CN112592793A - Dendrobium officinale extract fermented wine and preparation method thereof - Google Patents
Dendrobium officinale extract fermented wine and preparation method thereof Download PDFInfo
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- CN112592793A CN112592793A CN202110010042.3A CN202110010042A CN112592793A CN 112592793 A CN112592793 A CN 112592793A CN 202110010042 A CN202110010042 A CN 202110010042A CN 112592793 A CN112592793 A CN 112592793A
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- 239000000284 extract Substances 0.000 title claims abstract description 47
- 241001076416 Dendrobium tosaense Species 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000000047 product Substances 0.000 claims abstract description 51
- 239000004365 Protease Substances 0.000 claims abstract description 40
- 241001523681 Dendrobium Species 0.000 claims abstract description 31
- 108091005804 Peptidases Proteins 0.000 claims abstract description 30
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 29
- 238000000855 fermentation Methods 0.000 claims abstract description 20
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/026—Preparation of other alcoholic beverages by fermentation with health-improving ingredients, e.g. flavonoids, flavones, polyphenols or polysaccharides, added before or during the fermentation stage; with flavouring ingredients added before or during the fermentation stage
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention provides a preparation method of dendrobium officinale extract fermented wine, belonging to the technical field of beverage food and processing thereof, comprising the steps of raw material treatment, water extraction, enzymolysis and fermentation, wherein fresh dendrobium officinale is cleaned, dried, crushed and sieved; performing sealed heating leaching on the treated dendrobium with water as a solvent to obtain water extract; adding compound protease into the water extract, adjusting the pH and temperature of the water extract to adapt to the compound protease, sealing, preserving heat, performing enzymolysis, performing enzyme deactivation treatment after the enzymolysis is finished, and performing centrifugal separation on supernatant to obtain an enzymolysis solution; adjusting sugar degree and pH, sterilizing at high temperature, inoculating Saccharomyces cerevisiae, and fermenting at constant temperature to obtain herba Dendrobii extract fermented wine; according to the method, active components in the dendrobium officinale are fully dissolved out by a water extraction method and then subjected to enzymolysis and fermentation, so that the utilization rate of the dendrobium officinale and the nutritional value of a product are improved.
Description
Technical Field
The invention relates to the technical field of beverage food and processing thereof, and particularly relates to dendrobium officinale extract fermented wine and a preparation method thereof.
Background
Dendrobium officinale (Dendrobium officinale Kimura et Migo), Orchidaceae, Dendrobium, perennial herb, is one of the traditional and rare Chinese herbal medicine representatives in China, and has high medicinal value. The Shen nong Ben Cao Jing mentions that the disease is the main cause of injury; removing arthralgia, descending qi, tonifying the internal organs, strengthening yin, and nourishing the stomach after long-term use, namely promoting the production of body fluid, moistening the lung, removing heat, nourishing yin, and nourishing the stomach, is praised as "gold in medicine" and "the first of Jiudao immortal grass". Modern researches show that the dendrobium officinale mainly contains various components such as polysaccharide, phenanthrene, alkaloid, bibenzyl, phenols, volatile oil, amino acid, mineral elements and the like, and has pharmacological effects of resisting oxidation, reducing blood pressure, reducing blood sugar, resisting tumors and the like.
At present, the eating method of dendrobium officinale mainly comprises fresh eating, tea making, soup cooking, wine soaking, juice squeezing and the like. Wherein, dendrobium officinale wine is directly infused by high-concentration alcohol, has the problems of stimulating taste and the like, and is unacceptable for most consumers. The fermented wine prepared by biological fermentation has lower alcoholic strength and fresh taste, and compared with strong wine soaking, the fermented wine has simple production process, can retain more abundant nutritional ingredients, has more health-care functional value, and is deeply loved by people.
