CN101629200B - Method for producing acteoside by fresh herba cistanches - Google Patents
Method for producing acteoside by fresh herba cistanches Download PDFInfo
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Abstract
The invention provides a method for producing acteoside by fresh herba cistanches, comprising the following steps: carrying out such system processes as enzyme deactivation at high temperature, cell disruption, size extraction and separation and purification on fresh herba cistanches fleshy stems to obtain herba cistanches extract, taking the obtained herba cistanches extract as substrates for catalytic reaction and beta-glucosidase, an industrial immobilized enzyme, as a biocatalyst to catalyze echinacoside in hydrolysis substrates to enable the ends of glucosyl ligands to be broken and the echinacoside to be directionally converted into high-activity monomeric compounds of the acteoside, and obtaining powder or dry products abundant in the acteoside by carrying out the processes of concentration and drying on the herba cistanches extract undergoing catalytic reaction, wherein, by adjusting the conditions of the separation and purification processes, the percentage content of phenylethanoid glycosides in the obtained herba cistanches extract (calculated by dry substances) can reach 54.3-93%. The percentages content of the acteoside in the obtained powder or dry products can reach 38.6-89.2%.
Description
Technical field
The present invention relates to a kind of is raw material with the fresh herba cistanches, carries out the method that catalyzed conversion is produced mullein glucoside monomeric compound through immobilized enzyme.
Background technology
Herba Cistanches (Herba Cistanches) is famous traditional tonic medicine, effects such as having beneficial vital essence, tonify the kidney and support yang and delay senility.Analysis revealed, the main active ingredient of Herba Cistanches are phenylethyl alcohol glycoside, phenylcarbinol glycoside, iridoid glycosides, lignanoid's glycoside, oligosaccharides verivate, D-N.F,USP MANNITOL and trimethyl-glycine etc.Phenylethyl alcohol glycoside (Phenylethanoid Glycosides; Ph Gs) comprises echinacoside (Echinacoside), verbascoside (Acteoside) isoreactivity material; Discover through bacterium metabolism in the intestines; Benzyl carbinol glycosides staple echinacoside moving process need micro bioenzyme catalysis in large intestine in gi tract is converted into the metabolism passage of verbascoside, is to cause oral artifact availability and curative effect to have the principal element of significant difference.Mullein glucoside monomeric compound has small molecules and low polar characteristic, in drug absorption, shows good specific absorption and special pharmacologically active.
Summary of the invention
The purpose of this invention is to provide a kind of fresh herba cistanches raw material that utilizes, carry out the method that catalyzed conversion is produced mullein glucoside monomeric compound through system process processing and industrial immobilized enzyme reactor.
Method of the present invention is, with the fresh herba cistanches chylocaulous, goes out through high temperature that enzyme, cytoclasis, slurries extract, process for separating and purifying is handled, and obtains the Herba Cistanches extracting solution; Again with the Herba Cistanches extracting solution as the catalyzed reaction substrate; With beta-glucosidase industrial enzyme preparation immobilized enzyme as biological catalyst; Carry out catalyzed reaction, echinacoside makes the terminal fracture of glucone part, orientation be converted into highly active mullein glucoside monomeric compound in the catalytic hydrolysis substrate; The catalytic reaction process temperature is 38 ℃～60 ℃, and pH is 4.0～5.8; Time is 4h～8h; Beta-glucoside enzyme activity in the catalytic reaction process reactant is 20U/g～1000U/g; Herba Cistanches extracting solution that will be after catalyzed reaction through concentrating and drying process, obtains to be rich in the meal or the dry thing of verbascoside composition again.
