WO2020177420A1 - 一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法 - Google Patents

一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法 Download PDF

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WO2020177420A1
WO2020177420A1 PCT/CN2019/123517 CN2019123517W WO2020177420A1 WO 2020177420 A1 WO2020177420 A1 WO 2020177420A1 CN 2019123517 W CN2019123517 W CN 2019123517W WO 2020177420 A1 WO2020177420 A1 WO 2020177420A1
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cordyceps militaris
cordyceps
cordycepin
extraction
polysaccharide
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French (fr)
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张慜
石浩
易思富
郭小玲
侯丽华
阳武雄
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Jiangnan University
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Jiangnan University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/70—Harvesting
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0211—Solvent extraction of solids in combination with an electric or magnetic field
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
    • B01D11/0265—Applying ultrasound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00

Definitions

  • the invention relates to an ultrasonic composite acid water extraction method of cordyceps polysaccharide and cordycepin in Cordyceps militaris, and belongs to the technical field of food processing.
  • Cordyceps militaris is a traditional Chinese edible medicinal mushroom, commonly known as Cordyceps militaris. It belongs to the phylum Ascomycota and pyrethroid fungi. It has been proven to have similar pharmacological effects to Cordyceps.
  • Cordyceps militaris has been used in many Asian countries, especially China for hundreds of years. It is rich in special natural ingredients with biological and pharmacological activities, such as cordycepin (3′-deoxyadenosine), adenosine, cordycepic acid, SOD enzyme And Cordyceps polysaccharide.
  • Traditional Chinese medicine believes that water extracts of traditional Chinese medicine can nourish the lungs and kidneys, relieve waist and knee pain, and help patients recover after illness.
  • cordycepin is an important immunomodulatory compound that affects the function of immune cells and cytokine network, and has the effect of resisting ischemia-reperfusion injury.
  • Cordycepin can inhibit the growth of mouse melanoma cells and improve testicular damage without side effects.
  • Cordyceps polysaccharide can effectively improve immune function by protecting the body from oxidative stress, and its blood sugar lowering effect is even stronger than that of Cordyceps polysaccharide.
  • the extraction techniques often used mainly include hot water reflux extraction, ultrasonic assisted extraction, microwave assisted extraction, enzymatic extraction, ultra-high pressure extraction and supercritical liquid extraction.
  • the main factors affecting the water extract of Cordyceps include the type of extractant, extraction time, extraction temperature, material-to-liquid ratio, extraction times, and auxiliary technical parameters.
  • Most studies have adopted one or two extraction methods to obtain cordycepin or cordyceps polysaccharide active ingredients that are relatively single, and the extraction rate is low.
  • impurities such as chloroform, n-butanol, ethyl acetate, non-food grade chemical reagents will be introduced in the separation and purification process, which will bring troubles to the removal of active ingredients of Cordyceps militaris, and at the same time bring greater hidden dangers to food safety.
  • ethanol, deionized water, petroleum ether, chloroform, PEG, n-butanol and other solvents are mainly used in the selection of Cordyceps militaris fruiting body extractants. These extractants have their own advantages and disadvantages.
  • petroleum ether is used as an extractant, a large amount of fat-soluble substances in Cordyceps will be precipitated and it is difficult to separate polar substances such as cordycepin and cordyceps polysaccharides.
  • Organic solvents such as chloroform are toxic, affecting the health of operators and require more protective measures. The cost of supporting facilities increases. Therefore, deionized water or ethanol aqueous solution is generally used as the extractant.
  • Freeze-thaw extraction technology is an extraction method that uses a physical freezing-dissolution process to destroy the cell structure and increase the dissolution of active ingredients.
  • the intracellular solution especially the aqueous solution
  • forms intracellular ice crystals which causes the cell volume to increase, causing the rupture of the hydrophobic bond structure of the cell membrane, which in turn leads to the rupture of the cell membrane and cell wall;
  • thawing a large temperature difference occurs between the inside and outside of the cell wall, causing shock heat The stress again ruptured the cell wall.
  • Repeated cycles of freezing and dissolving process cause serious damage to the structure of the cell wall of the material, and ultimately increase the release, diffusion and dissolution of intracellular substances, and promote the dissolution of active water-soluble components.
  • Ultrasonic extraction technology mainly uses ultrasonic mechanical crushing and cavitation to destroy tissue structure and accelerate the diffusion of extracted materials from raw materials to solvents.
  • the liquid medium continuously receives stretching and compression to form cavitation bubbles.
  • the cavitation bubbles continue to increase until they rupture, causing multiple effects such as cavitation effect, high shear force, crushing and stirring in an instant. .
  • the cavitation effect not only damages the cell wall, but also causes the microflow inside the cell to generate shearing force.
  • the cavitation effect can also generate H ⁇ radicals and ⁇ OH radicals in the water medium to attack the chemical structure of the cell wall. Decreasing its mechanical strength is more easily broken and the content flows out.
  • the combined freeze-thaw cycle extraction technology can effectively improve the dissolution and acquisition of active ingredients in the material.
  • Deng Hua discloses a process for extracting cordycepin from Cordyceps militaris, through alcohol extraction, concentration, dewaxing, purification, liquid membrane extraction, refining and drying.
  • the present invention has the simplest process, short production cycle, small amount of organic solvent, reusable use, and high product yield.
  • the extraction method is more complicated, the liquid membrane preparation cost is higher, and there are certain requirements for the extraction operator, and the equipment maintenance cost in the production process is higher.
  • Zhang Yaozhou et al. (201410041853.X) provide a method for extracting cordycepin components from Cordyceps militaris, which includes the following steps: take the fruiting body of Cordyceps militaris and pulverize to obtain ultrafine powder; the ultrafine powder is extracted with ultrapure water Then take the supernatant and dry it, then use ethanol for alcohol precipitation treatment, centrifuge, collect the alcohol precipitation supernatant, spin the alcohol precipitation supernatant and dry to obtain a crude extract; use 15% methanol-water solution for the crude extract Dissolved to 100mg/mL, separated by preparative liquid phase DAC reverse phase chromatography column, reverse phase separation packing is C18, elution method is first 5%-15% methanol-water solution gradient elution, and then 15% methanol-water solution isocratic After elution and detection wavelength of 260nm, each component is screened for anti-tumor activity in vitro, and then the most active component is selected
  • the method is simple and controllable, with good reproducibility, high cordycepin content in the extracted components, and stable batches.
  • methanol reagent is introduced during the separation and purification process and the subsequent crystallization is not performed after elution and separation by preparative chromatography.
  • the resulting solution is not easy to store.
  • the water extract crystallizes into powder after freeze-drying, which is convenient for packaging the finished product.