The existing research shows that the content of polysaccharide in dendrobium is generally between 20 and 30 percent, and the polysaccharide component of dendrobium has close relation with the pharmacological actions of enhancing the body immune function, resisting tumor, resisting aging, resisting fatigue, reducing blood sugar and the like. The dendrobium officinale not only has rich polysaccharide components and indirect anti-tumor effect through antioxidation or enhancement of the cellular immune function of an organism, but also has direct anti-cancer active substances, namely phenanthrene compounds and bibenzyl compounds, which have influence on the cell cycle of tumor cells and the expression of cyclin and inhibit the proliferation of the cancer cells. The dendrobium polysaccharide is easy to dissolve in water and insoluble in ethanol, so the dendrobium polysaccharide cannot be fully leached by directly soaking the dendrobium polysaccharide in wine and cannot be fully utilized; chinese patent CN108841501A discloses a brewing method of dendrobium wine, which is specifically prepared by grinding dendrobium into pulp, performing enzymolysis treatment by pectinase and cellulase, and inoculating saccharomycetes for alcohol fermentation, wherein on one hand, the pectinase and the cellulase can only singly hydrolyze a pectin layer in a cell wall, but cannot hydrolyze other proteins, and cannot fully utilize nutrient substances in dendrobium officinale; on the other hand, the polysaccharide is synchronously dissolved out during enzymolysis and fermentation, the dissolving-out condition is limited by the enzymolysis and fermentation conditions, and the dendrobium polysaccharide cannot be fully dissolved out and utilized in the dendrobium wine.
Disclosure of Invention
Aiming at the problems, the invention provides a preparation method of dendrobium officinale extract fermented wine capable of maximally extracting and retaining polysaccharides in dendrobium officinale.
The purpose of the invention is realized by adopting the following technical scheme:
a preparation method of dendrobium officinale extract fermented wine comprises the following steps:
s1, processing raw materials
Cleaning fresh Dendrobium officinale, drying, crushing, and sieving to obtain Dendrobium powder;
s2, water extraction
Performing sealed heating leaching on the dendrobium powder by using water as a solvent, and performing centrifugal separation after leaching to obtain water extract and dendrobium residue;
s3, enzymolysis
Adding compound protease into the water extract, adjusting the pH and temperature of the water extract to adapt to the compound protease, sealing, preserving heat, performing enzymolysis, performing enzyme deactivation treatment after the enzymolysis is finished, and performing centrifugal separation on supernatant to obtain an enzymolysis solution;
s4, fermentation
Adjusting the sugar degree and the pH value of the enzymolysis liquid to adapt to fermentation of saccharomyces cerevisiae, inoculating saccharomyces cerevisiae after high-temperature sterilization, and fermenting at constant temperature to obtain the dendrobium officinale extracting solution fermented wine; the pH value of the enzymolysis liquid is adjusted by citric acid and potassium dihydrogen phosphate.
Preferably, in the step S2, the liquid-to-material ratio of the dendrobium powder to water is 20-100ml/g, and the leaching conditions are as follows: heating at 20-80 deg.C, extracting for 20-100 min/time for 1-4 times; the centrifugation conditions were: the centrifugation time is 10min, the centrifugation temperature is 10 ℃, the centrifugation speed is 10000rpm, and the centrifugation times are 2 times.
Preferably, the compound protease in step S3 is a combination of alcalase and bromelain, and the mass ratio of alcalase to bromelain is 1: (1-9), the enzyme activity of the alkaline protease and the bromelain is not less than 20000U/g, and the enzymolysis conditions are as follows: the adding amount of the compound protease is 5g/L, the pH of an aqueous extract is adjusted to 6-9, the temperature of the aqueous extract is adjusted to 35-65 ℃, and the enzymolysis time is 1-5 h; the centrifugation conditions were: the centrifugation time is 10min, the centrifugation temperature is 4 ℃, and the centrifugation speed is 10000 rpm.