The concrete steps of the inventive method comprise:
1, selects the fresh herba cistanches chylocaulous, carry out the high temperature enzyme that goes out, place the broken milling device of common industry that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells; Adopt common industrial pressure filter press filtration or industrial centrifugal machine to separate then, obtain Herba Cistanches cell slurries; Can be again with filter residue through once adding the technical pure water purification to (being generally 1～3 time) for several times; Once to further being milled into soup compound through common industrial broken runner milling for several times; Also can adopt common industrial ultrasonic extraction device that smudge cells is further extracted simultaneously, further obtain Herba Cistanches cell slurries through industrial pressure filter press filtration or industrial centrifugal machine separation again;
2, with the above-mentioned Herba Cistanches cell slurries that obtain, adopt common industrial ultra-filtration membrane to separate, macromole impurity such as removing polysaccharide, protein and impurity such as particulate and submicron are sieved and held back to selectivity; Adopt common industrial nanofiltration membrane separation again, small molecular weight impurities such as removing amino acid, D-N.F,USP MANNITOL, trimethyl-glycine is sieved and held back to selectivity; Also can adopt common industrial macroporous resin adsorption separating technology again; Or/and adopt common industrial chromatography column separating purification technology; Or/and adopt common separation system of simulated moving bed chromatography or the further separation and purification of common multi-functional chromatogram separating technology, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through adjusting above process for separating and purifying condition, (in dry-matter) benzyl carbinol glycosides constituents degree can reach 54.3%～93% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt the absorption method fixed form in the common immobilized enzyme industrial production; The beta-glucosidase industrial enzyme preparation immobilized enzyme that obtains is as biological catalyst; Carry out discontinuous or continous way catalyzed reaction through common industrial immobilized enzyme reactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 38 ℃～60 ℃, and pH is 4.0～5.8, and the time is 4h～8h, and the beta-glucoside enzyme activity in the catalytic reaction process reactant is 20U/g～1000U/g; Through measuring, the transformation efficiency that echinacoside is converted into verbascoside in the substrate reaches 78.5%～96%;
4, will be through the Herba Cistanches extracting solution after step 3 catalyzed reaction, through common industrial vacuum concentration technology or industrial film evaporating and concentrating process, the acquisition solid content is 30%～50% enriched material; Through industrial drying process with atomizing or other drying meanss, obtain to be rich in the meal or the dry thing of mullein glucoside monomeric compound composition again.Hair stamen glucosides percentage composition content can reach 38.6%～89.2% in the meal of results or the dry thing.
In the inventive method, catalytic reaction process can add K again
+, Na
+, Mn
2+Or Co
2+To promote the activity of enzyme, the activator of interpolation concentration in initial reactant is 0.1mol/L～2.5mol/L as the activator of catalyzed reaction, and optimum concn is 0.5mol/L～1.5mol/L.
The fresh herba cistanches chylocaulous that the inventive method relates to; Can be the fresh herba cistanches chylocaulous of new results (without refrigeration or freeze preservation), or place the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber or place the quick-frozen Herba Cistanches chylocaulous of industrial freezer refrigeration; Or the section of above-mentioned fresh herba cistanches chylocaulous and particle.Herba Cistanches of the present invention is to meet orobanchaceae plant cistanche Cistanche deserticola Y.C.Ma or Cistanche Tubulosa Cistanche tubulosa (Schrenk) Wight that Pharmacopoeia of the People's Republic of China version in 2005 is recorded.
It is 1 ℃～13 ℃ that the fresh-keeping Ku Wen of industrial refrigerator chamber that the inventive method relates to is generally, and the Ku Wen of industrial freezer refrigeration is generally and is-52 ℃～-10 ℃.Each amount that adds the technical pure water purification is 100%～400% (weight ratio) of Herba Cistanches smudge cells or filter residue.
The go out temperature of enzyme of the high temperature that the inventive method relates to is 85 ℃～130 ℃, and the time is 5min～10min.
