CN100439511C - Process for catalytic extraction of yam saponin by using modified cellulase - Google Patents
Process for catalytic extraction of yam saponin by using modified cellulase Download PDFInfo
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- CN100439511C CN100439511C CNB2006101292286A CN200610129228A CN100439511C CN 100439511 C CN100439511 C CN 100439511C CN B2006101292286 A CNB2006101292286 A CN B2006101292286A CN 200610129228 A CN200610129228 A CN 200610129228A CN 100439511 C CN100439511 C CN 100439511C
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- cellulase
- cyanuric chloride
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- yam
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Abstract
The invention discloses a method for extracting cinnamonvine saponin with modified cellulase, and belongs to the technique of extracting cinnamonvine saponin from cinnamonvine plant. The invention comprises the steps of: dissolving cyanuric chloride in benzene, recrystallisating, carrying out a reaction with polyglycol (treated by molecular sieve and exsiccated sodium carbonate in benzene) according to proper rate to obtain activated polyglycol, carrying out a reaction with natural cellulose at proper ratio in citric acid-sodium citrate buffer, and separating with ultrafiltration membrane to obtain modified cellulose, adding into aqueous soak of the raw materials, catalyzing, hydrolyzing, neutralizing, extracting, and crystallisating to obtain the product of cinnamonvine saponin. The invention improves the resistant ability of the modified cellulase against environment and temperature. The modified cellulase has high catalysis activity in a long time, obvious catalysis effect, and can be used for the extracting effective components from natural product rich in cellulose, and has wide versatility.
Description
Technical field
The present invention relates to a kind of method of utilizing modified cellulose enzyme extraction diosgenin.Belong to technology by Chinese yam plant extract diosgenin.
Background technology
Diosgenin is a diosgenin, is the important intermediate of steroidal pharmaceutical industry, is that raw material gets through acid hydrolysis, organic solvent extraction by the yam rhizome.Originally the production of diosgenin be that to be raw material through direct acid hydrolysis extract makes by Chinese yam plant rhizome, i.e. the Rothrok method.This method is at home and abroad continued to use always, and China begins to adopt this method to set up diosgenin factory at the end of the fifties, even still is extensive use of now in some small businesss of China.It is lower that but this method but exists the yield of the diosgenin that makes on the one hand, can only extract in the plant composition of 45% diosgenin; Make that on the other hand a large amount of effective constituent is discarded in the plant, cause the serious waste of resource and the pollution of environment.
In order to improve the yield of diosgenin, people once carried out number of research projects and kinds of processes and improved, one of wherein important discovery is that the people such as Paseshnichenko of the Canadian Hardman of the 60 to 70's and the Soviet Union are fermented plant material before acid hydrolysis after for some time in advance, can reach the purpose that improves the diosgenin yield.But this method elapsed time is longer, and fermenting process can produce other by product simultaneously.
In recent years, development along with biocatalysis technology, more domestic scholars adopt cellulase, amylase, polygalacturonase etc. to carry out biocatalysis respectively and handle the collaborative acid hydrolysis in back and study, and the result can make in the plant 65%～70% diosgenin composition extract.But, carrying out along with the biological enzyme reaction, the product that degradation of substrates generates can produce restraining effect to biological enzyme, add that raw material contains more impurity, make the biological enzyme vigor reduce greatly, be unfavorable in the plant effective degraded and fracture with dioscin bonded polymer substance, then acid-hydrolyzed resistance effectively do not reduced, thereby the yield and the quality that have limited diosgenin improve.
Summary of the invention
The method of the diosgenin in the Chinese yam plant is extracted in the modified cellulose enzyme catalysis that the object of the present invention is to provide a kind of utilization to have greater activity.This method has diosgenin extraction yield height, save energy and the low characteristics of environmental pollution.
For achieving the above object, the present invention is realized by following technical proposals.A kind of method of catalytic extraction of yam saponin by using modified cellulase is characterized in that comprising following process:
1. the purifying of cyanuric chloride, preparation mass concentration are 6%～15% cyanuric chloride benzole soln, and recrystallization twice obtains the cyanuric chloride of purifying.