Preferably, in the step S4, white granulated sugar is used for adjusting the sugar degree, the sugar degree is adjusted to 10-35%, the pH is adjusted to 3-7, the inoculation amount of the saccharomyces cerevisiae is 1-4%, and the constant-temperature fermentation conditions are as follows: the constant temperature is 28 ℃, and the constant temperature fermentation time is 7 d.
Preferably, the solvent in step S2 contains neohesperidin dihydrochalcone in 0.1-0.2 wt%.
Preferably, the complex protease is supported on a porous carrier.
Preferably, the porous carrier is a silica hollow microsphere with a double-shell layer and a plurality of pores.
Preferably, the preparation method of the double-shell porous silica hollow microsphere comprises the following steps:
s1, weighing 0.5g of potassium persulfate and 0.06g of sodium styrene sulfonate, adding 250ml of ethanol aqueous solution, stirring and dissolving, wherein the volume ratio of ethanol to water in the ethanol aqueous solution is 4:1, heating the solution to 60-70 ℃ after fully stirring and dissolving, adding 2ml of styrene and 10 microliter of divinylbenzene in the nitrogen protective atmosphere, adding 30ml of nano zinc oxide particle suspension aqueous solution, stirring and reacting for 1-2 hours in the protective atmosphere, centrifuging, collecting products, washing with absolute ethanol and water for several times respectively, and re-dispersing in 200ml of water to obtain suspension A; wherein the particle size of the nano zinc oxide is 50-100nm, and the mass concentration is 0.01 g/ml;
s2, adding 0.55ml of vinyl trimethoxy silane into 10ml of water, performing ultrasonic dispersion until the solution is transparent, adding 150ml of suspension A, adding 4ml of ammonia water solution with the mass fraction of 5% while stirring, continuing stirring and reacting at room temperature for 3-4h after the addition is finished, centrifuging, collecting a product, washing with absolute ethyl alcohol for several times, and drying to obtain a product A;
s3, ultrasonically dispersing the product A in 0.5-0.6mol/L sodium carbonate solution according to a liquid-material ratio of 1000ml/g, ultrasonically treating at 60-70 ℃ for 200 Wx 5min, centrifugally separating the product, washing the product with distilled water for several times, adding 2mol/L calcium chloride solution into the product according to a liquid-material ratio of 50-100ml/g, stirring and reacting for 1-2h, centrifugally separating the product, adding 2mol/L potassium phosphate solution into the product according to a liquid-material ratio of 50-100ml/g, stirring and reacting for 1-2h, and centrifugally separating the product to obtain a product B;
s4, weighing 4g of P123, dissolving the P123 in 50ml of absolute ethyl alcohol, adding 1.7ml of ammonia water with the mass fraction of 5-10%, 20ml of ethyl orthosilicate, 60ml of absolute ethyl alcohol and 6ml of distilled water, stirring uniformly, adding the product B prepared in S3, heating the solution to 50 ℃, keeping the temperature, stirring and reacting for 1-2 hours, centrifuging and collecting the product, and washing with absolute ethyl alcohol and water for several times respectively to obtain a product C;
s5, washing the product C with an acid solution with hydrogen ion concentration of 1mol/L, deionized water, tetrahydrofuran and absolute ethyl alcohol in sequence, and drying to obtain the porous silicon dioxide hollow microsphere with double shell layers.
Preferably, the acid solution is hydrochloric acid or sulfuric acid.
The invention has the beneficial effects that:
(1) different from the existing direct grinding and fermentation mode, the method has the advantages that the active components in the dendrobium are fully dissolved out by a water extraction method, and then the water extraction liquid is used for fermentation, so that the utilization rate of the dendrobium and the nutritional value of the product are improved; different from the existing enzymolysis wall breaking, the water extract is subjected to enzymolysis by preferably selecting the compound protease, so that the content of nutrient substances of the water extract is further improved, and particularly the content of free amino acid is greatly improved.
