A Method Of Producing Mullein By Biocatalytic Transformation Of Fresh Cistanche
Jun 27, 2023
Abstract
The present invention proposes a method for producing mullein by the biocatalytic transformation of fresh Cistanche. The method of the present invention uses fresh Cistanches as raw material, through the system process of cell crushing, slurry extraction and high-temperature enzyme inactivation process, the inactivated Cistanches cell slurry is used as the catalytic reaction substrate, and the β-glucosidase industrial enzyme immobilized enzyme is used as the biocatalyst to carry out intermittent or continuous catalytic reaction to catalyze the hydrolysis of pineal glucoside in the substrate to make it Then, through the separation and purification process and concentration and drying process, the mullein monomer compound is harvested; by adjusting the conditions of the separation and purification process, the purity percentage of the harvested mullein monomer compound can reach 50%-90%. The monomeric compound of mullein has the characteristics of a small molecule and low polarity, which exhibits a good absorption rate and special pharmacological activity in drug absorption.
Click Here To Get 8-16% Acteoside Cistanche Extract
Description
A method of producing mullein by biocatalytic transformation of fresh Cistanches
Technical Field
The present invention relates to a method of producing mullein monomeric compounds by biocatalytic transformation of fresh Cistanches.
Background Technology
Herba Cistanches, a perennial parasitic herb, is a famous tonic Chinese medicine with the effects of benefiting essence, tonifying the kidneys, and delaying aging. Modern analysis shows that the main active ingredients of Herba Cistanches are phenylethanol glycosides, benzyl alcohol glycosides, cyclic enol ether terpene glycosides, lignan glycosides, oligosaccharide derivatives, D-mannitol and betaine. Phenylethanoid Glycosides (Ph Gs) include active substances such as Echinacoside and Acteoside, and it was found through intestinal bacterial metabolism that the movement process of Phenylethanoid Glycosides in the gastrointestinal tract needs to be catalyzed by microbial enzymes in the large intestine to convert into Acteoside. The metabolic channel of pineal glycoside in the gastrointestinal tract is the main factor leading to the significant difference in bioavailability and efficacy after oral administration. The monomeric compound of mullein has the characteristics of small molecule and low polarity, demonstrating good absorption rate and exceptional pharmacological activity in drug absorption.
Content of the Invention
The purpose of the present invention is to provide a method for the production of mullein glycoside monomeric compounds from fresh Cistanches as raw material by systematic process treatment and catalytic conversion in an industrial immobilized enzyme reactor.
The method of the present invention is to use fresh Cistanche as raw material, treat it by cell crushing, slurry extraction process and high-temperature enzyme inactivation process, use the inactivated Cistanche cell slurry as the catalytic reaction substrate, immobilize the enzyme by β-glucosidase industrial enzyme preparation as a biocatalyst, catalyze the hydrolysis of pineal glucoside in the substrate to break the end of glucosyl ligand and convert it to highly active marocain monomer Then, through the separation and purification process, and the concentration and drying process, the mullein monomer compound was harvested; the catalytic reaction process temperature was 38℃～60℃, pH was 4.0～5.8, time was 4h～8h, and the β-glucosidase activity in the reaction product was 2U/g～1000U/g; by adjusting the separation and purification process conditions, the harvested mullein monomer compound The percentage purity of the harvested mullein monomers could reach 50%-90%.