  • Zhang Yaozhou et al. (201410041854.4) provided a method for extracting anti-tumor active components from Cordyceps militaris: Ultra-low temperature pulverization of Cordyceps militaris fruiting bodies; extraction of superfine powder in water and extraction with acetone after precipitation; liquid chromatography after the obtained precipitate was dissolved.
  • the elution reagents are 100% n-hexane, 25% ethyl acetate-n-hexane, 100% ethyl acetate; the most active component is selected for liquid chromatography, and the elution reagents are 100% n-hexane and 10% ethyl acetate- N-hexane, 15% ethyl acetate-n-hexane, 30% ethyl acetate-n-hexane, 60% ethyl acetate-n-hexane; select the most active component for liquid chromatography, and the elution reagent is 100% dichloromethane , 25% dichloromethane-methanol, the most active component is selected for liquid chromatography, the elution reagent is 100% n-hexane, 5% ethanol-n-hexane, and the most active component is selected as the target component of the present invention.
  • organic reagent organic reagent
  • Keming Kou and Yanmei Peng invented a method for extracting cordycepin and cordyceps polysaccharides from Cordyceps militaris by using a macroporous adsorption resin, including crushing the fruiting bodies of Cordyceps militaris, extracting with water, concentrating the extract, and then adding ethanol to stand still The cordyceps polysaccharide is separated out and the supernatant is obtained. The supernatant is passed through a macroporous adsorption resin column to obtain an eluate, and the eluate is concentrated and dried to obtain a cordycepin extract.
  • the product obtained by the method of the present invention has few harmful solvent residues, but the desorption capacity of the resin filler has not been studied during the elution process of the macroporous resin, and the problem of filler blockage and aging during the repeated use of the macroporous resin has been affected, which affects the life of the resin.
  • this method has lower requirements on the extraction container and is suitable for industrial production.
  • Li Fuquan et al. (201210014073.7) invented a method for extracting cordycepin and cordyceps polysaccharides from Cordyceps militaris, including water extraction, alcohol extraction, column extraction supernatant and column extraction sediment steps.
  • the temperature of the water is set between 55-58°C, which can fully dissolve the active ingredients in the cordyceps as much as possible, while also avoiding the decomposition of cordycepin. Therefore, the water temperature is used to extract cordycepin And Cordyceps polysaccharide will not cause waste of resources.
  • the method extracts both cordycepin and cordyceps polysaccharide at the same time, which has reference significance for the higher utilization rate of the active substance of Cordyceps militaris. Compared with the method of the present invention, the extraction time is longer and the extraction process is cumbersome, which needs to be further optimized.
  • Hua Chun et al. (201410002259.X) proposed a method for extracting grass matrix polysaccharides from waste Cordyceps militaris and the application of the extract.
  • the method realizes the extraction of cordyceps matrix polysaccharides from waste cordyceps militaris by enzymatic hydrolysis, microwave, centrifugal separation and ethanol precipitation, and then through experimental steps of starch removal, protein removal, gel column purification and the like.
  • the obtained polysaccharide extract has high purity and is an effective waste utilization for the waste culture of Cordyceps militaris, but also loses the rate of active substances such as cordycepin during the extraction process. Compared with this method, the degree of waste utilization is further improved. Space.
  • the present invention discloses a method for enzymatically assisting extraction of cordycepin from waste Cordyceps militaris culture medium.
  • the invention uses waste Cordyceps militaris culture medium as a raw material, first undergoes high pressure treatment and then undergoes enzymatic hydrolysis with neutral protease. Then heat the water to kill enzyme extraction, remove impurities by resin adsorption, dialysis and concentration, ice bath crystallization and freeze-drying, thereby obtaining a method for extracting cordycepin from the Cordyceps militaris culture medium by enzymatic hydrolysis.
  • the invention reuses the waste culture medium and saves production cost. Compared with the invention, this method does not have a high degree of utilization of protease, so it will increase another part of the raw material cost.
  • the present invention discloses a method for extracting and continuously enriching polysaccharides from Cordyceps militaris culture medium.
  • the steps include drying, superfine pulverization, and enzymatic hydrolysis of Cordyceps militaris rice culture medium. Centrifugal filtration, reflux control, multifunctional membrane concentration and enrichment, spray drying, qualitative and quantitative, established a fast, efficient and economical separation method of Cordyceps polysaccharide.
  • the invention effectively utilizes the waste Cordyceps militaris culture medium and saves resources and costs.
  • the invention is suitable for large-scale industrial production.
  • the application of ultrafine pulverization and membrane separation technology has a great effect on the dissolution of effective substances and the collection of cordycepin components, which has reference value for the improvement of the present invention.
  • Su Liuhua discloses a method for extracting cordycepin and polysaccharides from Cordyceps militaris. Crush the raw material of Cordyceps militaris and put it into a subcritical extraction kettle, pass in deionized water, and extract 10-60 minutes countercurrently at a temperature of 100 ⁇ 200°C and a pressure of 1 ⁇ 15MPa to obtain an extract; after the extract is concentrated, add 5 -6 times the volume of 95% ethanol, fully stirred and fully precipitated, the precipitate was dissolved in water again, added with 3%-10% trichloroacetic acid to remove the protein, and then precipitated with alcohol and dried to obtain Cordyceps polysaccharide; combine the two alcohol precipitation solutions, Concentrate under reduced pressure until there is no alcohol, add activated carbon column for adsorption, elution with ethanol solution, concentrate the eluate under reduced pressure, place to crystallize, and dry the crystal to obtain cordycepin.
  • the invention has a higher utilization rate of Cordyceps militaris, and the yield of cordyceps polysaccharide and cordycepin is improved.
  • the subcritical extraction technology requires higher extraction equipment and operators, which will increase the production cost to a certain extent.
  • Dong Chao 201610605098.2 disclosed a method for extracting cordycepin from Cordyceps militaris.
  • the main steps are: petroleum ether degreasing, high-concentration ethanol extraction, ethyl acetate extraction, and D1400 resin twice purification to obtain ⁇ 95% Purity cordycepin.
  • the invention has the advantages of simple operation and high cordycepin extraction rate, but only the cordycepin in Cordyceps militaris is extracted, and the cordycepin polysaccharide and other substances are lost greatly.
  • Hongfei Liu discloses a preparation method of Cordyceps militaris extract rich in biologically active substances, which belongs to the field of functional foods.
  • the Cordyceps militaris biotransformation complex enzyme system containing superoxide dismutase, glycosidase, transferase, hydrolase, isomerase and other biological enzymes
  • the Cordyceps militaris complex enzyme system hydrolysis reaction is carried out, and then the Cordyceps militaris biologically active substance is highly effective Dynamic cycle extraction, microporous membrane filtration, vacuum decompression concentration and other steps to obtain Cordyceps militaris extract rich in biologically active substances.