(2) Neohesperidin Dihydrochalcone (NHDC), a sweetener, is a flavonoid derivative extracted from plants such as natural oranges and tangerines through hydrogenation, has high sweetness, small heat, fresh and cool mouthfeel, lasting aftertaste and excellent bitter shielding effect, has strong synergistic effect when being compounded with sucralose, polyol sweeteners, saccharin and the like, and can improve the sweetness.
(3) The content of free amino acid in the polysaccharide extracting solution can be further improved by enzymolysis, but the directly added biological enzyme is difficult to recycle; in addition, the enzyme deactivation treatment after enzymolysis is easy to damage the active structure of the polysaccharide; in order to improve the utilization rate and reduce the production cost, an immobilized enzyme technology is provided; at present, the carrier materials of immobilized enzymes mainly comprise the following types: the method comprises the steps of preparing an enzyme immobilization carrier from inorganic carriers (such as glass, silica gel, montmorillonite and the like), magnetic polymer microspheres (such as polystyrene magnetic microspheres), natural polymer carriers (such as sodium alginate, chitosan, synthetic polymer carriers (such as polyacrylamide and polystyrene) and the like), wherein immobilized enzymes prepared from the materials have the problems of low enzyme activity and low stability, the method has the characteristics of adsorption and embedding by preparing the double-shell porous silica hollow microspheres as the enzyme immobilization carrier, so that the compound protease can be adsorbed on the inner surface and the outer surface of the enzyme immobilization carrier, the adsorption immobilization rate and the storage stability of the compound protease are improved, meanwhile, the double-shell porous silica hollow microspheres can promote the rapid diffusion of substrates and products, the enzymatic reaction rate is improved, higher enzyme carrying amount and activity are shown, and particularly, the zinc oxide particles tend to move to the surface by utilizing the hydrophilic difference between the nano zinc oxide particles and the polystyrene, preparing polystyrene particles with zinc oxide inlaid on the surfaces, selectively growing a layer of vinyl silicon dioxide on hydrophobic polystyrene parts of the polystyrene particles through vinyl trimethoxy silane with hydrophobic groups to obtain a silicon dioxide layer, forming a calcium phosphate layer on the silicon dioxide layer through mesoporous, calcium ionization and phosphorylation of the silicon dioxide, generating an outer porous silicon dioxide layer on the calcium phosphate layer, and removing the calcium phosphate layer, the zinc oxide and the polystyrene which are used as templates in an acid solution and a tetrahydrofuran solution to obtain the silicon dioxide hollow microsphere with the double-shell porous structure.
Detailed Description
The invention is further described with reference to the following examples.
The embodiment of the application relates to a preparation method of dendrobium officinale extract fermented wine, which comprises the following steps:
s1, processing raw materials
Cleaning fresh Dendrobium officinale, drying, crushing, and sieving to obtain Dendrobium powder;
s2, water extraction
Performing sealed heating leaching on the dendrobium powder by using water as a solvent, and performing centrifugal separation after leaching to obtain water extract and dendrobium residue;
s3, enzymolysis
Adding compound protease into the water extract, adjusting the pH and temperature of the water extract to adapt to the compound protease, sealing, preserving heat, performing enzymolysis, performing enzyme deactivation treatment after the enzymolysis is finished, and performing centrifugal separation on supernatant to obtain an enzymolysis solution;
s4, fermentation
Adjusting the sugar degree and the pH value of the enzymolysis liquid to adapt to fermentation of saccharomyces cerevisiae, inoculating saccharomyces cerevisiae after high-temperature sterilization, and fermenting at constant temperature to obtain the dendrobium officinale extracting solution fermented wine; the pH value of the enzymolysis liquid is adjusted by citric acid and potassium dihydrogen phosphate.
