CN102558379A - Method for extraction and deproteinization of hop polysaccharide - Google Patents
Method for extraction and deproteinization of hop polysaccharide Download PDFInfo
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- CN102558379A CN102558379A CN2012100072443A CN201210007244A CN102558379A CN 102558379 A CN102558379 A CN 102558379A CN 2012100072443 A CN2012100072443 A CN 2012100072443A CN 201210007244 A CN201210007244 A CN 201210007244A CN 102558379 A CN102558379 A CN 102558379A
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Abstract
The invention provides a method for extraction of hop polysaccharide from hop residues. The hop polysaccharide is extracted via microwave assistance and water extraction from the hop residues generated in production of CO2 hop extract or solvent-extracted hop extract, and fine hop polysaccharide is prepared via carrying out removal of impurities, bleaching, enzyme-chemical treatment and deproteinization on obtained coarse polysaccharide, and can also be subject to DEAE (diethylaminoethyl) cellulose column chromatography to collect polysaccharide components. The high-purity hop polysaccharide can be prepared via alcohol extraction, centrifugation and drying.
Description
Technical field
The present invention relates to a kind of extraction and Deproteinated method of hops polysaccharide.More specifically, the present invention relates to the liquid CO of a kind of employing
2, subcritical CO
2, supercritical CO
2Or the hops extract remainder after lower alcohol (comprising methyl alcohol, ethanol, the propyl alcohol) extraction is raw material, adopts the microwave-assisted water extracting alcohol to analyse method, extraction separation hops Crude polysaccharides after centrifugal, dry; To the hops Crude polysaccharides, through taking off after impurity, decolouring and the coupling of enzyme-chemically method remove protein, analyse, obtain purified hops polysaccharide after centrifugal, the drying through alcohol; To the hops polysaccharide after refining; Further adopt the DEAE-cellulose chromatography, collect the solution that contains polysaccharide fraction, concentrate after dialysis tubing is dialysed, alcohol is analysed, spinning; Obtain highly purified hops polysaccharide after the drying, belong to active components of plants and extract the field.
Background technology
Hops, the formal name used at school golden hop (
Humulus LupulusL.), be the Moraceae Humulus herbage that overgrows for many years, be mainly used in brewage.Discovering in recent years; In the hops except that odor component that contains influential beer flavor quality and bitter taste resene composition; Polyphenol wherein and flavonoid class material also have very important pharmacologically active effect, therefrom separate and have identified hundreds of compounds.Research shows; Polysaccharose substance is not only energy derive or structured material in vivo; The more important thing is that it has participated in various important vital movements; Have the informational function that can compare mutually with protein, nucleic acid, the same with protein, nucleic acid, carbohydrate also relates to one of three types of biomacromolecules of vital movement essence.Early stage people generally believe that polysaccharose substance itself does not have activity; But along with going deep into of research; The structure of polysaccharose substance and activity are studied personnel gradually and disclose; Vegetable polysaccharides class material has all shown very strong biological activity, and like: reducing blood-fat, hypoglycemic, antitumor, strengthening immunity, effect such as anti-oxidant and antibacterial, part has been used for the auxiliary and rehabilitation of tumour, hepatitis and cardiovascular disorder etc. clinically by medical circle; Therefore, the research to vegetable polysaccharides has become one of current focus of research both at home and abroad.We find in the research to the hops activeconstituents, and the polysaccharose substance in the hops also has very strong biological activity such as anti-oxidant, but the separation and Extraction research of polyose in pair hops is not arranged in the present research as yet.In addition; In the process for processing of hops; Usually only extract wherein fat-soluble bitter taste resene composition, volatile essential oil class component and flavonoid component wherein; Discard and the polyose composition in the hops still remains in the waste residue after the extraction usually, be not utilized effectively.
