CN110129824B - Method for preparing Sparassis crispa ergosterol by electric field-semi-bionic technology - Google Patents
Method for preparing Sparassis crispa ergosterol by electric field-semi-bionic technology Download PDFInfo
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- CN110129824B CN110129824B CN201910536884.5A CN201910536884A CN110129824B CN 110129824 B CN110129824 B CN 110129824B CN 201910536884 A CN201910536884 A CN 201910536884A CN 110129824 B CN110129824 B CN 110129824B
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- 208000001132 Osteoporosis Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- MECHNRXZTMCUDQ-UHFFFAOYSA-N Vitamin D2 Natural products C1CCC2(C)C(C(C)C=CC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C MECHNRXZTMCUDQ-UHFFFAOYSA-N 0.000 description 1
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- DNVPQKQSNYMLRS-APGDWVJJSA-N ergosterol group Chemical group [C@@H]1(CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)[C@H](C)\C=C\[C@H](C)C(C)C DNVPQKQSNYMLRS-APGDWVJJSA-N 0.000 description 1
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
-
- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
- C25B3/00—Electrolytic production of organic compounds
- C25B3/20—Processes
- C25B3/25—Reduction
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Materials Engineering (AREA)
- Metallurgy (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a method for preparing Sparassis crispa ergosterol by an electric field-semi-bionic technology, which comprises the following steps: (1) taking sparassis crispa sporocarp, grinding and sieving to obtain sparassis crispa powder; (2) under the condition of an external electric field, taking a strong alkaline solution with the pH of 9-10 as a raw material, and performing saponification reaction at 90 ℃ to obtain ergosterol; (3) electrolytically reducing the heavy metal ions desorbed from the denatured proteins in the step (2); (4) taking the mixed solution obtained in the step (3), and performing centrifugal separation to obtain bottom sediment, middle layer flocculate and supernatant; (5) taking the middle layer flocculate, taking an ethanol water solution as an extracting solution, adjusting the pH to 3-4 with weak acid, extracting in a water bath, and filtering to obtain a filtrate; (6) mixing the supernatant and the filtrate, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, and vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol, which has high extraction rate, can remove heavy metal and Sparassis crispa protein, has stable property, and can be absorbed well via gastrointestinal tract.
Description
Technical Field
The invention relates to the field of sparassis crispa, and in particular relates to a method for preparing sparassis crispa ergosterol by an electric field-semi-bionic technology.
Background
Ergosterol is also called ergosterol, is a characteristic alcohol contained in fungi, has large and stable content of sparassis crispa in edible fungi, is often used as a main raw material for producing vitamin D2, can promote human body to absorb Pin, phosphorus and the like, is favorable for bone formation, and prevents rickets in children, adult osteoporosis and osteomalacia, and meanwhile, ergosterol is an important component of microbial cell membranes, plays an important role in ensuring the integrity of the cell membranes, the activity of membrane-bound enzymes, the fluidity of membranes, the cell activity, the transportation of cell substances and the like, but is easily oxidized into yellow when meeting sunlight and air, and has unstable property.
In the prior art, most ergosterol is extracted by an organic solvent extraction method, a large amount of irrelevant substances are extracted while the ergosterol is extracted, a large amount of organic solvent is consumed, the ergosterol finally plays a relevant health care role through oral administration, the adaptability of an extract obtained by the organic solvent extraction method to intestinal tracts of a human body is poor, and meanwhile, the heavy metal content in the extract obtained by the organic solvent extraction method is possibly over-standard because edible fungi are easily enriched with heavy metal.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preparing Sparassis crispa ergosterol by an electric field-semi-bionic technology, the extraction rate of the ergosterol is high, the Sparassis crispa polysaccharide components are mixed through the electric field-semi-bionic extraction, heavy metals and Sparassis crispa protein are removed, the property is stable, the Sparassis crispa polysaccharide components are not easy to oxidize, and the Sparassis crispa ergosterol is well absorbed by gastrointestinal tracts.