Example 1
A preparation method of dendrobium officinale extract fermented wine comprises the following steps:
s1, processing raw materials
Cleaning fresh Dendrobium officinale, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain Dendrobium powder;
s2, water extraction
Sealing, heating and leaching the dendrobium powder by using water as a solvent, and performing centrifugal separation to obtain a water extract and dendrobium residue; wherein, the feed liquid ratio of dendrobium and water is 1: 40(g/ml), the extraction conditions were: heating to 35 deg.C, extracting for 60 min/time, and extracting for 4 times; the centrifugation conditions were: centrifuging for 10min at 10 deg.C and 10000rpm for 2 times; the feed-liquid ratio refers to the ratio of the volume of the liquid serving as the leaching solution to the mass of the solid feed;
s3, enzymolysis
Adding composite protease into the water extract, wherein the composite protease is alkaline protease and bromelain, the mass ratio of the alkaline protease to the bromelain is 3:2, the addition amount of the composite protease is 5g/L, adjusting the pH to 9, heating to 55 ℃, sealing, preserving heat, performing enzymolysis for 3h, performing enzyme deactivation treatment in a hot water bath at 90 ℃ for 1h after the enzymolysis is finished, and performing centrifugal separation on supernatant, wherein the centrifugal conditions are as follows: centrifuging for 10min at 4 deg.C and 10000rpm to obtain enzymolysis solution;
s4, fermentation
Adjusting the sugar degree of the enzymolysis liquid to 25% by using white granulated sugar, adjusting the pH of the enzymolysis liquid to 5 by using citric acid and potassium dihydrogen phosphate, inoculating saccharomyces cerevisiae after high-temperature sterilization at 121 ℃ for 30min, wherein the inoculation amount is 3%, and fermenting at the constant temperature of 28 ℃ for 7d to obtain the dendrobium officinale extract fermented wine.
Example 2
A preparation method of dendrobium officinale extract fermented wine comprises the following steps:
s1, processing raw materials
Cleaning fresh Dendrobium officinale, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain Dendrobium powder;
s2, water extraction
Taking neohesperidin dihydrochalcone water solution with the mass fraction of 0.1-0.2% as a solvent to carry out sealed heating extraction on the dendrobium powder, and carrying out centrifugal separation to obtain water extract and dendrobium residue; wherein, the feed liquid ratio of dendrobium and water is 1: 40(g/ml), the extraction conditions were: heating to 35 deg.C, extracting for 60 min/time, and extracting for 4 times; the centrifugation conditions were: centrifuging for 10min at 10 deg.C and 10000rpm for 2 times;
s3, enzymolysis
Adding composite protease into the water extract, wherein the composite protease is alkaline protease and bromelain, the mass ratio of the alkaline protease to the bromelain is 3:2, the addition amount of the composite protease is 5g/L, adjusting the pH to 9, heating to 55 ℃, sealing, preserving heat, performing enzymolysis for 3h, performing enzyme deactivation treatment in a hot water bath at 90 ℃ for 1h after the enzymolysis is finished, and performing centrifugal separation on supernatant, wherein the centrifugal conditions are as follows: centrifuging for 10min at 4 deg.C and 10000rpm to obtain enzymolysis solution;
s4, fermentation
Adjusting the sugar degree of the enzymolysis liquid to 25% by using white granulated sugar, adjusting the pH of the enzymolysis liquid to 5 by using citric acid and potassium dihydrogen phosphate, inoculating saccharomyces cerevisiae after high-temperature sterilization at 121 ℃ for 30min, wherein the inoculation amount is 3%, and fermenting at the constant temperature of 28 ℃ for 7d to obtain the dendrobium officinale extract fermented wine.