  • the method greatly increases the content of cordycepin, cordycepic acid, cordyceps polysaccharide, adenosine and other biologically active substances in the extract.
  • the composite enzyme system prepared in the present invention has a complicated process and harsh environmental requirements, so it will greatly increase the manufacturing cost of extraction equipment and the cost of enzyme preparations.
  • Tang Qingjiu et al. (201510585474.1) disclosed a method for preparing Cordyceps militaris active polysaccharides: the fruiting bodies of Cordyceps militaris were superfinely crushed and then extracted with boiling water, and the supernatant was collected; the supernatant was concentrated and then precipitated with ethanol, and the precipitation site was passed through a DEAE-sepharose column , 0-0.5N site desalination and freeze-drying.
  • the method is simple and has reference value.
  • Yin Ming et al. (201710788899.1) disclosed a method for extracting cordycepin from Cordyceps militaris, including the following: first soak the Cordyceps militaris in an aqueous citric acid solution for 35-40 minutes, then microwave drying, and then mix it with an appropriate amount of purified water and Cellulase is mixed with enzymatic hydrolysis, and the filtrate is filtered out; finally, it is obtained by acidification, alcohol extraction, and spray drying.
  • the invention adopts citric acid soaking and microwave drying, and is similar to the invention in terms of improving the extraction rate of cordycepin and the dissolution of active substances of Cordyceps militaris, but the environmental conditions such as temperature and pressure in the enzymatic hydrolysis process have strict requirements and need to be further optimized.
  • Kang Wenyi discloses a method for extracting polysaccharides from Cordyceps militaris. It decocts Cordyceps militaris in water and filters. The filtrate is added with absolute ethanol to a final concentration of 70 ⁇ 5V%. The precipitate is Cordyceps militaris polysaccharide.
  • the invention consumes a huge volume of ethanol and only uses a boiling water bath for extraction, which takes a long time and has low efficiency.
  • the extracted product contains high content of other components except cordyceps polysaccharide, and the purity of the polysaccharide is low.
  • Sun Junshe and Pei Haisheng disclosed a method for extracting and purifying walnut green peel polyphenols with acidic electrolyzed water, which mainly includes the following steps: mixing fresh walnut green peel with acidic electrolyzed water and beating, homogenizing, to obtain a slurry; The slurry liquid is stirred and leached to obtain a stirred extract; the stirred extract is subjected to slag-liquid separation, and the separated solid slag is added to acidic electrolyzed water for a second stirring leaching; the separated liquid after two stirring leaching After the phases are mixed, the flow rate is controlled through the macroporous adsorption resin.
  • the acidic water extraction of Cordyceps militaris in this research is rich in active ingredients, which reduces the purification and impurity removal process for a single active ingredient, and is suitable for the extraction of large-scale crude cordyceps water extracts in factories and reduces costs for subsequent downstream processing.
  • Ding Zhansheng (application number: 201110365289.3) and others disclosed a method for efficient separation, extraction and stable storage of bayberry polyphenols.
  • the invention uses acidic water with pH 3.5-4.0 as the extract, and the bayberry raw materials are beaten and centrifuged many times.
  • Extract polyphenols from bayberry pomace and raw juice It mainly includes the steps of macroporous adsorption resin adsorption, evaporation and concentration to remove alcohol, adding maltodextrin, freeze drying and other steps.
  • the invention uses water as the extraction liquid, which reduces the cost of impurity removal in the subsequent purification project.
  • the extraction rate of active extracts such as cordycepin and cordycepin polysaccharides in the water extract obtained by the acidic aqueous solution extraction in this achievement is higher, the operation process is simple, and the production cost is lower.
  • Yang Jie (application number: 201711021626.0) and others provided a method for extracting Tremella fuciformis polysaccharides with acidic buffer solution, which includes the following steps: under heating conditions, leaching Tremella fuciformis powder with an acidic buffer solution to obtain an extract liquid; Solid-liquid separation is performed on the extract liquid to obtain a liquid component containing Tremella fuciformis polysaccharide; the liquid component is subjected to alcohol precipitation, and the obtained precipitate is Tremella fuciformis polysaccharide.
  • This invention has a high content of polysaccharides in extracting Tremella fuciformis, so it also proves the feasibility of extracting polysaccharides from herbs or edible fungi by acidic aqueous solution.
  • the invention uses acidic aqueous solution combined with ultrasonic to assist in extracting cordycepin and cordyceps polysaccharide in Cordyceps militaris.
  • Such as water-soluble active ingredients the process is simple and suitable for industrial applications.
  • Zhu Zhou (application number: 201710126525.3) and others disclosed a method for extracting potato anthocyanins, which includes the following steps: a) in the presence of ascorbic acid, mixing a potato with a first acid solution to prepare a potato slurry; b) making a potato slurry The slurry is subjected to the first filtration, the addition of enzyme preparations to reduce the viscosity, the first centrifugation to remove the precipitate, and the second filtration; c) The crude anthocyanin extract is adsorbed by a macroporous adsorption resin and eluted with water and ethanol respectively And d) Concentrating and drying the obtained desorption liquid in sequence to obtain anthocyanins.
  • ascorbic acid is used as the acid solvent to extract the anthocyanin components in potatoes, which improves the stability of potato anthocyanins in the mixed extract.
  • the present invention uses citric acid or edible hydrochloric acid as the acid extractant , It is more economical in production cost, and added within the scope of national standards, without any side effects on the human body.
  • the purpose of the present invention is to provide an ultrasonic composite acidic water extraction method for the water extract of Cordyceps militaris fruiting bodies to solve the problems existing in the prior art.
  • Ultra-low temperature freezing-microwave thawing cycle add distilled water to the defatted cordyceps powder according to the material-to-liquid ratio of 1:40 (w:v) to obtain the extract, and titrate the 1mol/L dilute acid solution to make the pH of the extract reach 4.8 ⁇ 0.2, freeze the extract at -80°C for 2h, after taking it out, thaw it under 300 ⁇ 400W power microwave conditions for 2min, repeat the freeze-thaw operation 3 times to obtain the Cordyceps militaris primary extract mixture;
  • Evaporative concentration The crude water extract of Cordyceps militaris fruit body is concentrated to 1% of the original volume by rotary evaporation under the conditions of 60-65°C and 0.095MPa, and finally becomes a viscous thick slurry, then the concentrated solution is collected,- Freeze at 70°C for 24h;
  • Freeze-drying powder freeze-dry the frozen Cordyceps militaris concentrate at a cold trap temperature of -55°C for 3 hours to obtain a water-extracted dry powder of Cordyceps militaris.
  • the components of the solid medium include rice, wheat, glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, vitamin B1 and vitamin B2.