The relative molecular mass of the ultra-filtration membrane that the inventive method relates to is 1000～4500, and the nf membrane relative molecular mass is 200～300; Ultra-filtration membrane is selected the series membranes model of CA or CTA, PAN, PS, PSA, PES, PVDF, PEK, SPS for use; Nf membrane is selected the series membranes model of 4040-UHT-ESNA or 8540-UHY-ESNA, ESNA-FREE650, ESNA-FREE1700, NTR7450HG, NTR729HG, NF-CA film, NF-CA wound element and tubular fibre spare for use; Macroporous adsorbent resin is selected XAD for use; Diaion; SP; Posapak and Chromosorb and non-ionic type polymeric sorbent AB-8; CHA-III; CAD-40; D101; D301; D296; D396R; D4006; D4020; D3520; DA201; DM301; D130; GDX104; HPD100; HPD450; HPD500; HPD600; HPD8; H107; JD-KW; LD601; LD605; ME-1; ME-2; ME-3; NKA-2; NKA-9; R-A; S8; SIP; WLD and X-5 type serial model No.; Separation system of simulated moving bed chromatography and multi-functional chromatogram separation system can select for use MB, MD, ME, MF preparative scale chromatography to separate the chromatographic fractionation system of chromatograph, SMBC experiment type, SMBC pilot scale type, XZ12E-4L type, XZ20Z-2L type and the continuous preparative hplc type of SMBC industriallization.The ultra-filtration membrane that the present invention relates to, nf membrane, macroporous adsorbent resin, non-ionic type polymeric sorbent, industrial chromatography column, moving bed chromatographic fractionation system, multi-functional chromatogram separation system can be selected usually external and domestic goods for use.
Absorption method fixed form in the industrial production that the inventive method relates to obtains immobilized enzyme; Normally beta-glucosidase industrial enzyme preparation enzyme liquid is contacted with sorbent material; Remove non-adsorbable enzyme liquid through washing again and just can make immobilized enzyme, comprise physisorphtion, ionic adsorption method; Sorbent material comprises micropore glass, Win 40350, cellophane, macroporous type synthetic resins, special cermacis, DEAE-Mierocrystalline cellulose, DEAE-polydextran gel, Amberlite IRA-93, IRA-410, IRA-900, Dowex-50, Amberlite CG-50, IRC-50, IR-120, can select usually external and domestic goods for use.
The beta-glucosidase industrial enzyme preparation that the inventive method relates to comprises beta-glucosidase and is rich in cellulase, plant extract prozyme, the plant hydrolyzed prozyme of beta-glucosidase.
The industrial immobilized enzyme reactor that the inventive method relates to can be common industrial stirred tank, immobilization bed bioreactor or fluidized-bed reactor.
The industrial vacuum concentration technology that the inventive method relates to or the temperature of industrial film evaporating and concentrating process are 42 ℃～80 ℃; The drying tower EAT of industry drying process with atomizing is 125 ℃～285 ℃.
The method of other industrially drying that the inventive method relates to comprises common industrial roller drying, air stream drying, fluidised bed drying, microwave vacuum drying, microwave drying, vacuum-drying or heat-wind circulate drying.
Meal that is rich in the mullein glucoside monomeric compound composition or dry thing that the inventive method is produced; Can directly be used for medical material; Or, also can be used as raw material or functional health-care foods such as compound raw material production oral liquid, capsule, tablet, granule, electuary, pill or teabag as cosmetic material.The promotion and implementation of the inventive method are with the health that this famous and precious Chinese medicine of Herba Cistanches is brought benefit to the mankind better.
The per-cent of the various amounts that relate in the inventive method (%) except other has explanation, all is weight percentage.
Embodiment
Below in conjunction with embodiment the present invention is done further explain.