The specific steps of the method of the present invention include:
1. selecting fresh fleshy stems of cistanche tubulosa, placing them in a washing tank and rinsing them with cold water or industrial pure water; picking them up, placing them in a usual industrial crushing and grinding device to crush and grind the fleshy stems of cistanche tubulosa, adding industrial pure water or pre-chilled industrial pure water to wash the broken cells to become cistanche tubulosa crushed cell pulp; then using a usual industrial filter press to press and filter or an industrial centrifuge to separate them and obtain cistanche tubulosa cell pulp; may then The filter residue can be added to industrial pure water or pre-cooled industrial pure water once or several times (generally 1 to 3 times) and further ground into pulp by the usual industrial crushing and grinding machine, and the crushed cells can be extracted by the usual industrial ultrasonic extraction device at the same time, and then separated by the industrial filter press or industrial centrifuge to obtain cistanche tubulosa cell pulp. process cistanche tubulosa crushed cell pulp and cell pulp are controlled at a low temperature below 20℃ (usually 4-20℃);
2, the cistanche cell pulp obtained from step 1 is inactivated at high temperature by usual industrial heating device, or industrial ultra-high temperature instantaneous inactivation is carried out; the inactivated cistanche cell pulp is used as the catalytic reaction substrate, and the β-glucosidase industrial enzyme preparation immobilized enzyme obtained by the usual immobilized enzyme industrial production of adsorption method is used as the biocatalyst, and the reaction is carried out by the usual industrial immobilized enzyme reactor Intermittent or continuous catalytic reaction, catalytic hydrolysis to remove a glucose group at the end of the glucosyl ligand of the substrate pineal glucoside, directional conversion to mullein monomer compound; catalytic reaction process temperature is 38 ℃ ~ 60 ℃, the best temperature is 42 ℃ ~ 48 ℃; pH is 4.0 ~ 5.8, the best pH is 4.5 ~ 5.3; time is 4h ~ 8h, the best time is 5h ~ 6h. The β-glucosidase activity of the reaction product in the catalytic reaction process was 2U/g～1000U/g, and the best enzyme activity was 25U/g～150U/g; the conversion rate of pineal glucoside into myricetin monomer compound in the reaction product in the catalytic reaction process was measured to be 78%～95%
3, the reaction solution after the reaction of step 2, using the usual industrial ultrafiltration membrane separation, selective sieving and retention of polysaccharides, proteins and other large molecule impurities, as well as particulate and sub-particulate impurities; and then using the usual industrial nanofiltration membrane separation, selective sieving and retention of amino acids, D-mannitol, betaine and other small molecule impurities; can also be used again using the usual industrial macroporous resin adsorption separation process, or / and using the usual industrial chromatographic column separation and purification process, or/and the usual analog moving bed chromatography separation system process or the usual multifunctional chromatography separation process for further separation and purification; by adjusting the above separation and purification process conditions, different purity extracts of mullein monomer compounds can be obtained; and
4, the extract of mullein monomer compound obtained in step 3, through the usual industrial vacuum concentration process or industrial thin film evaporation concentration process, to obtain a solids content of 30% to 50% of the concentrate; and then through the industrial spray drying process, or the usual other industrial drying methods, to obtain the powder or dry matter of mullein monomer compound. The percentage purity content of the harvested mullein monomer compounds can reach 50% to 90%.
The fresh Cistanche raw material involved in the method of the present invention can be freshly harvested (not refrigerated or frozen and preserved) fleshy stems of Cistanche, or fresh fleshy stems of Cistanche preserved in industrial freezers, or fresh fleshy stems of Cistanchefrozen in industrial freezers; or sliced or shredded fleshy stems of fresh Cistanche as mentioned above.
The Cistanche described in the present invention is cistanche tubulosa Y.C. Ma or Cistanche tubulosa (Schrenk) Wight of the family Lietangidae, which is included in the 2005 edition of the Pharmacopoeia of the People's Republic of China.
The method of the invention involves the use of industrial cold storage preservation of the reservoir temperature is generally 1 ℃ ~ 13 ℃, the best cold storage preservation of the reservoir temperature is 4 ℃ ~ 10 ℃; the use of industrial freezer refrigeration of the reservoir temperature is generally -52 ℃ ~ -10 ℃, the best freezer refrigeration of the reservoir temperature is -36 ℃ ~ -18 ℃; the use of cold water placed in the water tank water temperature is 1 ℃ ~ 20 ℃, the best cold water temperature is 4 ℃ ~ 10 ℃ The amount of industrial pure water or industrial pure water added by pre-cooling each time is 100% to 400% (weight ratio) of the crushed cells or filter residue of cistanche tubulosa, and the best addition is 200% to 300% (weight ratio); the water temperature of industrial pure water by pre-cooling is 1°C to 20°C, and the best water temperature is 4°C to 10°C.