Example 3
The preparation method of the dendrobium officinale extract fermented wine is the same as the step of the example 1, and is characterized in that: the compound protease is loaded on a porous carrier; the porous carrier is a double-shell porous silicon dioxide hollow microsphere, and the preparation method of the double-shell porous silicon dioxide hollow microsphere comprises the following steps:
s1, weighing 0.5g of potassium persulfate and 0.06g of sodium styrene sulfonate, adding 250ml of ethanol aqueous solution, stirring and dissolving, wherein the volume ratio of ethanol to water in the ethanol aqueous solution is 4:1, heating the solution to 60-70 ℃ after fully stirring and dissolving, adding 2ml of styrene and 10 microliter of divinylbenzene in the nitrogen protective atmosphere, adding 30ml of nano zinc oxide particle suspension aqueous solution, stirring and reacting for 1-2 hours in the protective atmosphere, centrifuging, collecting products, washing with absolute ethanol and water for several times respectively, and re-dispersing in 200ml of water to obtain suspension A; wherein the particle size of the nano zinc oxide is 50-100nm, and the mass concentration is 0.01 g/ml;
s2, adding 0.55ml of vinyl trimethoxy silane into 10ml of water, performing ultrasonic dispersion until the solution is transparent, adding 150ml of suspension A, adding 4ml of ammonia water solution with the mass fraction of 5% while stirring, continuing stirring and reacting at room temperature for 3-4h after the addition is finished, centrifuging, collecting a product, washing with absolute ethyl alcohol for several times, and drying to obtain a product A;
s3, ultrasonically dispersing the product A in 0.5-0.6mol/L sodium carbonate solution according to a liquid-material ratio of 1000ml/g, ultrasonically treating at 60-70 ℃ for 200 Wx 5min, centrifugally separating the product, washing the product with distilled water for several times, adding 2mol/L calcium chloride solution into the product according to a liquid-material ratio of 50-100ml/g, stirring and reacting for 1-2h, centrifugally separating the product, adding 2mol/L potassium phosphate solution into the product according to a liquid-material ratio of 50-100ml/g, stirring and reacting for 1-2h, and centrifugally separating the product to obtain a product B;
s4, weighing 4g of P123, dissolving the P123 in 50ml of absolute ethyl alcohol, adding 1.7ml of ammonia water with the mass fraction of 5-10%, 20ml of ethyl orthosilicate, 60ml of absolute ethyl alcohol and 6ml of distilled water, stirring uniformly, adding the product B prepared in S3, heating the solution to 50 ℃, keeping the temperature, stirring and reacting for 1-2 hours, centrifuging and collecting the product, and washing with absolute ethyl alcohol and water for several times respectively to obtain a product C; the P123 is a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer with the molecular formula: PEO-PPO-PEO;
s5, washing the product C with 1mol/L hydrochloric acid solution, deionized water, tetrahydrofuran and absolute ethyl alcohol in sequence, and drying to obtain the double-shell porous silicon dioxide hollow microspheres;
soaking the prepared double-shell porous silica hollow microspheres in absolute ethyl alcohol for 12h, washing with deionized water, weighing 1 part after drying, adding the weighed part into a reactor, adding a compound protease solution, keeping the temperature at 45-50 ℃ in a water bath, stirring and adsorbing for 4-6h, filtering out a product, washing with a buffer solution, and refrigerating for later use.
Examples of the experiments
1. Influence of enzymolysis on free amino acids in the dendrobium officinale extract
The content of free amino acids in the extract before and after the enzymatic hydrolysis in example 1 was measured, and the measurement results are shown in Table 1.
TABLE 1 comparison table of amino acid types and contents before and after enzymolysis
2. Biological activity of water extract
Selecting 40 healthy KM mice, feeding normally for 1 week, fasting for 12h, drinking normally, randomly dividing the mice into 10 mice/group, which are respectively a normal group, a control group, a first experimental group and a second experimental group, weighing the mice of the control group, the first experimental group and the second experimental group respectively, injecting 1% alloxan into the abdominal cavity at 300mg/kg after the mice are fed normally, feeding blood from the tail vein after 72h, measuring the fasting blood sugar at 0 week, feeding the water extract of example 1 at 200mg/kg (by polysaccharide content) per day for the first experimental group, feeding the water extract of example 2 at 200mg/kg (by polysaccharide content) per day for the second experimental group at 200mg/kg (by polysaccharide content), feeding the control group at equal volume of physiological saline, feeding normally for the normal group, taking the venous blood from the tail veins of each group of mice after two weeks and four weeks, measuring the fasting blood sugar, the results are shown in Table 2.