Embodiment one:
1, select that to place the storehouse temperature be 4 ℃ the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber, carry out the high temperature enzyme that goes out through the industrial microwave heating unit, temperature is 85 ℃, and the time is 5min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 200% of a smudge cells weight; Adopt industrial pressure filter press filtration then, obtain Herba Cistanches cell slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 3000 ultra-filtration membrane separates; Adopting relative molecular mass again is that 250 nf membrane is carried out nanofiltration; Adopt the industrial macroporous adsorbent resin of AB-8 type to carry out fractionation by adsorption then; And through industrial chromatography column separating purification technology, and adopt the multi-functional chromatogram separation system of XZ12E-4L type further to separate purification, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 93% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt physisorphtion Win 40350 in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common immobilization bed bioreactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; Be added in that concentration is the Na of 2.5mol/L in the initial reactant
+As the activator of catalyzed reaction, the catalytic reaction process temperature is 47 ℃, and pH is 4.9, and every batch of catalyzed reaction time is 5.5h, carries out 5 batches of catalyzed reactions continuously; Beta-glucoside enzyme activity in the catalytic reaction process reactant is 45U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 96%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial vacuum concentration technology, temperature is 60 ℃, the acquisition solid content is 40% enriched material; Further through the industrial vacuum drying process, drying temperature is 58 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 89.2%.
Embodiment two:
1, select that to place the storehouse temperature be 10 ℃ the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 130 ℃, and the time is 10min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 150% of a smudge cells weight; Adopt industrial centrifugal machine to separate then, obtain Herba Cistanches cell slurries; And again filter residue is added the technical pure water purification; The add-on of water is 200% of a filter residue weight; Further be milled into soup compound through common industrial broken runner milling, and adopt the ultrasonic extraction device that smudge cells is extracted, separate through industrial centrifugal machine again and further obtain Herba Cistanches cell slurries;
2, with the above-mentioned Herba Cistanches cell slurries that obtain; The employing relative molecular mass is 3500 ultra-filtration membrane separation; Adopting relative molecular mass again is that 300 nf membrane is carried out nanofiltration, adopts the industrial macroporous adsorbent resin of XAD type to carry out fractionation by adsorption then, and through industrial chromatography column separating purification technology; And the multi-functional chromatogram separation system of XZ20Z-ZL type further separates purification, obtains being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 91.3% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt physisorphtion cellophane in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common industrial immobilization reactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; Be added in that concentration is the Mn of 0.1mol/L in the initial reactant
2+Activator as catalyzed reaction; The catalytic reaction process temperature is 43 ℃, and pH is 4.6, and every batch of catalyzed reaction time is 6.0h, carries out 10 batches of catalyzed reactions continuously; Beta-glucoside enzyme activity in the catalytic reaction process reactant is 100U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 93.2%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial film evaporating and concentrating process, temperature is 55 ℃, the acquisition solid content is 43% enriched material; Further through industrial roller drying technology, drying temperature is 55 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 88.9%.
Embodiment three:
1, select and place the fresh herba cistanches chylocaulous of storehouse temperature for-32 ℃ industrial freezer refrigeration, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 125 ℃, and the time is 10min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 300% of a smudge cells weight; Adopt industrial pressure filter press filtration then, obtain Herba Cistanches cell slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 4500 ultra-filtration membrane separates; Adopting relative molecular mass again is that 250 nf membrane is carried out nanofiltration; Adopt the industrial macroporous adsorbent resin of NKA-2 type to carry out fractionation by adsorption then; And further separate purification through industrial chromatography column separating purification technology, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 76.5% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt physisorphtion macroporous type synthetic resins in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common industrial fluidized bed reactor drum, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 42 ℃, and pH is 4.9, and every batch of catalyzed reaction time is 8h, carries out 8 batches of catalyzed reactions continuously; The beta-glucoside enzyme activity that catalyzed reaction is crossed in the reactant is 80U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 88.5%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial vacuum concentration technology, temperature is 60 ℃, the acquisition solid content is 42% enriched material; Further through industrial drying process with atomizing, the drying tower EAT is 185 ℃, obtains to be rich in the meal of mullein glucoside monomeric compound composition; Through assay determination, in the meal of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 73.1%.