  • the height of the Cordyceps militaris fruiting body is ⁇ 7 cm.
  • step (3) when crushing, it is first cut into 3-4 cm segments, and then crushed.
  • the rotation speed of the centrifugation is 4000 r/min, and the centrifugation time is 20 min.
  • the dilute acid solution is dilute hydrochloric acid, citric acid or acetic acid.
  • This method adopts food-grade hydrochloric acid dilute solution to adjust the pH value of the extractant.
  • the solution conditions of the most suitable dissolution isoelectric point of cordycepin and cordycepin polysaccharides in Cordyceps militaris are obtained, and the water-soluble activity of Cordyceps militaris at the same temperature is increased.
  • the solubility of the ingredient Appropriate freezing and thawing can rupture the hydrophobic bond structure of the cell membrane and destroy the cell membrane and cell wall.
  • the increase in the volume of water crystals in the cell causes the cell to swell and rupture. After thawing, the intracellular substance is dissolved, thereby improving the water-soluble components in the Cordyceps militaris cell.
  • Dissolution rate Supplemented by low-frequency ultrasonic technology can increase the breaking effect of the powder during the extraction process, and can effectively reduce the polymerization effect of the powder in the solvent, thereby increasing the yield of the active water extract in the final Cordyceps militaris fruiting body, and effectively saving the extraction time .
  • freeze-drying the dry powder of Cordyceps militaris water extract can effectively prevent the decomposition and denaturation of some heat-sensitive active ingredients, thereby retaining the biological activity of the extracted product.
  • the invention improves the extraction method of general Chinese herbal medicine or medicinal fungi. Compared with the hot water reflux extraction method, the extraction time is shortened by about 33%.
  • the extraction yields of cordyceps polysaccharide and cordycepin are respectively 8.72g/100g and 4.422mg/g , Increased to 10.13g/100g, 5.794 mg/g, while reducing the problem of powder aggregation during the water extraction process of Cordyceps militaris, and effectively retaining the active ingredients in Cordyceps.
  • ultrasonic waves combined with acidic water are mainly used to extract water-soluble substances such as cordycepin and cordyceps polysaccharides in Cordyceps militaris.
  • water-soluble substances such as cordycepin and cordyceps polysaccharides in Cordyceps militaris.
  • it can reduce the incorporation of organic impurities in the water-extracted product and reduce the manpower and material costs in the subsequent cleaning process;
  • use ultra-low temperature freezing-microwave thawing technology to increase the damage of Cordyceps cell structure and increase the dissolution of active ingredients; Melting and breaking the wall combined with ultrasonic assisted extraction technology, multiple mechanical and physical effects, effectively avoid the introduction and residue of organic chemical reagents.
  • It is of reference significance for solving the problems of low extraction rate and many impurities in the extract in the current water extraction process of Cordyceps militaris.
  • Example 1 Using dilute hydrochloric acid solution as extractant combined with ultra-low temperature freeze-thaw and low-frequency ultrasonic technology to assist extraction of cordyceps polysaccharides and cordycepin from Cordyceps militaris fruiting bodies
  • the extraction yields of cordyceps polysaccharide and cordycepin in the fruiting bodies of Cordyceps militaris were 10.13g/100g and 5.794. mg/g,
  • Example 1 Comparative Example: Extraction of Cordyceps polysaccharide and cordycepin from Cordyceps militaris fruiting body with dilute hydrochloric acid solution as extractant combined with ultra-low temperature freeze-thaw
  • the final extraction rate of Cordyceps militaris polysaccharide and cordycepin from the entity were 9.09g/100g and 4.854mg/g, respectively, and the effect was significantly lower than the yield of active ingredients in Example 1.
  • Example 2 Extraction of Cordyceps polysaccharides and cordycepin from Cordyceps militaris fruiting bodies using citric acid solution as extractant combined with ultra-low temperature freeze-thaw and low-frequency ultrasonic technology
  • the final extraction yields of Cordyceps polysaccharide and cordycepin in the fruiting bodies of Cordyceps militaris were 10.09g/100g and 5.721 mg/g, respectively.
  • Example 2 Comparative Example: Extraction of Cordyceps polysaccharide and cordycepin from Cordyceps militaris fruiting body using citric acid solution as extractant combined with low-frequency ultrasonic technology
  • Example 3 Extraction of Cordyceps polysaccharides and cordycepin from Cordyceps militaris fruiting bodies using acetic acid solution as extractant combined with ultra-low temperature freeze-thaw and low-frequency ultrasonic technology
  • the final extraction yields of Cordyceps polysaccharide and cordycepin in Cordyceps militaris fruiting bodies were 10.17g/100g and 5.682 mg/g, respectively.
  • Example 3 Comparative Example: Extraction of Cordyceps polysaccharides and cordycepin from Cordyceps militaris fruiting bodies using acetic acid solution as extractant combined with low-frequency ultrasonic technology
  • the extraction yields of cordyceps polysaccharide and cordycepin in the fruiting bodies of Cordyceps militaris were 9.08g/100g respectively And 4.782 mg/g, significantly lower than the yield of each active ingredient in Example 3.