TABLE 2 biological Activity of aqueous extracts on diabetes model mice
| Average blood glucose level | 0 week | 2 weeks | 4 weeks |
| Normal group | 5.1mmol/L | 5.2mmol/L | 5.1mmol/L |
| Control group | 20.2mmol/L | 19.7mmol/L | 18.5mmol/L |
| First experimental group | 20.1mmol/L | 14.3mmol/L | 9.2mmol/L |
| Second Experimental group | 20.2mmol/L | 12.8mmol/L | 6.9mmol/L |
3. Loading of Complex proteases
The existing hollow mesoporous silica microspheres (microspheres 1) and microspheres (microspheres 2) of the product A obtained in example 3 after washing with tetrahydrofuran are respectively used as porous carriers to carry out load comparison on the composite protease, and the load capacity, load activity and temperature stability of the composite protease load product prepared in example 3 (microspheres 3) are measured, wherein the load capacity, load activity and temperature stability are measured according to bromelain.
And (3) respectively placing each load microsphere and free protease with the same load amount as the load microsphere in PBS at 75 ℃, and testing the enzyme activity every 4h by taking the initial free protease enzyme activity as 100%.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of dendrobium officinale extract fermented wine is characterized by comprising the following steps:
s1, processing raw materials
Cleaning fresh Dendrobium officinale, drying, crushing, and sieving to obtain Dendrobium powder;
s2, water extraction
Performing sealed heating leaching on the dendrobium powder by using water as a solvent, and performing centrifugal separation after leaching to obtain water extract and dendrobium residue;
s3, enzymolysis
Adding compound protease into the water extract, adjusting the pH and temperature of the water extract to adapt to the compound protease, sealing, preserving heat, performing enzymolysis, performing enzyme deactivation treatment after the enzymolysis is finished, and performing centrifugal separation on supernatant to obtain an enzymolysis solution;
s4, fermentation
Adjusting the sugar degree and the pH value of the enzymolysis liquid to adapt to fermentation of saccharomyces cerevisiae, inoculating saccharomyces cerevisiae after high-temperature sterilization, and fermenting at constant temperature to obtain the dendrobium officinale extracting solution fermented wine; the pH value of the enzymolysis liquid is adjusted by citric acid and potassium dihydrogen phosphate.
2. The preparation method of the dendrobium officinale extract fermented wine according to claim 1, wherein the liquid-material ratio of the dendrobium officinale powder to water in step S2 is 20-100ml/g, and the leaching conditions are as follows: heating at 20-80 deg.C, extracting for 20-100 min/time for 1-4 times; the centrifugation conditions were: the centrifugation time is 10min, the centrifugation temperature is 10 ℃, the centrifugation speed is 10000rpm, and the centrifugation times are 2 times.
3. The preparation method of the dendrobium officinale extract fermented wine according to claim 1, wherein the compound protease in the step S3 is a combination of alkaline protease and bromelain, and the mass ratio of the alkaline protease to the bromelain is 1: (1-9), the enzyme activity of the alkaline protease and the bromelain is not less than 20000U/g, and the enzymolysis conditions are as follows: the adding amount of the compound protease is 5g/L, the pH of an aqueous extract is adjusted to 6-9, the temperature of the aqueous extract is adjusted to 35-65 ℃, and the enzymolysis time is 1-5 h; the centrifugation conditions were: the centrifugation time is 10min, the centrifugation temperature is 4 ℃, and the centrifugation speed is 10000 rpm.