Embodiment four:
1, select that to place the storehouse temperature be 13 ℃ the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 130 ℃, and the time is 9min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 100% of a smudge cells weight; Adopt industrial pressure filter press filtration to separate then, obtain Herba Cistanches cell slurries; And respectively the filter residue warp being added the technical pure water purification 2 times again, the add-on of each water is 100% of a filter residue weight, further is milled into soup compound through common industrial broken runner milling again, further obtains Herba Cistanches cell slurries through industrial pressure filter press filtration separation again;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 4000 ultra-filtration membrane separates; Adopting relative molecular mass again is that 300 nf membrane is carried out nanofiltration; Adopt the industrial macroporous adsorbent resin of CAD-40 type to carry out fractionation by adsorption then; And further separate purification through industrial chromatography column separating purification technology, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 79.1% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt ionic adsorption method DEAE-polydextran gel in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common industrial stirred tank, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 47 ℃, and pH is 5.0, and every batch of catalyzed reaction time is 5.0h, carries out 13 batches of catalyzed reactions continuously; The beta-glucoside enzyme activity that catalyzed reaction is crossed in the reactant is 100U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 90.1%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial film evaporating and concentrating process, temperature is 60 ℃, the acquisition solid content is 48% enriched material; Further through industrial roller drying technology, drying temperature is 55 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 70.1%.
Embodiment five:
1, select and place the fresh herba cistanches chylocaulous of storehouse temperature for-18 ℃ industrial freezer refrigeration, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 130 ℃, and the time is 10min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 400% of a smudge cells weight; Adopt industrial pressure filter press filtration then, obtain Herba Cistanches cell slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 1000 ultra-filtration membrane separates; Adopting relative molecular mass again is that 250 nf membrane is carried out nanofiltration; Adopt the industrial macroporous adsorbent resin of DM301 type to carry out fractionation by adsorption then; And further separate purification through industrial chromatography column separating purification technology, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 78.9% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt ionic adsorption method IRC-50 in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common industrial stirred tank, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 45 ℃, and pH is 4.2, and every batch of catalyzed reaction time is 7h, carries out 8 batches of catalyzed reactions continuously, and the beta-glucoside enzyme activity that catalyzed reaction is crossed in the reactant is 120U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 95%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial vacuum concentration technology, temperature is 56 ℃, the acquisition solid content is 30% enriched material; Further through the industrial vacuum drying process, drying temperature is 56 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 72.3%.
Embodiment six:
1, select that to place the storehouse temperature be 10 ℃ the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 130 ℃, and the time is 10min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 250% of a smudge cells weight; Adopt industrial pressure filter press filtration then, obtain Herba Cistanches cell slurries; And again filter residue being added the technical pure water purification, the add-on of water is 200% of a filter residue weight, further is milled into soup compound through common industrial broken runner milling, separates through industrial pressure filter press filtration and further obtains Herba Cistanches smudge cells slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 1500 hollow fiber ultrafiltration membrane is separated; Adopting relative molecular mass again is that 200 nf membrane is carried out nanofiltration; Adopt the industrial macroporous adsorbent resin of X-5 type to carry out fractionation by adsorption then; And further separate purification through industrial chromatography column separating purification technology, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 82.3% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt ionic adsorption method IRC-120 in the common industrial production as the fixed form of sorbent material; Obtain plant hydrolyzed complex enzyme immobilization enzyme as biological catalyst; Carry out the discontinuous catalyzed reaction through common industrial stirred tank, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 45 ℃, and pH is 4.9, and the catalyzed reaction time is 6h; The beta-glucoside enzyme activity that catalyzed reaction is crossed in the reactant is 400U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 88.6%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial film evaporating and concentrating process, temperature is 70 ℃, the acquisition solid content is 38% enriched material; Further through industrial roller drying technology, drying temperature is 60 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 71.2%.