At present, in the extraction separation of most plants and fungus polysaccharide, traditional method is that water extracting alcohol is analysed method, and this method has cheapness, characteristics easily, but extraction time is longer, and polysaccharide extract rate efficient is lower.Most of method for removing protein can be divided into biological enzyme and chemical method; Biological enzyme mainly is that the certain condition of control adopts protease treatment; Chemical method is mainly Sevag method, salt acid system, trichloroacetic acid method or elder generation after hydrochloric acid, trichoroacetic acid(TCA) are handled, again with methods such as propyl carbinol processing.Like Chinese patent 200510012557.8,92109345.4,02114216.5,200810142918.4 grade all discloses extraction, separation and the process for purification to different plants or fungus polysaccharide.But because the specificity characteristics of enzyme make the albumen decreasing ratio variant because of the difference of raw material, effect is also unstable, and the albumen decreasing ratio is unsatisfactory.Chemical method has higher albumen decreasing ratio, but in subtractive process, needs to use a large amount of organic solvent repeated treatments, and process is complicated, and the rate of loss of polysaccharide is bigger.
Summary of the invention
Be not utilized effectively in normal processing to the hops polysaccharide fraction; The polysaccharide extraction time that exists in the prior art is long; Biological enzyme processing protein decreasing ratio is low, chemical Treatment solvent usage quantity is big; Number of processes is many, and polysaccharide loss rate high-technology defective, and technical problem to be solved by this invention provides a kind of extraction and Deproteinated method of hops polysaccharide.The objective of the invention is with liquid CO
2, subcritical CO
2, supercritical CO
2Or the hop residues after lower alcohol (comprising methyl alcohol, ethanol, the propyl alcohol) extraction is raw material, adopts microwave-assisted to extract the polyose component in the hops, and adopts that alcohol is analysed, obtains the hops Crude polysaccharides after filtration or centrifugal analysis, the drying; The hops Crude polysaccharides is decoloured, and the coupling of enzyme-chemically method removes protein wherein, and alcohol is analysed, obtained refining hops polysaccharide after filtration or spinning, the drying once more; The refining polysaccharide of hops is dissolved in a small amount of zero(ppm) water or the deionized water,, collects the solution that contains polysaccharide fraction, concentrate after dialysis tubing dialysis, alcohol are analysed, obtain highly purified hops polysaccharide after centrifugal, the drying through the DEAE-cellulose chromatography.
For achieving the above object, the present invention adopts following technical scheme to be achieved:
A kind ofly from hops, extract the hops polysaccharide and remove proteic method, it is characterized in that may further comprise the steps:
(1) to adopt CO
2, organic solvent is that the waste residue that extraction agent extracted Hops Extract is a raw material; The control extraction temperature is 25 ~ 95 ℃, with water extraction 1 ~ 3 time; Solid-liquid ratio is 1:3 ~ 1:20, and the extraction time is 1 ~ 10 hour, makes soluble saccharide components dissolved in the hops in water; During extracting, adopt microwave auxiliary extraction 5 ~ 30min;
(2) aqueous solution is isolated in filtration or centrifugal, and the operation of filter residue repetition above-mentioned steps (1) once; Merge twice extraction solution, and be concentrated to 10% ~ 30% of former extracting solution TV, in liquid concentrator, add the high concentration ethanol of certain volume; Make the alcohol concn of solution reach 30% ~ 85%; Leave standstill 12 ~ 24h, polyose component wherein precipitates from solution to be separated out, and filtration or spinning go out deposition wherein; Solution adds the high concentration ethanol solution of certain volume once more after appropriateness concentrates, make ethanolic soln wherein reach 30 ~ 85%; Leave standstill 12 ~ 24h, filtration or spinning go out deposition wherein once more; Solution discards, and merges the deposition that obtains for twice;
(3) deposition that obtains in the above-mentioned steps (2) is dissolved in the zero(ppm) water or deionized water of certain volume once more; Filtration or spinning go out insolubles wherein; Solution adds the ethanol of high density once more; Make the alcohol concn in the solution reach 30-85%, after the hold over night, spinning goes out deposition wherein; After the washing with alcohol of deposition with a small amount of high density, be lower than vacuum-drying or lyophilize under 50 ℃ the temperature, obtaining the bullion of hops polysaccharide;
The bullion of the hops polysaccharide that (4) obtains in the above-mentioned steps (3) is dissolved in zero(ppm) water or the deionized water, earlier with gac or hydrogen peroxide decolouring; Adopt the coupling of enzyme-chemically method to carry out deproteinated then and handle, removed proteinic polysaccharide soln, analyse through alcohol again; Spinning; A small amount of high concentration ethanol washing after vacuum-drying or the lyophilize, obtains the highly finished product of hops polysaccharide;
The highly finished product of the hops polysaccharide that (5) obtains in the above-mentioned steps (4); With a small amount of zero(ppm) water or deionized water dissolving; Again behind DEAE-cellulose column column chromatography, collect the solution that contains polysaccharide fraction, after concentrating again through dialysis tubing dialysis, alcohol analyse, spinning; After being lower than 50 ℃ temperature vacuum-drying or lyophilize, obtain highly purified hops polysaccharide.