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Abstract

一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，属于食品加工技术领域。将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草干粉，然后再将其浸入配置好的稀盐酸溶液中，超低温冷冻-微波解冻循环3次，同时采用低频超声波辅助搅拌提取，再对提取液进行蒸发浓缩处理，最后对其冷冻干燥收集水提产物干粉。所述的方法中使用的添加剂用量符合国内外食品添加剂的使用要求，操作简便易行，采用化学与物理方法相结合，省时高效。

Description

一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法 技术领域
本发明涉及一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，属于食品加工技术领域。
 背景技术
蛹虫草(Cordyceps militaris，CM)是中国传统的食用药用蘑菇，俗称北冬虫夏草，属子囊菌门和拟除虫菊科真菌，已被证明具有与冬虫夏草相似的药理作用。蛹虫草在亚洲许多国家，特别是中国已应用数百年，它含有丰富的具有生物和药理活性的特殊天然成分，如虫草素(3′-脱氧腺苷)、腺苷、虫草酸、SOD酶和虫草多糖。中医学认为，中药水提物具有滋养肺肾，缓解腰膝疼痛，帮助患者病后康复等功效。近年来的研究也证实了这些观点，在这些天然活性化合物中，虫草素是影响免疫细胞和细胞因子网络作用的重要免疫调节活性化合物，具有抗缺血再灌注损伤的作用。虫草素可以抑制小鼠黑色素瘤细胞的生长以及改善睾丸损伤而没有副作用。虫草多糖通过保护机体免受氧化应激而有效地改善免疫功能，其降血糖作用甚至比冬虫夏草多糖更强。
在大部分草本植物活性成分提取工艺中，经常采用的提取技术主要包括热水回流提取、超声波辅助提取、微波辅助提取、酶解提取以及超高压提取和超临界液体提取等多种方法。其中影响虫草水提物（主要是虫草素和虫草多糖）的主要因素包括提取剂种类，提取时间，提取温度，料液比，提取次数和辅助技术参数等。大部分研究采取一种或两种提取方法得到虫草素或者虫草多糖活性成分较为单一，且提取得率较低。同时在分离纯化过程中会引入氯仿、正丁醇、乙酸乙酯、非食品级化学试剂等杂质，给蛹虫草活性成分的除杂带来麻烦，同时给食用安全带来较大隐患。
从文献报道来看，在蛹虫草子实体提取剂选取上主要用到乙醇、去离子水、石油醚、三氯甲烷、PEG、正丁醇等溶剂，这些提取剂各有优缺点。利用石油醚做提取剂时会导致虫草中的脂溶性物质大量析出而难以分离虫草素虫草多糖等极性物质；三氯甲烷等有机溶剂具有毒性，影响操作者的身体健康同时对防护措施要求更高，配套设施成本增加，故一般采用去离子水或者乙醇水溶液做提取剂，在获取单一成分时由于溶出物质中成分较复杂需要后续进行分离纯化工作，工艺繁复且成本较高，但对于获取蛹虫草中水溶性物质用水或醇作溶剂恰恰能提高子实体中活性成分析出。研究发现与酸热提取法、微波提取法、酸热微波提取法、丙酮提取法相比，酸热超声波提取法提取蛹虫草中的胡萝卜素等物质得率最高。根据相似相容及等电点原理，利用酸溶液提取蛹虫草子实体能通过增加极性水溶性物质在提取溶剂中的溶解度来提高最终水溶活性物质的得率。
冻融提取技术是一种利用物理冷冻-溶解过程破坏细胞结构增加有效成分溶出的提取方法。物料在低温条件下，细胞内溶液尤其是水溶液形成胞内冰晶导致细胞体积增大，引起细胞膜疏水键结构破裂，进而导致细胞膜和细胞壁破裂；解冻时细胞壁内外两侧产生较大温差，产生冲击热应力，再次使得细胞壁破裂。多次冷冻-溶解过程循环进行造成物料细胞壁等结构严重破坏，最终增加了胞内物质的释放扩散和溶解，促进活性水溶性成分溶出。
超声波提取技术主要利用超声波的机械破碎和空化作用破坏组织结构，加速提取物质从原料向溶剂扩散。当超声波在液体介质中传播时，液体介质不断收到拉伸和压缩作用形成空化泡，空化泡继续增大直至破裂，瞬间产生空化效应、高剪切力、破碎以及搅拌等多重效应。其中空化效应除了会对细胞壁产生损伤外同时使细胞内部的微流产生剪切力，另外空化效应还能在水介质中又到产生H·自由基和·OH自由基攻击细胞壁化学结构从而降低其机械强度更易破碎使内容物流出。同时联合冻融循环提取技术，有效提升物料体内活性成分的溶解和获取。
邓华（专利申请号201410199236.2）公开了一种从蛹虫草中提取虫草素的工艺方法，通过醇提、浓缩、脱蜡、纯化、液膜萃取、精制和干燥。与现有技术相比，本发明工艺最为简单、生产周期短，有机溶剂用量少且可重复使用，产品得率高。与本发明相比，该提取方法较为复杂，液膜制备成本较高，且对提取操作人员有一定要求，且生产过程中设备维护成本较高。
张耀洲等人（201410041853.X）提供一种从蛹虫草中提取虫草素组分的方法，它包括以下步骤：取蛹虫草子实体超微粉碎，得到超微粉；超微粉用超纯水进行水提后取上清液干燥，然后用乙醇进行醇沉处理，离心，收集醇沉上清液，将醇沉上清液旋蒸后干燥，得到粗提物；将粗提物用15%甲醇-水溶液溶解至100mg/mL，经制备液相DAC反相色谱柱进行分离，反相分离填料为C18，洗脱方式为先5%-15%甲醇-水溶液梯度洗脱、再15%甲醇-水溶液等度洗脱，检测波长260nm，得到各组分经体外抗肿瘤活性筛选后，选取活性最强的组分即为目的虫草素组分。该方法简单可控，重复性好，提取得到的组分中虫草素含量高，批次稳定，但在分离纯化过程中会引入甲醇试剂且经制备色谱洗脱分离后并未做后一步结晶处理，所得溶液不易储存。而本方法中经冷冻干燥后水提液结晶成粉，便于包装成品。
张耀洲等人（201410041854.4）提供一种从蛹虫草中提取抗肿瘤活性组分的方法：蛹虫草子实体超低温粉碎；得到超微粉水煮提取，沉淀再丙酮提取；得到沉淀溶解后过液相色谱，洗脱试剂为100%正己烷、25%乙酸乙酯-正己烷、100%乙酸乙酯；选取活性最强组分过液相色谱，洗脱试剂为100%正己烷、10%乙酸乙酯-正己烷、15%乙酸乙酯-正己烷、30%乙酸乙酯-正己烷、60%乙酸乙酯-正己烷；选取活性最强组分过液相色谱，洗脱试剂为100%二氯甲烷、25%二氯甲烷-甲醇，选取活性最强组分过液相色谱，洗脱试剂为100%正己烷、5%乙醇-正己烷，选取活性最强组分即为本发明目的组分。该方法重复性虽好但在提取和洗脱过程中引入了正己烷、乙酸乙酯等有机试剂，为接下来的除杂增加难度。
寇克明和彭艳梅（201310592568.2）发明了一种利用大孔吸附树脂从蛹虫草中提取虫草素及虫草多糖的方法，包括将蛹虫草子实体粉碎、用水提取，将提取液浓缩，然后加入乙醇静置析出虫草多糖并得到上清液，将上清液过大孔吸附树脂柱得洗脱液，将洗脱液浓缩干燥得虫草素浸膏。本发明方法所得产品有害溶剂残留少，但在大孔树脂洗脱过程中并未研究树脂填料的解吸附能力，在大孔树脂重复利用过程中存在填料堵塞老化问题，影响树脂寿命。而本方法中对提取容器要求较低，适宜于工业生产。