Embodiment seven:
1, select the Herba Cistanches particle, carry out the high temperature enzyme that goes out through the industrial microwave heating unit, temperature is 95 ℃, and the time is 6min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 200% of a smudge cells weight; Then Herba Cistanches cell soup compound is adopted industrial pressure filter press filtration, obtain Herba Cistanches cell slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 3000 ultra-filtration membrane separates; Adopting relative molecular mass again is that 250 nf membrane is carried out nanofiltration; And further separate purification through industrial chromatography separation purifying technique, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 83.1% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt physisorphtion Win 40350 in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common immobilization bed bioreactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 52 ℃, and pH is 5.8, and every batch of catalyzed reaction time is 8h, carries out 12 batches of catalyzed reactions continuously, and the beta-glucoside enzyme activity in the catalytic reaction process reactant is 1000U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 85.6%;
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial vacuum concentration technology, temperature is 60 ℃, the acquisition solid content is 50% enriched material; Further through the industrial vacuum drying process, drying temperature is 58 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 78.1%.
Embodiment eight:
1, select that to place the storehouse temperature be 8 ℃ the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 128 ℃, and the time is 10min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells, the add-on of water is 100% of a smudge cells weight; Adopt industrial centrifugal machine to separate then, obtain Herba Cistanches cell slurries; And respectively the filter residue warp being added the technical pure water purification 2 times again, the add-on of each water is 150% of a filter residue weight, further is milled into soup compound through common industrial broken runner milling, further obtains Herba Cistanches smudge cells slurries through the industrial centrifugal machine separation again;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 3000 ultra-filtration membrane separates; Adopting relative molecular mass again is that 300 nf membrane is carried out nanofiltration; Adopt the industrial macroporous adsorbent resin of R-A type to carry out fractionation by adsorption then, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 61.9% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt ionic adsorption method DEAE-Mierocrystalline cellulose in the common industrial production as the fixed form of sorbent material; Obtain the cellulase immobilization enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common fluidized-bed reactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 50 ℃, and pH is 5.0, and every batch of catalyzed reaction time is 4h, carries out 7 batches of catalyzed reactions continuously; Beta-glucoside enzyme activity in the catalytic reaction process reactant is 10U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 80.2%
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial film evaporating and concentrating process, temperature is 61 ℃, the acquisition solid content is 38% enriched material; Further through industrial roller drying technology, drying temperature is 65 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 52.1%.
Embodiment nine:
1, adopt fresh Herba Cistanches chylocaulous, carry out the high temperature enzyme that goes out through industrial heating unit, temperature is 130 ℃, and the time is 10min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, the add-on that adds technical pure water washing smudge cells water is 250% of a filter residue weight; Then Herba Cistanches cell soup compound is adopted industrial pressure filter press filtration, obtain Herba Cistanches cell slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 3500 ultra-filtration membrane separates, and adopting relative molecular mass again is that 250 nf membrane is carried out nanofiltration, obtains being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 54.3% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt ionic adsorption method DEAE-polydextran gel in the common industrial production as the fixed form of sorbent material; Obtain the beta-glucosidase immobilized enzyme as biological catalyst; Carry out the continous way catalyzed reaction through common immobilization bed bioreactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 60 ℃, and pH is 4.6, and every batch of catalyzed reaction time is 3.5h, carries out 12 batches of catalyzed reactions continuously, and the beta-glucoside enzyme activity in the catalytic reaction process reactant is 24U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 78.5%
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial vacuum concentration technology, temperature is 60 ℃, the acquisition solid content is 38% enriched material; Further through industrial drying process with atomizing, the drying tower EAT is 185 ℃, obtains to be rich in the meal of mullein glucoside monomeric compound composition; Through assay determination, in the meal of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 38.6%.