4. The preparation method of the dendrobium officinale extract fermented wine according to claim 1, wherein the sugar degree is adjusted by white granulated sugar in step S4 to 10-35%, the pH is adjusted to 3-7, the inoculation amount of saccharomyces cerevisiae is 1-4%, and the constant-temperature fermentation conditions are as follows: the constant temperature is 28 ℃, and the constant temperature fermentation time is 7 d.
5. The method for preparing the dendrobium officinale extract fermented wine according to claim 1, wherein the solvent in the step S2 contains 0.1-0.2% by mass of neohesperidin dihydrochalcone.
6. The method for preparing the dendrobium officinale extract fermented wine according to claim 1, wherein the compound protease is loaded on a porous carrier.
7. The method for preparing the dendrobium officinale extract fermented wine according to claim 1, wherein the porous carrier is a silica hollow microsphere with double shells and pores.
8. The preparation method of the dendrobium officinale extract fermented wine according to claim 1, wherein the preparation method of the double-shell porous silica hollow microspheres comprises the following steps:
s1, weighing 0.5g of potassium persulfate and 0.06g of sodium styrene sulfonate, adding 250ml of ethanol aqueous solution, stirring and dissolving, wherein the volume ratio of ethanol to water in the ethanol aqueous solution is 4:1, heating the solution to 60-70 ℃ after fully stirring and dissolving, adding 2ml of styrene and 10 microliter of divinylbenzene in the nitrogen protective atmosphere, adding 30ml of nano zinc oxide particle suspension aqueous solution, stirring and reacting for 1-2 hours in the protective atmosphere, centrifuging, collecting products, washing with absolute ethanol and water for several times respectively, and re-dispersing in 200ml of water to obtain suspension A; wherein the particle size of the nano zinc oxide is 50-100nm, and the mass concentration is 0.01 g/ml;
s2, adding 0.55ml of vinyl trimethoxy silane into 10ml of water, performing ultrasonic dispersion until the solution is transparent, adding 150ml of suspension A, adding 4ml of ammonia water solution with the mass fraction of 5% while stirring, continuing stirring and reacting at room temperature for 3-4h after the addition is finished, centrifuging, collecting a product, washing with absolute ethyl alcohol for several times, and drying to obtain a product A;
s3, ultrasonically dispersing the product A in 0.5-0.6mol/L sodium carbonate solution according to a liquid-material ratio of 1000ml/g, ultrasonically treating at 60-70 ℃ for 200 Wx 5min, centrifugally separating the product, washing the product with distilled water for several times, adding 2mol/L calcium chloride solution into the product according to a liquid-material ratio of 50-100ml/g, stirring and reacting for 1-2h, centrifugally separating the product, adding 2mol/L potassium phosphate solution into the product according to a liquid-material ratio of 50-100ml/g, stirring and reacting for 1-2h, and centrifugally separating the product to obtain a product B;
s4, weighing 4g of P123, dissolving the P123 in 50ml of absolute ethyl alcohol, adding 1.7ml of ammonia water with the mass fraction of 5-10%, 20ml of ethyl orthosilicate, 60ml of absolute ethyl alcohol and 6ml of distilled water, stirring uniformly, adding the product B prepared in S3, heating the solution to 50 ℃, keeping the temperature, stirring and reacting for 1-2 hours, centrifuging and collecting the product, and washing with absolute ethyl alcohol and water for several times respectively to obtain a product C;
s5, washing the product C with an acid solution with hydrogen ion concentration of 1mol/L, deionized water, tetrahydrofuran and absolute ethyl alcohol in sequence, and drying to obtain the porous silicon dioxide hollow microsphere with double shell layers.
9. The method for preparing the dendrobium officinale extract fermented wine according to claim 8, wherein the acid solution is hydrochloric acid or sulfuric acid.
10. A dendrobium officinale extract fermented wine, which is characterized by being prepared by the preparation method of the dendrobium officinale extract fermented wine in any one of claims 1-9.
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