2. the activation of polyoxyethylene glycol, by the mass ratio of Macrogol 4000 and 4A molecular sieve and anhydrous sodium carbonate is 1: 2～6: 4～10 Macrogol 4000,4A molecular sieve and anhydrous sodium carbonate to be added in the solvent benzol, after the stirring at normal temperature 30～60 minutes, filter, remove 4A molecular sieve and yellow soda ash and obtain filtrate, be that the cyanuric chloride of 2～2.5: 1 purifying that step 1 is made joins in the filtrate according to the mol ratio of the cyanuric chloride of Macrogol 4000 and purifying then, normal temperature condition reaction 20～50 hours down.Solution through concentrating, filters again, and cryodrying obtains activated polyglycol.
3. the modification of cellulase, the activated polyethylene glycol that cellulase and step 2 are made are that 1: 2～6 to join pH be in 5.0～7.0 citric acid-sodium citrate buffer according to mass ratio, and controlled temperature is 0 ℃～8 ℃, reacts 1～5 hour.Adopting molecular weight cut-off is that 5000～10000 ultra-filtration membrane is isolated cellulase, and frozen drying obtains using modified cellulase.
4. the conversion of diosgenin is extracted, is the Rhizome of Peltate Yam of pulverizing or Dioscorea nipponica Mak. Ningpo Yam Rhizome raw material to add at 1: 3 to soak 30～60 minutes in the entry according to mass ratio, the using modified cellulase that step 3 is made according to the ratio with Rhizome of Peltate Yam or Dioscorea nipponica Mak. Ningpo Yam Rhizome mass ratio 2.0 ‰ adds in the soak solution again, 50 ℃～60 ℃ following katalysis reactions of controlled temperature 4～7 hours, acid concentration with sulfuric acid or hydrochloric acid regulation system is 2mol/L～5mol/L then, temperature is 98～108 ℃ of following hydrolysis, and hydrolyzed solution adopts 5% Na
2CO
3Solution washing places 60～80 ℃ of baking ovens dry filter cake to neutral filtration, and dried solids utilizes extraction agent 120# gasoline to extract in cable type extractor according, and extraction liquid filters through concentrating, and gets the diosgenin product after the cryodrying.
The invention has the advantages that having improved cellulase resists the ability that envrionment conditions causes enzyme deactivation, thereby improved the catalytic activity of enzyme, in reaction process, remain advantages of high catalytic activity, reduced the resistance of material subsequent disposal, the dioscin that helps in the yam is converted into diosgenin, thereby improved the diosgenin extraction yield, increased output, and waste discharge is low.
Embodiment
Embodiment one: the 20.0g cyanuric chloride is dissolved in the 300mL benzene, behind twice in the recrystallization, obtains the cyanuric chloride 17.9g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 5.0g, the 10.0g anhydrous sodium carbonate, 500mL benzene and 20.0g Macrogol 4000, stirring at normal temperature is after 30 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.4613g of purifying then in there-necked flask, normal temperature condition reacted 36 hours down.Solution is through concentrating, and B filters, and is-14 ℃ of following cryodryings in temperature then, obtains activated polyglycol 4000.The activated polyethylene glycol 4000 and the pH that add 4.0g in the flask of 500mL are 6.8 citric acid-sodium citrate buffer 100mL, attemperation is 4 ℃, activated polyethylene glycol 4000 fully adds the 1.0g cellulases in the dissolving back, react that to utilize molecular weight cut-off after 2 hours be that 5000 polysulphones hyperfiltration membrane separation obtains using modified cellulase.