Embodiment ten:
1, select that to place the storehouse temperature be 13 ℃ the fresh-keeping fresh herba cistanches chylocaulous of industrial refrigerator chamber, through the industrial high temperature device enzyme that goes out, temperature is 125 ℃, and the time is 9min; Place industrial broken milling device that the fragmentation of Herba Cistanches chylocaulous is milled then, the add-on that adds technical pure water washing smudge cells water is 100% of a filter residue weight; Adopt industrial centrifugal machine to separate then, obtain Herba Cistanches cell slurries; And again filter residue being added the technical pure water purification, the add-on of water is 400% of a filter residue weight, further is milled into soup compound through common industrial broken runner milling, separates through industrial centrifugal machine again, further obtains Herba Cistanches smudge cells slurries;
2, the above-mentioned Herba Cistanches cell slurries that obtain being adopted relative molecular mass is that 1000 ultra-filtration membrane separates; Adopting the SP macroporous resin adsorption to separate again purifies; And be that 200 nf membrane is carried out nanofiltration through relative molecular mass, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents; Through detecting, (in dry-matter) benzyl carbinol glycosides constituents degree is 55.2% in the Herba Cistanches extracting solution of results;
3, with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt ionic adsorption method IR-120 in the common industrial production as the fixed form of sorbent material; Obtain plant extract complex enzyme immobilization enzyme as biological catalyst; Carry out the discontinuous catalyzed reaction through common industrial stirred tank, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 38 ℃, and pH is 4.0, and the catalyzed reaction time is 6h, carries out 12 batches of catalyzed reactions continuously, and the beta-glucoside enzyme activity in the catalytic reaction process reactant is 32U/g; Through detecting, the transformation efficiency that echinacoside is converted into mullein glucoside monomeric compound in the substrate reaches 79.2%
4, will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through the industrial film evaporating and concentrating process, temperature is 49 ℃, the acquisition solid content is 43% enriched material; Further through industrial roller drying technology, drying temperature is 65 ℃, obtains to be rich in the dry thing of mullein glucoside monomeric compound composition; Through assay determination, in the dry thing of being gathered in the crops (in dry-matter), the degree of mullein glucoside monomeric compound is 46.3%.
Claims (11)
1. method of utilizing fresh herba cistanches to produce verbascoside with the fresh herba cistanches chylocaulous, goes out through high temperature that enzyme, cytoclasis, slurries extract, process for separating and purifying is handled, and obtains the Herba Cistanches extracting solution; Again with the Herba Cistanches extracting solution as the catalyzed reaction substrate; As biological catalyst, echinacoside makes the terminal fracture of glucone part, orientation be converted into highly active mullein glucoside monomeric compound in the catalytic hydrolysis substrate with beta-glucosidase industrial enzyme preparation immobilized enzyme; The catalytic reaction process temperature is 38 ℃～60 ℃, and pH is 4.0～5.8; Time is 4h～8h, and the beta-glucoside enzyme activity in the catalytic reaction process reactant is 20U/g～1000U/g; Herba Cistanches extracting solution that will be after catalyzed reaction through concentrating and drying process, obtains to be rich in the meal or the dry thing of verbascoside composition again.
2. according to the described method of claim 1, it is characterized in that the concrete steps of this method comprise:
(1) selects the fresh herba cistanches chylocaulous,, place the broken milling device of common industry that the fragmentation of Herba Cistanches chylocaulous is milled then, add technical pure water washing smudge cells through carrying out the high temperature enzyme that goes out; Adopt common industrial pressure filter press filtration or industrial centrifugal machine to separate then, obtain Herba Cistanches cell slurries;
(2) with the above-mentioned Herba Cistanches cell slurries that obtain, adopt common industrial ultra-filtration membrane to separate, adopt common industrial nanofiltration membrane separation again, obtain being rich in the Herba Cistanches extracting solution of benzyl carbinol glycosides constituents;
(3) with the above-mentioned Herba Cistanches extracting solution that obtains as the catalyzed reaction substrate; Adopt the absorption method fixed form in the common immobilized enzyme industrial production; The beta-glucosidase industrial enzyme preparation immobilized enzyme that obtains is as biological catalyst; Carry out catalyzed reaction through common industrial immobilized enzyme reactor, glucone, an orientation that catalytic hydrolysis removes echinacoside glucone part end in the substrate are converted into mullein glucoside monomeric compound; The catalytic reaction process temperature is 38 ℃～60 ℃, and pH is 4.0～5.8, and the time is 4h～8h, and the beta-glucoside enzyme activity in the catalytic reaction process reactant is 20U/g～1000U/g;
(4) will be through the Herba Cistanches extracting solution after the above-mentioned catalyzed reaction, through common industrial vacuum concentration technology or industrial film evaporating and concentrating process, the acquisition solid content is 30%～50% enriched material; Through industrial drying process with atomizing or other drying meanss, obtain to be rich in the meal or the dry thing of mullein glucoside monomeric compound composition again.