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method, preferably extracting temperature is 80 ~ 90 ℃, and extraction time is 1 ~ 2h; The microwave action time is 12 ~ 15min;
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method, the ethanol of high density is content greater than 90% ethanolic soln or absolute ethyl alcohol;
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method; The coupling of enzyme-chemically method remove proteic method for the dissolving crude product of hops polysaccharide in zero(ppm) water or deionized water; Adopt 0.5-3.0mg/mL bromeline or papoid to handle earlier; The temperature of control solution is that ratio is 0.5 ~ 5.0% at the bottom of 35 ~ 65 ℃, enzyme, the pH value is 5.0 ~ 9.0, and enzymolysis time is 0.5 ~ 4h; After question response is accomplished, between pH value to 2.0 ~ 4.0 with hydrochloric acid conditioning solution, hold over night under the low temperature, centrifugal, discard deposition and collect clear liquid; Add the propyl carbinol reagent that is equivalent to liquor capacity 1/3 ~ 1/5 in the clear liquid, behind the stirring 20-30min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln.
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method; The coupling of enzyme-chemically method remove proteic method for the dissolving crude product of hops polysaccharide in zero(ppm) water or deionized water; Adopt earlier 0.5-3.0mg/mL bromeline or papoid to handle, the temperature of control solution is that ratio is 0.5 ~ 5.0% at the bottom of 35 ~ 65 ℃, enzyme, the pH value is 5.0 ~ 9.0, enzymolysis time is 0.5 ~ 4h; After question response is accomplished, between pH value to 2.0 ~ 3.0 with the trichoroacetic acid(TCA) regulator solution, hold over night under the low temperature, centrifugal, discard deposition and collect clear liquid; In clear liquid, add the propyl carbinol reagent that is equivalent to liquor capacity 1/3 ~ 1/5 then, behind the stirring 20-30min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln.
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method; When adopting the 1mg/mL bromeline; The deproteinated technology of said optimization is that ratio is 4.5% at the bottom of the enzyme of bromeline, and the pH value is 5.5, and hydrolysis temperature is that 45 ℃, enzymolysis time are 0.5h; After enzymolysis was accomplished, adopting the pH value of trichoroacetic acid(TCA) regulator solution was 2.0, and after 4 ℃ of hold over night, spinning discards deposition, collected clear liquid; In clear liquid, add the propyl carbinol reagent that is equivalent to liquor capacity 1/5 then, behind the stirring 20min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln; After this handled, the albumen decreasing ratio of hops Crude polysaccharides was about 90%, and the polysaccharide retention rate is about 80%.
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method; When adopting the 1mg/mL papoid; The deproteinated technology of said optimization is that ratio is 4.5% at the bottom of the enzyme of papoid, and the pH value is 5.5, and hydrolysis temperature is that 55 ℃, enzymolysis time are 0.5h; After enzymolysis was accomplished, adopting the pH value of trichoroacetic acid(TCA) regulator solution was 2.0, and after 4 ℃ of hold over night, spinning discards deposition and collects clear liquid; In clear liquid, add the propyl carbinol reagent that is equivalent to liquor capacity 1/5 then, behind the stirring 20min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln; After this handled, the albumen decreasing ratio of hops Crude polysaccharides was about 90%, and the polysaccharide retention rate is about 80%.
Describedly a kind ofly from hops, extract the hops polysaccharide and remove proteic method, the raw material that is used to extract the hops polysaccharide is for through liquid CO
2, subcritical CO
2, supercritical CO
2, methyl alcohol, ethanol, propyl alcohol, acetone for extract solvent extraction, separate the waste residue of hops bitter taste resin, volatile oil or Polyphenols component.Because the waste residue that in the production process of hops, is produced accounts for about 80% of raw material total amount; It is effectively utilized to extract the hops polysaccharide; Can improve the comprehensive utilization value of hops; Have characteristics such as cheapness, wide material sources, aboundresources, can be used as a kind of new resources of producing the vegetable active polysaccharide.