Then 100g 60～80 order Rhizome of Peltate Yam powder are placed the 500mL there-necked flask, add 300mL water and stirred 45 minutes down for 30 ℃, after fully immersion is expanded, add the cellulase that 0.2g modifies in temperature, 50 ℃ of control reaction temperature, modified cellulose enzymic catalytic reaction 5 hours; Measuring the reaction solution volume then is 330mL, and adding 98% vitriol oil 64.3mL adjusting hydrolyzation system acid concentration is 3mol/L, carries out acid hydrolysis 2 hours in 101 ℃; Rhizome of Peltate Yam powder after the hydrolysis is used 5% Na again
2CO
3Solution washing is to neutral, and is then dry in 60 ℃ of baking ovens, and stirring frequently prevents that material too; Dried hydrolyzate 21.6g was extracted in cable type extractor according 7 hours with the 120# gasoline of 500mL, reclaim gasoline with rotatory evaporator then, concentrated extracting solution, product crystallization under-4 ℃ of cold condition, sand core funnel filters, dry colourless needle crystal 2.2537g in-15 ℃ of freeze drying box at last, extraction yield can reach more than 96%.Product is through infrared measurement, result (IR (KBr): v cm
-13452,1049,978,918,898,866) with the document (IR (KBr): v cm that matches
-13450,1050,980,918,898,864); Fusing point is 197 ℃～198 ℃; Results of elemental analyses is C:77.98%, and H:10.19% is consistent with calculated value (78.21% and 10.21%); The high-performance liquid chromatogram determination result is that product purity is up to 98.6%.
Embodiment two: the 10.0g cyanuric chloride is dissolved in the 150mL benzene, and recrystallization twice obtains the cyanuric chloride 8.7g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 2.5g, the 8.0g anhydrous sodium carbonate, 400mL benzene and 10.0g Macrogol 4000, stirring at normal temperature is after 40 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.2306g of purifying then in there-necked flask, normal temperature condition reacted 38 hours down.Solution is through concentrating, and B filters, and temperature is-14 ℃ of following cryodryings, obtains activated polyglycol 4000.Adding 8.0g activated polyethylene glycol 4000 and pH are 7.0 citric acid-sodium citrate buffer 200mL in the flask of 500mL, attemperation is 4 ℃, activated polyethylene glycol 4000 fully adds the 2.0g cellulases in the dissolving back, and react after 2 hours with molecular weight cut-off is that 4000 polysulphones hyperfiltration membrane separation obtains using modified cellulase.
50.0g80～100 order Rhizome of Peltate Yam powder are placed the 250mL there-necked flask, add 150mL water and stirred 45 minutes down for 25 ℃, after fully immersion is expanded, add the 0.1g using modified cellulase, 50 ℃ of control reaction temperature, modified cellulose enzyme catalysis 6 hours in temperature; Working method such as the hydrolysis of back, neutralization, extraction are according to embodiment one then, concentrated extracting solution, product crystallization under-4 ℃ of low temperature, sand core funnel filters, dry colourless needle crystal 1.2365g in-15 ℃ of freeze drying box at last, extraction yield can reach more than 97%.Product is through infrared measurement, result (IR (KBr): v cm
-13450,1050,979,918,897,867) with the document (IR (KBr): v cm that matches
-13450,1050,980,918,898,864); Fusing point is 198 ℃～200 ℃; Results of elemental analyses is C:77.95%, and H:10.20% is consistent with calculated value (78.21% and 10.21%); The high-performance liquid chromatogram determination result is that purity is up to 97.9%.
Embodiment three: the 20.0g cyanuric chloride is dissolved in the 300mL benzene, and recrystallization twice obtains the cyanuric chloride 18.3g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 8.0g, the 12.0g anhydrous sodium carbonate, 600mL benzene and 30.0g Macrogol 4000, stirring at normal temperature is after 60 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.5535g of purifying then in there-necked flask, normal temperature condition reacted 40 hours down.Solution is through concentrating, and B filters, and temperature is-14.2 ℃ of following cryodryings, obtains activated polyglycol 4000.Adding 8.0g activated polyethylene glycol 4000 and pH are 6.8 citric acid sodium citrate buffer 200mL in the flask of 500mL, attemperation is 4 ℃, activated polyethylene glycol 4000 fully adds the 2.0g cellulases in the dissolving back, react that to utilize molecular weight cut-off after 2 hours be that 4000 polysulphones hyperfiltration membrane separation obtains using modified cellulase.