3. according to claim 1 or 2 described methods, it is characterized in that in catalytic reaction process, adding K
+, Na
+, Mn
2+Or Co
2+As the activator of catalyzed reaction, the activator of interpolation concentration in initial reactant is 0.1mol/L～2.5mol/L.
4. according to the described method of claim 3, it is characterized in that the K that in catalytic reaction process, adds
+, Na
+, Mn
2+Or Co
2+Activator concentration in initial reactant is 0.5mol/L～1.5mol/L.
5. according to claim 1 or 2 described methods, it is characterized in that the go out temperature of enzyme of described high temperature is 85 ℃～130 ℃, the time is 5min～10min.
6. according to the described method of claim 2, it is characterized in that the amount of water of the adding technical pure water washing smudge cells described in the step (1) is 100%～400% of a Herba Cistanches smudge cells weight.
7. according to the described method of claim 2; It is characterized in that the common industrial pressure filter press filtration of the employing described in the step (1) or industrial centrifugal machine separate obtain Herba Cistanches cell slurries after; Again the filter residue warp is once extremely added for several times the technical pure water purification; Each amount that adds the technical pure water purification is 100%～400% of a filter residue weight; Once further be milled into soup compound through common industrial broken runner milling extremely for several times, perhaps simultaneously adopt common industrial ultrasonic extraction device that smudge cells is extracted again, further obtain Herba Cistanches cell slurries through industrial pressure filter press filtration or industrial centrifugal machine separation again.
8. according to the described method of claim 2, the relative molecular mass that it is characterized in that the ultra-filtration membrane described in the step (2) is 1000～4500, and the relative molecular mass of nf membrane is 200～300.
9. according to the described method of claim 2; It is characterized in that adopting again in the step (2) common industrial macroporous resin adsorption separating technology; Or/and adopt common industrial chromatography column separating purification technology; Or/and adopt common separation system of simulated moving bed chromatography technology or common multi-functional chromatogram separating technology, further separation and purification.
10. according to claim 1 or 2 described methods, it is characterized in that described beta-glucosidase industrial enzyme preparation, comprise beta-glucosidase or/and cellulase.
11. according to the described method of claim 2, the temperature that it is characterized in that industrial vacuum concentration technology described in the step (4) or industrial film evaporating and concentrating process is 42 ℃～80 ℃; The drying tower EAT of industry drying process with atomizing is 125 ℃～285 ℃.
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| CN101214281A (en) * | 2007-12-28 | 2008-07-09 | 中山大学 | Method for producing cistanche salsa extract |
| CN101214282A (en) * | 2007-12-28 | 2008-07-09 | 中山大学 | Method for extracting active component from cistanche salsa |
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| CN101214282A (en) * | 2007-12-28 | 2008-07-09 | 中山大学 | Method for extracting active component from cistanche salsa |
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| Title |
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| 蔡鸿等.鲜管花肉苁蓉加工工艺.《中国中药杂志》.2007,第32卷(第13期),1289-1291. * |
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