The beneficial effect that the present invention obtained is:
1. the present invention adopts microwave-assisted to extract the method for hops polysaccharide, because carry out microwave radiation heating efficient is high, the crushing effect of pair cell wall is big; Therefore, this method is compared with conventional water extraction, has the extraction yield height; The polyose component is easy to stripping, the advantage that extraction time is short.Separation and Extraction hops polysaccharide is a reported first from hops.
2. the present invention adopts the coupling of enzyme-chemically method to remove the protein in the hops Crude polysaccharides, has promptly utilized the selectivity of enzyme process high, survivable polysaccharide structures; The advantage that polysaccharide loss is less, the high advantage of deproteinated efficient of the chemical method that utilizes again, simultaneously; Because the combination of two kinds of methods makes that the treating processes of chemical method is more simple, number of processes is few; The solvent usage quantity is few, effectively raises the retention rate of polysaccharide.
3. the extraction raw material that the present invention selected for use is for extracting the extract remainder hop residues of Hops Extract, and the regenerated using that has improved resource is worth, and belongs to utilization of waste material.
Embodiment
Adopt the mode of embodiment to explain the present invention particularly below, but the present invention is not limited to embodiment.
Embodiment 1:
Get 100g through liquid CO
2Hop residues after the extraction adds 1000mL water, lixiviate 2h in 90 ℃ of water-baths, during adopt microwave auxiliary extraction 12min; Filter, isolate the aqueous solution, filter residue repeats the above-mentioned steps operation once, merges twice extraction solution; And be concentrated to about 100mL, in liquid concentrator, add the ethanol of 530mL95%, make the concentration of ethanol of solution reach 80%; Leave standstill 24h, polyose component wherein precipitates from solution to be separated out, and spinning goes out deposition wherein; This resolution of precipitate in the zero(ppm) water of 80mL, is filtered to isolate insolubles wherein, and solution adds the ethanol of 430mL95% once more, makes the alcohol concn in the solution reach 80%, and after the hold over night, spinning goes out deposition wherein; Deposition is with after a small amount of 95% the washing with alcohol, and vacuum-drying drying under 40 ℃ temperature obtains the bullion of 9.24g hops polysaccharide, and its polysaccharide content is 31.44%, and protein contnt is 45.5%.
Embodiment 2:
Get 500g through liquid CO
2Hop residues after the extraction adds 6000mL water, lixiviate 1h in 90 ℃ of water-baths, during adopt microwave auxiliary extraction 15min; Filter, isolate the aqueous solution, filter residue repeats the above-mentioned steps operation once, merges twice extraction solution; And be concentrated to about 1200mL, in liquid concentrator, add the ethanol of 6400mL95%, make the concentration of ethanol of solution reach 80%; Leave standstill 12h, polyose component wherein precipitates from solution to be separated out, and spinning goes out deposition wherein; This resolution of precipitate in the zero(ppm) water of 250mL, is filtered to isolate insolubles wherein, and solution adds the ethanol of 1330mL95% once more, makes the alcohol concn in the solution reach 80%, and after the hold over night, spinning goes out deposition wherein; Deposition is with after a small amount of 95% the washing with alcohol, and vacuum-drying under 50 ℃ temperature obtains the bullion of 51.37g hops polysaccharide, and its polysaccharide content is 28.55%, and protein contnt is 43.78%.
Embodiment 3
Get papoid 0.10g, use dissolved in distilled water, be settled to 100mL, obtaining enzyme liquid ultimate density is 1mg/mL, and the pH value is 7.0; 100mL Crude polysaccharides solution (2g/100mL) is put into 37 ℃ of water-bath preheating 10min, is that 55 ℃, pH value are to add the 4.5mL papoid at 5.5 o'clock in temperature, and enzymolysis 0.5h is centrifugal, gets supernatant.The hydrochloric acid soln that in this clear liquid, adds 2 mol/L is regulated pH value to 3.0,4 ° of C hold over night.The centrifugal 20min of 3000r/min next day discards deposition and collects clear liquid, in clear liquid, adds propyl carbinol 20mL again; Magnetic agitation 20min, the centrifugal 5min of 3000r/min discards protein layer; Get clear liquid; This clear liquid is for having removed proteinic polysaccharide soln, and its protein decreasing ratio is 82.81%, and the polysaccharide retention rate is 77.45%.With above-mentioned solution concentration, alcohol analyse, spinning precipitates and after vacuum-drying under 50 ℃ the temperature, obtains the refining polysaccharide 0.78g of hops, through analyzing, contains polysaccharide 55.32% in this sample, protein 12.15%.