李福泉等人（201210014073.7）发明了一种提取蛹虫草中虫草素和虫草多糖的方法，包括水提、醇提、柱提上清液和柱提沉淀物步骤。该种方法在水提步骤中，水的温度设置在55－58℃之间，即能让虫草中的活性成份尽量充分溶解，同时也可避免虫草素分解，所以，采用该种水温提取虫草素和虫草多糖不会造成资源浪费。本方法同时提取了虫草素和虫草多糖两种物质，对蛹虫草活性物质利用率较高具有借鉴意义，相比于本发明该方法提取时间较长，提取工艺繁琐，有待进一步优化。
华春等人（201410002259.X）提出了一种从废弃蛹虫草培养基质中提取草基质多糖的方法以及提取物的应用。本发明通过酶解、微波、离心分离后加乙醇沉淀，然后再经过除淀粉、除蛋白、凝胶柱纯化等实验步骤实现从废弃蛹虫草培养基质中提取虫草基质多糖。所得多糖提取物纯度较高且对于蛹虫草废弃培养基是一种有效废物利用，但在提取过程中也损失了率虫草素等活性物质，相较于本方法在废弃物利用程度上有进一步提升的空间。
仇颖超等人（201610123035.3）本发明公开了一种从废弃蛹虫草培养基中酶解辅助提取虫草素的方法，发明以废弃蛹虫草培养基为原料，首先经高压处理后经中性蛋白酶酶解，再加热水灭酶浸提，通过树脂吸附除杂后透析浓缩，冰浴结晶后冻干，从而得到从蛹虫草培养基中酶解辅助提取虫草素的方法。该发明对废弃培养基进行了再利用，节约了生产成本，与本发明相比该方法对于蛋白酶的利用程度不高，因此会增加另一部分原料成本。
高兆建等人（201710009796.0）本发明公开了一种从蛹虫草培养基中提取及连续富集多糖的方法，步骤包括蛹虫草大米残余培养基的干燥、超微粉碎、生物多酶体系酶法水解、离心过滤、回流控制、多功能膜浓缩富集、喷雾干燥、定性定量，建立了一条虫草多糖的快速、高效、经济的分离方法。该发明有效的利用了废弃的蛹虫草培养基，节约了资源和成本。本发明适合大规模工业化生产。且超微粉碎和膜分离技术的应用在有效物质溶出和虫草素组分收集上作用巨大，对于本发明的改进具有参考价值。
苏刘花（201210353679.3）公开了一种从蛹虫草提取虫草素和多糖的方法。将蛹虫草原料粉碎后装入亚临界萃取釜，通入去离子水，在温度100~200℃、压力1~15MPa条件，逆流提取10-60分钟，得提取液；提取液浓缩后，加入5-6倍体积95%乙醇，充分搅拌充分沉淀，沉淀物再次用水溶解，加入浓度3%-10%的三氯乙酸除去蛋白，再次醇沉，干燥即得虫草多糖；合并两次醇沉液，减压浓缩至无醇，加入活性炭柱吸附，乙醇溶液洗脱，洗脱液减压浓缩，放置结晶，结晶物干燥即得虫草素。该发明对于蛹虫草利用率较高，虫草多糖和虫草素得率得到提升，与本发明相比，亚临界萃取技术对提取设备以及操作人员要求较高，会在一定程度上增加生产成本。
董超（201610605098.2）公开了一种从蛹虫草中提取虫草素的方法，其主要步骤为：石油醚脱脂，高浓度乙醇提取，乙酸乙酯萃取，D1400树脂两次纯化，就可以获得≥95%纯度虫草素。该发明具有操作简便、虫草素提取率高的优点，但只提取了蛹虫草中的虫草素而对于虫草多糖及其他物质损失较大。
刘宏非（201610082656.1）公开了一种富含生物活性物质的蛹虫草提取物制备方法，属于功能食品领域。通过制备含有超氧化物歧化酶、糖苷酶、转移酶、水解酶、异构酶等生物酶的蛹虫草生物转化复合酶系，进行蛹虫草复合酶系水解反应，再通过蛹虫草生物活性物质高效动态循环提取，微孔滤膜过滤，真空减压浓缩等步骤，得到富含生物活性物质的蛹虫草提取物。与目前已公开的蛹虫草提取物制备方法相比，该方法大幅提高提取物中虫草素、虫草酸、虫草多糖、腺苷等生物活性物质的含量。但与本发明相比，该发明中制备的复合酶系统工艺复杂且对环境要求苛刻，因此会大大增加提取设备制造成本和酶制剂的成本。
唐庆九等人（201510585474.1）公开了一种蛹虫草活性多糖制备方法：蛹虫草子实体超细粉碎后用沸水提取，收集上清液；上清液浓缩后加入乙醇沉淀，沉淀部位过DEAE-sepharose柱，0-0.5N的部位脱盐后冷冻干燥获得。该方法操作过程简便具有参考价值。
殷铭等人（201710788899.1）公开了一种蛹虫草中虫草素的提取方法，包括以下内容：先将蛹虫草经过柠檬酸水溶液浸泡35‑40分钟，再微波烘干，然后与适量的纯净水以及纤维素酶混合酶解，过滤取滤液；最后经酸化、醇提、喷雾干燥即得。该发明采用柠檬酸浸泡及微波烘干，在提高虫草素的提取率提高蛹虫草活性物质溶出方面与本发明类似，但酶解过程温度压力等环境条件要求严格，有待进一步优化。
康文艺（201710033639.3）公开了一种蛹虫草多糖的提取方法，其将蛹虫草用水煎煮，过滤，滤液中加入无水乙醇至乙醇终浓度为70±5V%，静置，固液分离，所得沉淀即为蛹虫草多糖。该发明消耗乙醇体积巨大且仅用沸水浴提取耗时长效率低，提取产物除虫草多糖外其他成分含量较高，多糖纯度偏低。
孙君社和裴海生（申请号：201710322120.7）公开了一种酸性电解水提取纯化核桃青皮多酚的方法，主要包含以下步骤：将鲜核桃青皮与酸性电解水混合打浆，均质，得浆料液；将浆料液搅拌浸提，得搅拌提取液；将搅拌提取液进行渣液分离，分离后的固体渣料再次加入酸性电解水进行第二次搅拌浸提；将两次搅拌浸提分离后的液相混合后通过大孔吸附树脂，控制流速，经过大孔吸附树脂后用乙醇溶液将吸附在树脂上的多酚洗脱，洗脱液随后蒸馏回收乙醇，即可得到高纯度的核桃多酚。该发明针对核桃多酚进行提取和纯化工作，所得产物纯度较高。与该发明相比，本研究中酸性水提取蛹虫草活性成分丰富，减少了针对单一活性成分的纯化除杂工艺，适宜于工厂大规模粗虫草水提物提取，为后续下游加工降低成本。
 丁占生（申请号：201110365289.3）等人公开了一种杨梅多酚的高效分离提取及稳定贮藏的方法，该发明以pH3.5-4.0的酸性水为提取液，对杨梅原料经多次打浆、离心，从杨梅果渣、原汁中提取多酚。主要包括大孔吸附树脂吸附，蒸发浓缩去醇，添加麦芽糊精，冷冻干燥等步骤。该发明以水为提取液，降低了后续纯化工程中的除杂成本。与该发明相比，本成果中利用酸性水溶液提取所得水提物中活性提取物质例如虫草素和虫草多糖提取率较高，且操作工艺简单，生产成本较低。
 杨捷（申请号：201711021626.0）等人提供了一种酸性缓冲溶液提取银耳多糖的方法，包括以下步骤：在加热条件下，采用酸性缓冲溶液对银耳粉进行浸提，得到浸提料液；将所述浸提料液进行固液分离，得到液体组分，所述液体组分中含有银耳多糖；将所述液体组分进行醇沉，得到的沉淀为银耳多糖。该发明提取银耳多糖含量较高，因此也佐证了利用酸性水溶液提取草本植物或食用菌类中多糖成分的可行性，本发明正是采用酸性水溶液结合超声波辅助提取蛹虫草中的虫草素和虫草多糖等水溶性活性成分，工艺简便，适宜工业范围内应用。