50.0g80～100 order Dioscorea nipponica Mak. Ningpo Yam Rhizome powder are placed the 250mL there-necked flask, add 150mL water and stirred 60 minutes down for 30 ℃, after fully immersion is expanded, add the 0.1g using modified cellulase in temperature, 55 ℃ of control reaction temperature, modified cellulose enzyme catalysis 5.5 hours; Working method such as the hydrolysis of back, neutralization, extraction are according to embodiment then, last concentrated extracting solution, product crystallization under-4 ℃ of low temperature, sand core funnel filters, at last in-15 ℃ of freeze drying box in dry colourless needle crystal 1.3684g, extraction yield can reach more than 97%.Product is through infrared measurement, result (IR (KBr): vcm
-13450,1050,979,918,897,867) with the document (IR (KBr): v cm that matches
-13450,1050,980,918,898,864); Fusing point is 197 ℃～199 ℃; Results of elemental analyses is C:78.14%, and H:10.20% is consistent with calculated value (78.21% and 10.21%); The high-performance liquid chromatogram determination result is that purity is up to 95.6%.
Embodiment four: the 20.0g cyanuric chloride is dissolved in the 300mL benzene, and recrystallization twice obtains the cyanuric chloride 17.6g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 4.0g, the 9.0g anhydrous sodium carbonate, 500mL benzene and 16.0g Macrogol 4000, stirring at normal temperature is after 40 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.2952g of purifying then in there-necked flask, normal temperature condition reacted 40 hours down.Solution is through concentrating, and B filters, and temperature is-14.2 ℃ of following cryodryings, obtains activated polyglycol 4000.Adding 12.0g activated polyethylene glycol and pH are 7.0 citric acid-sodium citrate buffer 100mL in the flask of 500mL, attemperation is 4.0 ℃, activated polyethylene glycol 4000 fully adds the 3.0g cellulases in the dissolving back, react that to utilize molecular weight cut-off after 2 hours be that 5000 polysulphones hyperfiltration membrane separation obtains using modified cellulase.
100g60～80 purpose Dioscorea nipponica Mak. Ningpo Yam Rhizome powder are placed there-necked flask, add 300mL water and stirred 45 minutes down for 25 ℃, after fully immersion is expanded, add the cellulase that 0.2g modifies through Macrogol 4000 in temperature, 60 ℃ of control reaction temperature, cellulase catalysis 5 hours; Working method such as the hydrolysis of back, neutralization, extraction are according to embodiment one then, last concentrated extracting solution, product crystallization under-4 ℃ of cold condition, sand core funnel filters, dry colourless needle crystal 2.6957g in-60 ℃ of freeze drying box at last, extraction yield can reach more than 95%.Product is through infrared measurement, result (IR (KBr): v cm
-13450,1050,980,920,897,866) with the document (IR (KBr): v cm that matches
-13450,1050,980,918,898,864); Fusing point is 197 ℃～199 ℃; Results of elemental analyses is C:78.18%, and H:10.23% is consistent with calculated value (78.21% and 10.21%); The high-performance liquid chromatogram determination result is that purity is up to 97.8%.
Embodiment five: the 15.0g cyanuric chloride is dissolved in the 250mL benzene, and recrystallization twice obtains the cyanuric chloride 13.6g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 5.0g, the 10.0g anhydrous sodium carbonate, 600ml benzene and 16.0g Macrogol 4000, stirring at normal temperature is after 40 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.2952g of purifying then in there-necked flask, normal temperature condition reacted 40 hours down, and solution is through concentrating, and B filters, and temperature is-14.2 ℃ of following cryodryings, obtains activated polyglycol 4000.Adding 14.0g activated polyethylene glycol 4000 and pH are 7.0 citric acid-sodium citrate buffer 120mL in the flask of 500mL, attemperation is 5.0 ℃, activated polyethylene glycol 4000 fully adds the 4.0g cellulases in the dissolving back, react that to utilize molecular weight cut-off after 2 hours be 5000 polysulphones hyperfiltration membrane separation using modified cellulase.