Embodiment 4:
Get bromeline 0.05g, use dissolved in distilled water, be settled to 50mL, obtaining enzyme liquid ultimate density is 1mg/mL, and the pH value is 7.0.500mL Crude polysaccharides solution (2g/100mL) is put into 37 ℃ of water-bath preheating 10min, is that 45 ℃, pH value are to add the 25mL bromeline at 5.5 o'clock in temperature, and enzymolysis 0.5h is centrifugal, gets supernatant.The hydrochloric acid soln of adding 5% is regulated pH value to 2.0,4 ° of C hold over night in this clear liquid.The centrifugal 20min of 3000r/min next day discards deposition and collects clear liquid, in clear liquid, adds propyl carbinol 100mL; Magnetic agitation 20min, the centrifugal 5min of 3000r/min discards protein layer; Collect clear liquid; This clear liquid is for having removed proteinic polysaccharide soln, and its protein decreasing ratio is 90.06%, and the polysaccharide retention rate is 79.23%.With above-mentioned solution concentration, alcohol analyse, spinning precipitates and after vacuum-drying under 50 ℃ the temperature, obtains the refining polysaccharide 3.22g of hops, through analyzing, contains polysaccharide 65.32% in this sample, protein 2.31%.
Embodiment 5:
Get papoid 0.10g, use dissolved in distilled water, be settled to 100mL, obtaining enzyme liquid ultimate density is 1mg/mL, and the pH value is 7.0; 100mL Crude polysaccharides solution (2g/100mL) is put into 37 ℃ of water-bath preheating 10min, is that 55 ℃, pH value are to add the 4.5mL papoid at 5.5 o'clock in temperature, and enzymolysis 0.5h is centrifugal, gets supernatant.The trichoroacetic acid(TCA) solution that in this clear liquid, adds 2 mol/L is regulated pH value to 3.0,4 ° of C hold over night.The centrifugal 20min of 3000r/min next day discards deposition and collects clear liquid, in clear liquid, adds propyl carbinol 20mL again; Magnetic agitation 20min, the centrifugal 5min of 3000r/min discards protein layer; Get clear liquid; This clear liquid is for having removed proteinic polysaccharide soln, and its protein decreasing ratio is 76.81%, and the polysaccharide retention rate is 91.45%.With above-mentioned solution concentration, alcohol analyse, spinning precipitates and after vacuum-drying under 50 ℃ the temperature, obtains the refining polysaccharide 1.05g of hops, through analyzing, contains polysaccharide 48.25% in this sample, protein 16.39%.
Embodiment 6:
Get bromeline 0.05g, use dissolved in distilled water, be settled to 50mL, obtaining enzyme liquid ultimate density is 1mg/mL, and the pH value is 7.0.500mL Crude polysaccharides solution (2g/100mL) is put into 37 ℃ of water-bath preheating 10min, is that 45 ℃, pH value are to add the 25mL bromeline at 5.5 o'clock in temperature, and enzymolysis 0.5h is centrifugal, gets supernatant.The trichoroacetic acid(TCA) solution of adding 5% is regulated pH value to 2.0,4 ° of C hold over night in this clear liquid.The centrifugal 20min of 3000r/min next day discards deposition and collects clear liquid, in clear liquid, adds propyl carbinol 100mL; Magnetic agitation 20min, the centrifugal 5min of 3000r/min discards protein layer; Collect clear liquid; This clear liquid is for having removed proteinic polysaccharide soln, and its protein decreasing ratio is 80.06%, and the polysaccharide retention rate is 85.23%.With above-mentioned solution concentration, alcohol analyse, spinning precipitates and after vacuum-drying under 50 ℃ the temperature, obtains the refining polysaccharide 3.58g of hops, through analyzing, contains polysaccharide 57.69% in this sample, protein 7.15%.