The method of the invention involves the industrial production of adsorption method to obtain immobilized enzyme, usually β-glucosidase industrial enzyme preparation enzyme solution and adsorbent contact, and then wash to remove the non-adsorbed enzyme solution can be made immobilized enzyme; including physical adsorption method, ion adsorption method; adsorbent including microporous glass, hydroxyapatite, cellophane, macroporous synthetic resin, special ceramics, DEAE - cellulose , DEAE-dextran gel, Amberlite IRA-93, IRA-410, IRA-900, Dowex-50, Amberlite CG-50, IRC-50, IR-120, which can be selected from usual foreign and domestic commodities.
The method of the present invention involves β-glucosidase industrial enzyme preparation, including β-glucosidase and cellulase enriched with β-glucosidase, plant extract compound enzyme, plant hydrolysis compound enzyme.
The method of the present invention involves high-temperature inactivation of enzyme generally from 75℃ to 100℃, and the time is generally from 3min to 10min; the temperature of ultra-high temperature instantaneous inactivation of enzyme is generally from 135℃ to 141℃.
The industrial immobilized enzyme reactor involved in the method of the present invention can be a usual industrial stirred tank reactor, immobilized bed reactor or fluidized bed reactor.
The relative molecular mass of the ultrafiltration membrane involved in the method of the invention is 1000 to 4500, and the best relative molecular mass is 1500 to 3500; the relative molecular mass of the nanofiltration membrane is 200 to 300, and the best relative molecular mass is 250 to 280; the ultrafiltration membrane is selected from the series of membrane models of CA or CTA, PAN, PS, PSA, PES, PVDF, PEK, SPS For nanofiltration membranes, 4040-UHT-ESNA or 8540-UHY-ESNA, ESNA-FREE650, ESNA-FREE1700, NTR7450HG, NTR729HG, NF-CA membranes, NF-CA rolled elements and hollow fiber parts are used; for macroporous adsorption resins, XAD, Diaion, SP 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 series models; simulated moving bed chromatography separation system and multifunctional chromatography separation system, you can choose MB, MD, ME, MF preparation chromatographic separation chromatograph, SMBC experimental type, SMBC pilot type, XZ12E-4L type, XZ20Z-2L type and SMBC industrial continuous preparation chromatographic type of chromatographic separation system. The ultrafiltration membrane, nanofiltration membrane, macroporous adsorption resin, non-ionic polymer adsorbent, industrial chromatographic column, moving bed chromatographic separation system and multifunctional chromatographic separation system involved in the method of the present invention can be selected from the usual foreign and domestic commodities.
The method of the invention involves the industrial vacuum concentration process or industrial film evaporation concentration process, the temperature is generally 42 ℃ ~ 80 ℃, the best temperature is 45 ℃ ~ 65 ℃; industrial spray drying process used in the drying tower inlet air temperature is generally 125 ℃ ~ 285 ℃, the best inlet air temperature is 135 ℃ ~ 185 ℃.
Other methods of industrial drying involved in the method of the present invention include the usual industrial drum drying, or airflow drying, fluidized bed drying, microwave vacuum drying, microwave drying, vacuum drying or hot air circulation drying, the drying temperature is generally 50 to 100 ℃, the best is 58 to 80 ℃.
The powdered or dried mullein monomer compound produced by the method of the present invention can be used directly as pharmaceutical raw materials, or as cosmetic raw materials, or as raw materials or compound raw materials to produce functional health food such as oral liquid, capsule, tablet, granule, punch, pill or bagged tea. The promotion and implementation of the method of this invention will make cistanche tubulosa, a valuable Chinese medicine, better benefit human health.
The percentages (%) of various amounts involved in the method of the present invention are weight percentages unless otherwise stated.
Specific Embodiments
The present invention is described in further detail below in conjunction with embodiments.