朱洲（申请号：201710126525.3）等人公开了一种马铃薯花青素的提取方法，包括如下步骤：a）在抗坏血酸存在下，将马铃薯与第一酸性溶液混合制成马铃薯浆液；b）将马铃薯浆液依次进行第一次过滤、添加酶制剂降低粘性处理、第一次离心去除沉淀、及第二次过滤；c）将花青素粗提液经过大孔吸附树脂吸附，分别用水和乙醇洗脱；以及d）对所得解吸液依次进行浓缩和干燥以获得花青素。该发明中采用抗坏血酸作为酸溶剂提取马铃薯中的花青素成分，提高了马铃薯花青素在混合提取液中的稳定性，与之相比，本发明中利用柠檬酸或食用盐酸作为酸提取剂，在生产成本上更加节约，且在国家标准范围内添加，对人体无任何副作用。
 技术问题
本发明的目的是提供一种蛹虫草子实体水提物的超声波复合酸性水提取方法，以解决现有技术存在的问题。
 技术解决方案
本发明的技术方案：
一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的稀酸溶液中，超低温冷冻结合微波解冻多次冻融循环，同时采用低频超声波辅助搅拌提取，再对提取液进行蒸发浓缩处理，最后对其冷冻干燥收集水提产物干粉；具体过程如下：
（1）培养及采集：蛹虫草子实体通过固体培养基培养而成，培育40-45天后进行采集，采集后的新鲜子实体在4℃下冷藏储存；
（2）清洗干燥：将采集的蛹虫草子实体置于60-65℃热风干燥箱中干燥5h，干燥过程中蛹虫草子实体排布均匀保持通风，待干燥至恒重后收集并储藏于阴凉干燥处；
（3）粉碎；取蛹虫草干样进行破碎，过60目筛；
（4）脱脂：将粉碎好的样品置于回流装置中，用95%乙醇按料液比1:5（w:v）沸水浴下回流提取3h，离心，收集沉淀，60-65℃下烘干至恒重，获得脱脂虫草粉；
（5）超低温冷冻-微波解冻循环：按照料液比1:40（w:v）向脱脂虫草粉中加入蒸馏水，得到提取液，通过搅拌滴定1mol/L的稀酸溶液使提取液pH达到4.8±0.2，将提取液置于-80℃条件下冷冻2h，取出后在300~400W功率微波条件下解冻2min，重复冻融操作3次，得到蛹虫草初提混合液；
（6）超声波辅助提取：获得蛹虫草初提混合液后，辅以频率为20-30KHz，功率为300-400W的超声波，在65-70℃下提取35-40min。提取结束后4000r/min条件下离心15min，取上清液过滤得蛹虫草子实体水提物粗液；
（7）蒸发浓缩：将蛹虫草子实体水提物粗液在60-65℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，最后呈现粘稠浓浆状后，收集浓缩液，-70℃下冷冻24h；
（8）冷冻干燥制粉：将冷冻的蛹虫草浓缩液在冷阱温度-55℃条件下冻干3h，得到水提蛹虫草干粉。
所述步骤（1）中，固体培养基成分包括大米、小麦、葡萄糖、磷酸二氢钾、七水硫酸镁、维生素B1和维生素B2。
所述步骤（1）中，采集蛹虫草子实体的高度≥7cm。
所述步骤（3）中，破碎时先切割成3-4cm段状，再粉碎。
所述步骤（4）中，离心的转速为4000r/min，离心时间为20min。
所述步骤（6）中，稀酸溶液为稀盐酸溶液、柠檬酸酸溶液或醋酸溶液。
原理：本方法采取食品级盐酸稀溶液调节提取剂pH值，根据研究获取远离蛹虫草中虫草素及虫草多糖的最适溶解等电点的溶液条件，增大相同温度条件下蛹虫草水溶性活性成分的溶解度。适当次数冻融能使细胞膜的疏水键结构破裂破坏细胞膜和细胞壁，另外由于细胞内水结晶体积增大而引起细胞膨胀破裂，解冻后细胞内物质溶出，从而提高了蛹虫草细胞内水溶性成分的溶出率。辅以低频超声波技术更能增加提取过程中粉末的破壁效应，并且能够有效降低粉末在溶剂中的聚合效应，从而提升最终蛹虫草子实体中活性水提物的得率，且有效节约提取时间。最后冷冻干燥制得蛹虫草水提物干粉能有效防止某些热敏性活性成分的分解变性，从而保留提取产物的生物活性。本发明改进了一般中草药或药用菌类的提取方法，相较于热水回流提取法，缩短提取时间约33%，虫草多糖和虫草素提取得率分别从8.72g/100g，4.422mg/g,提升至10.13g/100g ，5.794 mg/g，同时减少了蛹虫草水提过程中的粉末聚集问题，且有效保留虫草中活性成分。
 有益效果
本发明的有益效果：本发明中主要采用超声波结合酸性水提取蛹虫草中虫草素、虫草多糖等水溶性物质。相较于传统有机溶剂提取能减少水提产物中有机杂质的掺入以及降低后续除杂工序中的人力物力成本；利用超低温冷冻-微波解冻技术增加虫草细胞结构破坏作用，增加活性成分溶出；冻融破壁联合超声波辅助提取技术，多重机械物理作用，有效避免有机化学试剂的引入与残留。对于解决目前蛹虫草水提工艺中存在的提取率低，提取物杂质成分多等不足具有借鉴意义。
 本发明的实施方式
下面将结合具体实施例对本发明的技术方案进行进一步的说明。
实施例1：以稀盐酸溶液为提取剂联合超低温冻融及低频超声波技术辅助提取蛹虫草子实体中的虫草多糖和虫草素
将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的稀盐酸溶液（pH=4.8）中，超低温冷冻（-80℃）-微波（400W，2min）解冻循环3次，同时采用低频超声波（频率为20-30KHz，功率为350W）辅助，在65℃下搅拌提取40min，再对提取液在60℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，收集浓缩液-70℃下冷冻24h，最后对其冷冻干燥收集水提产物干粉。最终蛹虫草子实体中虫草多糖和虫草素提取得率分别为10.13g/100g 和5.794. mg/g。
实施例1对比例：以稀盐酸溶液为提取剂联合超低温冻融提取蛹虫草子实体中的虫草多糖和虫草素
将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的稀盐酸溶液（pH=4.8）中，超低温冷冻（-80℃）-微波（400W，2min）解冻循环3次，再在65℃下搅拌提取40min，再对提取液在60℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，收集浓缩液-70℃下冷冻24h，最后对其冷冻干燥收集水提产物干粉。最终蛹虫草自实体中虫草多糖和虫草素提取得率分别为9.09g/100g和4.854mg/g，效果显著低于实施例1活性成分得率。
实施例2：以柠檬酸酸溶液为提取剂联合超低温冻融及低频超声波技术提取蛹虫草子实体中的虫草多糖和虫草素