100g80～100 order Dioscorea nipponica Mak. Ningpo Yam Rhizome powder are placed there-necked flask, add 300mL water and stirred 60 minutes down for 30 ℃, after fully immersion is expanded, add the cellulase that 0.2g modifies through Macrogol 4000 in temperature, 55 ℃ of control reaction temperature, cellulase catalysis 6 hours; Working method such as the hydrolysis of back, neutralization, extraction are according to embodiment one then, last concentrated extracting solution, product crystallization under-4 ℃ of cold condition, sand core funnel filters, dry colourless needle crystal 2.7153g in-60 ℃ of freeze drying box at last, extraction yield can reach more than 96%.Product is through infrared measurement, result (IR (KBr): v cm
-13450,1049,980,920,897,866) with the document (IR (KBr): v cm that matches
-13450,1050,980,918,898,864); Fusing point is 197 ℃～199 ℃; Results of elemental analyses is C:78.20%, and H:10.20% is consistent with calculated value (78.21% and 10.21%); The high-performance liquid chromatogram determination result is that purity is up to 96.9%.
Claims (1)
1. the method for a catalytic extraction of yam saponin by using modified cellulase is characterized in that comprising following process:
1). the purifying of cyanuric chloride, the preparation mass concentration is 6%～15% cyanuric chloride benzole soln, recrystallization twice obtains the cyanuric chloride of purifying;
2). the activation of polyoxyethylene glycol, by the mass ratio of Macrogol 4000 and 4A molecular sieve and anhydrous sodium carbonate is 1: 2～6: 4～10 with Macrogol 4000,4A molecular sieve and anhydrous sodium carbonate add in the solvent benzol, after the stirring at normal temperature 30～60 minutes, filter, remove 4A molecular sieve and yellow soda ash and obtain filtrate, be that the cyanuric chloride of 2～2.5: 1 purifying that step 1) is made joins in the filtrate according to the mol ratio of the cyanuric chloride of Macrogol 4000 and purifying then, normal temperature condition reacted 20～50 hours down, solution is again through concentrating, filter, cryodrying obtains activated polyglycol;
3). the modification of cellulase, with cellulase and step 2) activated polyethylene glycol that makes is that 1: 2～6 to join pH be in 5.0～7.0 citric acid-sodium citrate buffer according to mass ratio, controlled temperature is 0 ℃～8 ℃, reacted 1～5 hour, adopting molecular weight cut-off is that 5000～10000 ultra-filtration membrane is isolated cellulase, and frozen drying obtains using modified cellulase;
4). the conversion of diosgenin is extracted, is the Rhizome of Peltate Yam of pulverizing or Dioscorea nipponica Mak. Ningpo Yam Rhizome raw material to add at 1: 3 to soak 30～60 minutes in the entry according to mass ratio, the using modified cellulase that step 3) is made according to the ratio with Rhizome of Peltate Yam or Dioscorea nipponica Mak. Ningpo Yam Rhizome mass ratio 2.0 ‰ adds in the soak solution again, 50 ℃～60 ℃ following katalysis reactions of controlled temperature 4～7 hours, acid concentration with sulfuric acid or hydrochloric acid regulation system is 2mol/L～5mol/L then, temperature is 98～108 ℃ of following hydrolysis, and hydrolyzed solution adopts 5% Na
2CO
3Solution washing places 60～80 ℃ of baking ovens dry filter cake to neutral filtration, and dried solids utilizes extraction agent 120# gasoline to extract in cable type extractor according, and extraction liquid filters through concentrating, and gets the diosgenin product after the cryodrying.
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| CN110272936A (en) * | 2018-08-22 | 2019-09-24 | 张秀梅 | A method of improving hydrolysis saponin yield |
| CN111642730B (en) * | 2020-06-18 | 2023-06-06 | 安徽省农业科学院农产品加工研究所 | Method, product and equipment for recycling and eluting soy sauce residues |
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