Example 1:
1, select fresh fleshy stems of cistanche tubulosa placed in industrial cold storage at a temperature of 4°C, rinse them in a water washing tank with cold water at a temperature of 18°C, pick them up, place them in an industrial crushing and grinding device to crush and grind the fleshy stems of cistanche tubulosa, add industrial pure water to wash the crushed cells to become cistanche tubulosa crushed cell pulp, the amount of water added is 300% of the weight of the crushed cells; then use an industrial filter press The temperature of cistanche tubulosa crushed cell pulp and cell pulp during the crushing and grinding and separation process was tested to be 20℃;
2, high temperature inactivation of cistanche tubulosa cell slurry by industrial microwave heating device, the temperature is 100℃, the time is 3min; the inactivated cistanche tubulosa cell slurry is used as the catalytic reaction substrate, the usual industrial production of physical adsorption method hydroxyapatite as the adsorbent fixation method to obtain β-glucosidase immobilized enzyme as a biocatalyst, through the usual immobilized bed reactor for The catalytic reaction was carried out continuously in the immobilized bed reactor to catalyze the removal of a glucose group at the end of the glucosyl ligand of the substrate pinealoside and the targeted conversion to the monomeric compound of mullein; the catalytic reaction process was carried out at a temperature of 46℃, pH of 4.9, and the catalytic reaction time was 5.5h per batch, and 10 batches of catalytic reaction were carried out continuously; the β-glucosidase activity in the reactants during the catalytic reaction process was 10U/g; by testing that the conversion of pineal glucoside to myricetin monomer compound in the substrate reached 94%;
3. The reaction solution after the above catalytic reaction was separated by ultrafiltration membrane with a relative molecular mass of 4000, and then nanofiltration by nanofiltration membrane with a relative molecular mass of 250, followed by adsorption separation by industrial macroporous adsorption resin of type AB-8, and separation and purification by industrial chromatographic column separation and purification process, and then further separation and purification by XZ12E-4L multifunctional chromatographic separation system to obtain higher purity extract of mullein monomer compound;
4, the above extract was concentrated by industrial vacuum concentration process at a temperature of 60 ℃ to obtain a concentrate with 42% solids; further dried by industrial vacuum drying process at a temperature of 58 ℃ to obtain the dried mullein monomer compound; the percentage purity content of mullein monomer compound in the harvested dried material (in dry matter) was 87.5% as determined by analysis.
Example 2:
1. select fresh fleshy stems of cistanche tubulosa placed in industrial cold storage at a storage temperature of 10°C, rinse them in a water washing tank with cold water at a temperature of 4°C, pick them up, place them in an industrial crushing and grinding device to crush and grind the fleshy stems of cistanche tubulosa, add industrial pure water pre-chilled to a water temperature of 4°C to wash the crushed cells to become cistanche tubulosa crushed cell slurry, with the amount of water added being 200% of the weight of the crushed cells The slurry was then separated by an industrial centrifuge to obtain cistanche tubulosa cell slurry; and the filter residue was added to industrial pure water, with the amount of water added being 200% of the weight of the filter residue, and further ground into a slurry by the usual industrial crushing and grinding machine, and the crushed cells were extracted by an ultrasonic extraction device, and then separated by an industrial centrifuge to further obtain cistanche tubulosa cell slurry; after testing, the crushing and grinding and separation process The temperature of cistanche tubulosa crushed cell pulp and cell pulp was 18℃;
2, high-temperature inactivation of cistanche tubulosa cell slurry by industrial heating device, the temperature is 100℃, the time is 5min; the inactivated cistanche tubulosa cell slurry is used as the catalytic reaction substrate, the usual industrial production of physical adsorption method cellophane as the adsorbent fixation method, to obtain β-glucosidase immobilized enzyme as a biocatalyst, through the usual industrial immobilization reactor for continuous type The catalytic reaction was carried out at 45℃, pH 4.5, and the catalytic reaction time was 6.0h per batch, and 12 batches of catalytic reaction were carried out continuously. The conversion of pineal glucoside into myricetin monomeric compound in the substrate was detected as 95%;
3, the reaction solution after the above catalytic reaction was nanofiltration using a nanofiltration membrane with a relative molecular mass of 300, and then separated by adsorption using an industrial macroporous adsorption resin of XAD type, and purified by an industrial chromatographic column separation and purification process, and then further separated and purified using a multifunctional chromatographic separation system of XZ20Z-ZL type to obtain a higher purity extract of mullein monomer compound;
4, the above extract was passed through an industrial film evaporation concentration process at 55 ℃ to obtain the concentrate with 45% solids content; further passed through an industrial drum drying process at 55 ℃ to obtain the dried Myristica fragrans monomer compound; after analysis and determination, the percentage purity content of Myristica fragrans monomer compound in the harvested dried material (in dry matter) was 89.9%.
Ask for more details:
Email:wallence.suen@wecistanche.com
Whatsapp/Tel:+86 15292862950