Embodiment 7:
Get each 0.05g of bromeline and papoid, use dissolved in distilled water, be settled to 50mL, obtain content and be the 0.5mg/mL mixed enzyme solution.150mL Crude polysaccharides solution (2g/100mL) is put into 37 ℃ of water-bath preheating 10min, is that 50 ℃, pH value are to add the bromeline of 7.0mL and the mixed solution of papoid at 6.0 o'clock in temperature, and enzymolysis 1h is centrifugal, gets supernatant.The hydrochloric acid soln of adding 5% in this clear liquid is regulated pH value to 3.0,4 ° of C hold over night.The centrifugal 20min of 3000r/min next day discards deposition and collects clear liquid, in clear liquid, adds propyl carbinol 30mL; Magnetic agitation 20min, the centrifugal 5min of 3000r/min discards protein; Collect clear liquid; This clear liquid is and has removed proteinic polysaccharide soln, and its protein decreasing ratio is 86.34%, and the polysaccharide retention rate is 80.12%.With above-mentioned solution concentration, alcohol analyse, spinning precipitates and after vacuum-drying under 50 ℃ the temperature, obtains the refining polysaccharide 1.09g of hops, through analyzing, contains polysaccharide 59.77% in this sample, protein 4.57%.
Embodiment 8:
Get each 0.05g of bromeline and papoid, use dissolved in distilled water, be settled to 50mL, obtain content and be the 0.5mg/mL mixed enzyme solution.300mL Crude polysaccharides solution (2g/100mL) is put into 37 ℃ of water-bath preheating 10min, is that 50 ℃, pH value are to add the bromeline of 15mL and the mixed solution of papoid at 6.0 o'clock in temperature, and enzymolysis 1h is centrifugal, gets supernatant.The trichoroacetic acid(TCA) solution of adding 5% is regulated pH value to 2.0,4 ° of C hold over night in this clear liquid.The centrifugal 20min of 3000r/min next day discards deposition and collects clear liquid, in clear liquid, adds propyl carbinol 70mL; Magnetic agitation 20min, the centrifugal 5min of 3000r/min discards protein; Collect clear liquid; This clear liquid is for having removed proteinic polysaccharide soln, and its protein decreasing ratio is 83.45%, and the polysaccharide retention rate is 90.12%.With above-mentioned solution concentration, alcohol analyse, spinning precipitates, and after vacuum-drying under 50 ℃ the temperature, obtains the refining polysaccharide 2.27g of hops, through analyzing, contains polysaccharide 63.26% in this sample, protein 2.65%.
Embodiment 9:
Get the highly finished product 2g of hops polysaccharide, use a small amount of dissolved in distilled water, behind DEAE-cellulose column column chromatography; Collection contains the solution of polysaccharide fraction, concentrates the back with the dialysis tubing dialysis, again through alcohol analyse, after the spinning; The deposition lyophilize obtains highly purified hops polysaccharide 0.34g.Through analyzing, the polysaccharide content of this hops polysaccharide is 91.88%.
