=2.0,3 times are extracted with methyl acetate, removing lipid layer, non-lipid layer joins in H-103 macroporous resin column, first uses the ultrapure water wash-out of 4 times of volumes, then uses the ethanol elution of 10%, 30%, 60% successively, collect the ethanol eluate of 60%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 37.2 %.
Embodiment 6:
Spray the dextranase of 0.03% to the Blue berry leaf pulverized, at room temperature lucifuge is after 2 hours, adds propionic acid-45% butanols of pH=4.0 by solid-liquid ratio 1:5, continue stirring 6 hours, suction filtration, under reduced pressure rotate and reclaim propyl alcohol, obtaining vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the trifluoroacetic acid solution of appropriate pH=3.0,3 times are extracted with propyl acetate, removing lipid layer, non-lipid layer joins in S-8 macroporous resin column, first uses the ultrapure water wash-out of 4 times of volumes, then uses the ethanol elution of 10%, 30%, 60% successively, collect the ethanol eluate of 60%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 35.6 %.
Embodiment 7:
Spray the cellulase of 0.035% to the Blue berry leaf pulverized, at room temperature lucifuge is after 3 hours, adds hydrochloric acid-60% methyl alcohol of pH=2.0 by solid-liquid ratio 1:6, continue stirring 3 hours, suction filtration, filtrate under reduced pressure rotates recovery methyl alcohol, obtaining vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the formic acid solution of appropriate pH=4.0,3 times are extracted with methyl acetate, removing lipid layer, non-lipid layer joins in D101 macroporous resin column, first uses the ultrapure water wash-out of 3 times of volumes, then uses the ethanol elution of 10%, 25%, 50% successively, collect the ethanol eluate of 50%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 36.3 %.
Embodiment 8:
Cellulase-the polygalacturonase of 0.025% is sprayed to the Blue berry leaf pulverized, at room temperature lucifuge is after 3 hours, citric acid-75% butanols of pH=3.0 is added by solid-liquid ratio 1:5, continue stirring 4 hours, suction filtration, filtrate under reduced pressure rotates recovery butanols, obtains vaccinium oxycoccus pigment crude extract, is sorrel medicinal extract.
Above-mentioned vaccinium oxycoccus pigment crude extract is dissolved in the propionic acid solution of appropriate pH=3.5,3 times are extracted with propyl acetate, removing lipid layer, non-lipid layer joins in D4020 macroporous resin column, first uses the ultrapure water wash-out of 4 times of volumes, then uses the ethanol elution of 15%, 25%, 50% successively, collect the ethanol eluate of 50%, decompression rotates reclaims ethanol, and the product obtained carries out lyophilize, obtains the blueberry anthocyanin powder of puce.Adopt 2%HCl-Methanol edman degradation Edman, utilizing ultraviolet spectrophotometer to carry out the mensuration of anthocyanidin content, is 40.5 %.
Claims (10)
1. a method for high efficiency extraction vaccinium oxycoccus pigment, it extracts vaccinium oxycoccus pigment with blue berry or Blue berry leaf for raw material, it is characterized in that: it comprises the step extracting vaccinium oxycoccus pigment crude extract and purifying; It adopts first enzymolysis, then the method for alcohol extracting obtains vaccinium oxycoccus pigment crude extract, then carries out purifying to vaccinium oxycoccus pigment crude extract, by the vaccinium oxycoccus pigment obtaining refining after extraction grease removal, macroporous resin removal of impurities.
2. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 1, is characterized in that: described blue berry is the blueberry fresh fruit or low temperature storage the blue berry thawed newly plucked; Described Blue berry leaf is Blue berry leaf dried powder.
3. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 1 and 2, is characterized in that described extraction vaccinium oxycoccus pigment crude extract comprises the steps:
1) raw materials pretreatment: get the new blueberry fresh fruit plucked, or low temperature storage the blue berry thawed, cleaning removal of impurities is for subsequent use; Or get the Blue berry leaf after cleaning-drying, pulverize for subsequent use with pulverizer;
2) enzymolysis: add the biological enzyme of 0.005% ~ 0.5% in the blue berry or Blue berry leaf of step 1) process, at room temperature lucifuge leaves standstill 1 ~ 5 hour;
3) alcohol extracting: to step 2) in add acidity alcohol solution, at room temperature lucifuge stir 3 ~ 8 hours;
4) concentrated: by the vat liquor filter-cloth filtering of step 3), namely decompression filtrate recycling ethanol obtains vaccinium oxycoccus pigment crude extract.
4. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 1 and 2, is characterized in that described purifying comprises the steps:
5) degreasing: be dissolved in appropriate acidic aqueous solution by vaccinium oxycoccus pigment crude extract, adds appropriate ester and extracts 3 times, removing lipid layer;
6) macroporous resin removal of impurities: the non-lipid layer in step 5) is joined in macroporous resin column, presses gradient elution with ethanol water, collect suitable elutriant, decompression recycling ethanol;
7) dry: product step 6) obtained carries out lyophilize, obtains the vaccinium oxycoccus pigment refined.
5. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 3, is characterized in that: step 2) described in biological enzyme be the combination of one or two or more kinds in cellulase, polygalacturonase, fruit juice enzyme, compound pulp enzyme, dextranase.
6. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 3, it is characterized in that: in the acidity alcohol solution described in step 3), acid comprises the one of hydrochloric acid, sulfuric acid, trifluoroacetic acid, citric acid, tartrate, formic acid, acetic acid or propionic acid, and alcohol comprises the one in methyl alcohol, ethanol, propyl alcohol or butanols; PH=2 ~ 4 of acidity alcohol solution, alcohol content is 40% ~ 95%, and solid-liquid ratio is 1:2 ~ 20.
7. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 4, is characterized in that: the ester described in step 5) comprises the one in methyl acetate, ethyl acetate, propyl acetate or butylacetate; PH=1 ~ 4 of described acidic aqueous solution, acid wherein comprises the one in hydrochloric acid, sulfuric acid, trifluoroacetic acid, citric acid, tartrate, formic acid, acetic acid or propionic acid.
8. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 4, is characterized in that: the model of the macroporous resin described in step 5) comprises the one in D101, D3520, D4020, DA201, HPD-100, XAD-7, X-5, AB-8, H103, NKA-9, S-8.
9. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 4, is characterized in that: in the ethanol water described in step 6), the gradient concentration scope of ethanol is 0% ~ 80%.
10. the method for high efficiency extraction vaccinium oxycoccus pigment according to claim 4, is characterized in that: the suitable elutriant described in step 6) refers to that ethanol content is the elutriant of 25% ~ 75%.
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