将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的柠檬酸溶液（pH=4.8）中，超低温冷冻（-80℃）-微波（400W，2min）解冻循环3次，同时采用低频超声波（频率为20-30KHz，功率为350W）辅助，在65℃下搅拌提取40min，再对提取液在60℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，收集浓缩液-70℃下冷冻24h，最后对其冷冻干燥收集水提产物干粉。最终蛹虫草子实体中虫草多糖和虫草素提取得率分别为10.09g/100g 和5.721. mg/g。
实施例2对比例：以柠檬酸酸溶液为提取剂联合低频超声波技术提取蛹虫草子实体中的虫草多糖和虫草素
将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的柠檬酸溶液（pH=4.8）中，采用低频超声波（频率为20-30KHz，功率为350W）辅助，在65℃下搅拌提取40min，再对提取液在60℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，收集浓缩液-70℃下冷冻24h，最后对其冷冻干燥收集水提产物干粉。最终蛹虫草子实体中虫草多糖和虫草素提取得率分别为9.17g/100g 和4.911mg/g，明显低于实例2中虫草多糖和虫草素提取得率。
实施例3：以醋酸溶液为提取剂联合超低温冻融及低频超声波技术提取蛹虫草子实体中的虫草多糖和虫草素
将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的醋酸溶液（pH=4.9）中，超低温冷冻（-80℃）-微波（300W，3min）解冻循环3次，同时采用低频超声波（频率为20-30KHz，功率为350W）辅助，在在65℃下搅拌提取40min，再对提取液在60℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，收集浓缩液-70℃下冷冻24h，最后对其冷冻干燥收集水提产物干粉。最终蛹虫草子实体中虫草多糖和虫草素提取得率分别为10.17g/100g 和5.682 mg/g。
实施例3对比例：以醋酸溶液为提取剂联合低频超声波技术提取蛹虫草子实体中的虫草多糖和虫草素
将蛹虫草子实体通过采摘、清洗、干燥、粉碎、脱脂得到蛹虫草下脚料干粉，然后再将其浸入配置好的醋酸溶液（pH=4.9）中，采用低频超声波（频率为20-30KHz，功率为350W）辅助，在在65℃下搅拌提取40min，再对提取液在60℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，收集浓缩液-70℃下冷冻24h，最后对其冷冻干燥收集水提产物干粉。最终蛹虫草子实体中虫草多糖和虫草素提取得率分别为9.08g/100g 和4.782 mg/g，明显低于实例3中各活性成分得率。

Claims (10)

  1. 一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，具体过程如下：
    （1）培养及采集：蛹虫草子实体通过固体培养基培养而成，培育40-45天后进行采集，采集后的新鲜子实体在4℃下冷藏储存；
    （2）清洗干燥：将采集的蛹虫草子实体置于60-65℃热风干燥箱中干燥5h，干燥过程中蛹虫草子实体排布均匀保持通风，待干燥至恒重后收集并储藏于阴凉干燥处；
    （3）粉碎；取蛹虫草干样进行破碎，过60目筛；
    （4）脱脂：将粉碎好的样品置于回流装置中，沸水浴下用95%乙醇按料液比1:5（w:v）回流提取3h，离心，收集沉淀，60-65℃下烘干至恒重，获得脱脂虫草粉；
    （5）超低温冷冻-微波解冻循环：按照料液比1:40（w:v）向脱脂虫草粉中加入蒸馏水，得到提取液，用1mol/L的稀酸溶液搅拌滴定，使提取液pH达到4.8±0.2，将提取液置于-80℃条件下冷冻2h，取出后在300~400W功率微波条件下解冻2-3min，重复冻融操作3次，得到蛹虫草初提混合液；
    （6）超声波辅助提取：获得蛹虫草初提混合液后，辅以频率为20-30KHz，功率为300-400W的超声波，在65-70℃下提取35-40min；提取结束后4000r/min条件下离心15min，取上清液过滤得蛹虫草子实体水提物粗液；
    （7）蒸发浓缩：将蛹虫草子实体水提物粗液在60-65℃，0.095MPa条件下旋转蒸发浓缩至原体积的1%，最后呈现粘稠浓浆状后，收集浓缩液，-70℃下冷冻24h；
    （8）冷冻干燥制粉：将冷冻的蛹虫草浓缩液在冷阱温度-55℃条件下冻干3h，得到水提蛹虫草干粉。
  2. 根据权利要求1所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（1）中，固体培养基成分包括大米、小麦、葡萄糖、磷酸二氢钾、七水硫酸镁、维生素B1和维生素B2。
  3. 根据权利要求1或2所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（1）中，采集蛹虫草子实体的高度≥7cm。
  4. 根据权利要求1或2所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（3）中，破碎时先切割成3-4cm段状，再粉碎。
  5. 根据权利要求3所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（3）中，破碎时先切割成3-4cm段状，再粉碎。
  6. 根据权利要求1、2或5所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（4）中，离心的转速为4000r/min，离心时间为20min。
  7. 根据权利要求3所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（4）中，离心的转速为4000r/min，离心时间为20min。
  8. 根据权利要求4所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（4）中，离心的转速为4000r/min，离心时间为20min。
  9. 根据权利要求1、2、5、7或8所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（6）中，稀酸溶液为稀盐酸溶液、柠檬酸酸溶液或醋酸溶液。
  10. 根据权利要求6所述的一种蛹虫草中虫草多糖和虫草素的超声波复合酸性水提取方法，其特征在于，所述步骤（6）中，稀酸溶液为稀盐酸溶液、柠檬酸酸溶液或醋酸溶液。
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