Claims (8)
1. one kind is extracted the hops polysaccharide and removes proteic method from hops, it is characterized in that may further comprise the steps:
(1) to adopt CO
2, organic solvent is that the waste residue that extraction agent extracted Hops Extract is a raw material; The control extraction temperature is 25 ~ 95 ℃, with water extraction 1 ~ 3 time; Solid-liquid ratio is 1:3 ~ 1:20, and the extraction time is 1 ~ 10 hour, makes soluble saccharide components dissolved in the hops in water; During extracting, adopt microwave auxiliary extraction 5 ~ 30min;
(2) aqueous solution is isolated in filtration or centrifugal, and the operation of filter residue repetition above-mentioned steps (1) once; Merge twice extraction solution, and be concentrated to 10% ~ 30% of former extracting solution TV, in liquid concentrator, add the high concentration ethanol of certain volume; Make the alcohol concn of solution reach 30% ~ 85%; Leave standstill 12 ~ 24h, polyose component wherein precipitates from solution to be separated out, and filtration or spinning go out deposition wherein; Solution adds the high concentration ethanol solution of certain volume once more after appropriateness concentrates, make ethanolic soln wherein reach 30 ~ 85%; Leave standstill 12 ~ 24h, filtration or spinning go out deposition wherein once more; Solution discards, and merges the deposition that obtains for twice;
(3) deposition that obtains in the above-mentioned steps (2) is dissolved in the zero(ppm) water or deionized water of certain volume once more; Filtration or spinning go out insolubles wherein; Solution adds the ethanol of high density once more; Make the alcohol concn in the solution reach 30-85%, after the hold over night, spinning goes out deposition wherein; After the washing with alcohol of deposition with a small amount of high density, be lower than vacuum-drying or lyophilize under 50 ℃ the temperature, obtaining the bullion of hops polysaccharide;
The bullion of the hops polysaccharide that (4) obtains in the above-mentioned steps (3) is dissolved in zero(ppm) water or the deionized water, earlier with gac or hydrogen peroxide decolouring; Adopt the coupling of enzyme-chemically method to carry out deproteinated then and handle, removed proteinic polysaccharide soln, analyse through alcohol again; Spinning; A small amount of high concentration ethanol washing after vacuum-drying or the lyophilize, obtains the highly finished product of hops polysaccharide;
The highly finished product of the hops polysaccharide that (5) obtains in the above-mentioned steps (4); With a small amount of zero(ppm) water or deionized water dissolving; Again behind DEAE-cellulose column column chromatography, collect the solution that contains polysaccharide fraction, after concentrating again through dialysis tubing dialysis, alcohol analyse, spinning; After being lower than 50 ℃ temperature vacuum-drying or lyophilize, obtain highly purified hops polysaccharide.
2. method according to claim 1, it is characterized in that preferably extracting temperature is 80 ~ 90 ℃, extraction time is 1 ~ 2h; The microwave action time is 12 ~ 15min.
3. method according to claim 1, the ethanol that it is characterized in that described high density are content greater than 90% ethanolic soln or absolute ethyl alcohol.
4. method according to claim 1; It is characterized in that the coupling of described enzyme-chemically method remove proteic method for the dissolving crude product of hops polysaccharide in zero(ppm) water or deionized water; Adopt 0.5-3.0mg/mL bromeline or papoid to handle earlier; The temperature of control solution is that ratio is 0.5 ~ 5.0% at the bottom of 35 ~ 65 ℃, enzyme, the pH value is 5.0 ~ 9.0, and enzymolysis time is 0.5 ~ 4h; After question response is accomplished, between pH value to 2.0 ~ 4.0 with hydrochloric acid conditioning solution, hold over night under the low temperature, centrifugal, discard deposition and collect clear liquid; Add the propyl carbinol reagent that is equivalent to liquor capacity 1/3 ~ 1/5 in the clear liquid, behind the stirring 20-30min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln.
5. method according to claim 1; It is characterized in that the coupling of described enzyme-chemically method remove proteic method for the dissolving crude product of hops polysaccharide in zero(ppm) water or deionized water; Adopt earlier 0.5-3.0mg/mL bromeline or papoid to handle, the temperature of control solution is that ratio is 0.5 ~ 5.0% at the bottom of 35 ~ 65 ℃, enzyme, the pH value is 5.0 ~ 9.0, enzymolysis time is 0.5 ~ 4h; After question response is accomplished, between pH value to 2.0 ~ 3.0 with the trichoroacetic acid(TCA) regulator solution, hold over night under the low temperature, centrifugal, discard deposition and collect clear liquid; In clear liquid, add the propyl carbinol reagent that is equivalent to liquor capacity 1/3 ~ 1/5 then, behind the stirring 20-30min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln.
6. method according to claim 1 is characterized in that when adopting the 1mg/mL bromeline, and the deproteinated technology of said optimization is that ratio is 4.5% at the bottom of the enzyme of bromeline, and the pH value is 5.5, and hydrolysis temperature is that 45 ℃, enzymolysis time are 0.5h; After enzymolysis was accomplished, adopting the pH value of trichoroacetic acid(TCA) regulator solution was 2.0, and after 4 ℃ of hold over night, spinning discards deposition, collected clear liquid; In clear liquid, add the propyl carbinol reagent that is equivalent to liquor capacity 1/5 then, behind the stirring 20min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln; After this handled, the albumen decreasing ratio of hops Crude polysaccharides was about 90%, and the polysaccharide retention rate is about 80%.
7. method according to claim 1 is characterized in that when adopting the 1mg/mL papoid, and the deproteinated technology of said optimization is that ratio is 4.5% at the bottom of the enzyme of papoid, and the pH value is 5.5, and hydrolysis temperature is that 55 ℃, enzymolysis time are 0.5h; After enzymolysis was accomplished, adopting the pH value of trichoroacetic acid(TCA) regulator solution was 2.0, and after 4 ℃ of hold over night, spinning discards deposition and collects clear liquid; In clear liquid, add the propyl carbinol reagent that is equivalent to liquor capacity 1/5 then, behind the stirring 20min, spinning discards n-butanol layer, and water layer is and has removed proteinic polysaccharide soln; After this handled, the albumen decreasing ratio of hops Crude polysaccharides was about 90%, and the polysaccharide retention rate is about 80%.
8. method according to claim 1, the raw material that it is characterized in that extracting the hops polysaccharide is for through liquid CO
2, subcritical CO
2, supercritical CO
2, methyl alcohol, ethanol, propyl alcohol, acetone crosses the waste residue that separates hops bitter taste resin, volatile oil or Polyphenols component for extracting solvent extraction.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN103059162A (en) * | 2013-01-14 | 2013-04-24 | 胡玮 | Novel method for effectively extracting lentinan |
| CN104825349A (en) * | 2015-04-21 | 2015-08-12 | 浙江大学湖州市南太湖现代农业科技推广中心 | Method for producing kin care gel application agent from biomass raw material |
| CN107987180A (en) * | 2017-10-31 | 2018-05-04 | 海盐县凌特生物科技有限公司 | The method that polysaccharide is extracted from oyster mushroom |
| CN108498380A (en) * | 2018-05-30 | 2018-09-07 | 广州市康超信息科技有限公司 | A kind of hops facial mask and preparation method thereof |
| CN109535274A (en) * | 2018-11-30 | 2019-03-29 | 淮阴工学院 | A kind of method that common cattail polysaccharide takes off protein decolouring |
| CN110655586A (en) * | 2019-09-02 | 2020-01-07 | 广东药科大学 | Hop sugar polymer and preparation method and application thereof |
| CN113234552A (en) * | 2021-04-29 | 2021-08-10 | 浙江大学 | Hop polysaccharide nano particle and preparation method and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911277A (en) * | 2012-09-14 | 2013-02-06 | 广州市卫斯理日化实业有限公司 | Method for extracting and purifying garlic polysaccharides from supercritical CO2 raffinate and application of garlic polysaccharides in preparing yoghurt |
| CN102911277B (en) * | 2012-09-14 | 2015-08-05 | 广州市卫斯理化工科技有限公司 | Supercritical CO 2to extract in raffinate and the method for purified garlic polysaccharide and garlic polysaccharide are preparing the application in Yoghourt |
| CN103059162A (en) * | 2013-01-14 | 2013-04-24 | 胡玮 | Novel method for effectively extracting lentinan |
| CN103059162B (en) * | 2013-01-14 | 2015-08-19 | 胡玮 | A kind of novel method of high efficiency extraction lentinan |
| CN104825349A (en) * | 2015-04-21 | 2015-08-12 | 浙江大学湖州市南太湖现代农业科技推广中心 | Method for producing kin care gel application agent from biomass raw material |
| CN107987180A (en) * | 2017-10-31 | 2018-05-04 | 海盐县凌特生物科技有限公司 | The method that polysaccharide is extracted from oyster mushroom |
| CN108498380A (en) * | 2018-05-30 | 2018-09-07 | 广州市康超信息科技有限公司 | A kind of hops facial mask and preparation method thereof |
| CN109535274A (en) * | 2018-11-30 | 2019-03-29 | 淮阴工学院 | A kind of method that common cattail polysaccharide takes off protein decolouring |
| CN110655586A (en) * | 2019-09-02 | 2020-01-07 | 广东药科大学 | Hop sugar polymer and preparation method and application thereof |
| CN113234552A (en) * | 2021-04-29 | 2021-08-10 | 浙江大学 | Hop polysaccharide nano particle and preparation method and application thereof |
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