The technical scheme of the invention is to provide a method for preparing Sparassis crispa ergosterol by an electric field-semi-bionic technology, which comprises the following steps: (1) taking sparassis crispa sporocarp, grinding and sieving to obtain sparassis crispa powder; (2) under the condition of an external electric field, taking a strong alkaline solution with the pH of 9-10 as a raw material, and performing saponification reaction at 90 ℃ to obtain ergosterol; (3) electrolytically reducing the heavy metal ions desorbed from the denatured proteins in the step (2); (4) taking the mixed solution obtained in the step (3), and performing centrifugal separation to obtain bottom sediment, middle layer flocculate and supernatant; (5) taking the middle layer flocculate, taking an ethanol water solution as an extracting solution, adjusting the pH to 3-4 with weak acid, extracting in a water bath, and filtering to obtain a filtrate; (6) mixing the supernatant and the filtrate, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, and vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol.
Preferably, in the step (3), the cathode for electrolytic reduction is a stainless steel electrode plate, and the anode is a graphite electrode plate.
Preferably, in the step (2), the electric field intensity of an external electric field is 20-40kv/cm, the pulse is 6-12 μ s, and the material-liquid ratio is 1: 20.
Preferably, the strong base in step (2) is one of NaOH or KOH.
Preferably, the weak acid in step (5) is acetic acid.
Preferably, in the step (4), the centrifugation speed is 3000-4000r/min, and the centrifugation time is 2-3 min.
Preferably, the heavy metal ions in the step (3) are one or more of chromium, lead and arsenic.
Preferably, the volume ratio of ethanol to water in the extracting solution in the step (5) is 2: 8.
The high-voltage pulse electric field is a method for making liquid food as electrolyte be placed in a container, and making it pass through high-voltage current with two discharge electrodes at the edge of the container to produce electric pulse to make cell wall and cell membrane potential of treated cell be disordered instantaneously, and can change its permeability, and can even break down cell wall and cell membrane to make them produce irreversible damage and make the components in the cell flow out.
The ergosterol in Sparassis crispa fruiting body exists in two states, free state and combined state, the prior art generally adopts alcohol alkali for saponification, the purpose of saponifying mycelium is to remove fatty acid in mycelium, lower alcohol is added into alkali liquor as saponifying agent to increase the solubility of oil in saponification liquor, so that oil and alkali alcohol solution form homogeneous reaction system, thus improving the reaction rate of saponification reaction, then organic solvent such as petroleum ether and ethyl ether is used for extracting unsaponifiable matter, but the method not only uses a large amount of organic solvent, but also uses ethyl ether or petroleum ether and other organic solvent as extracting agent in the subsequent extraction process, the organic solvent has great potential safety hazard in industrial production, and the cost is high, in addition, because ergosterol is also dissolved in ethanol, the extraction rate of ergosterol is lost by alcohol alkali extraction, in the scheme, ergosterol is saponified by adopting an alkali solution in the environment of a pulse electric field, the pulse electric field acts on the sparassis crispa solution, so that the oscillation is intensified during reaction, the ergosterol is continuously dissolved out along with the pulse to the cell membrane, the saponification reaction is prevented from being carried out at the interface between two phases, the diffusion rate of the alkali solution to grease is accelerated, the bound ergosterol in the hypha can be changed into a free state, the subsequent extraction process is prevented from using organic solvents such as diethyl ether or petroleum ether as an extracting agent, the extraction safety of the ergosterol is improved, and in addition, the production cost is greatly reduced.
The extraction of the scheme also simulates the absorption of human gastrointestinal tract to carry out the first alkali liquor extraction and then the acid liquor, namely the semi-bionic extraction, wherein the pH value of the alkali liquor is close to the intestinal tract of the human body, the pH value of the acid liquor is close to the gastric acid of the human body, the alkali liquor in the saponification reaction is used as an extractant to extract the active ingredients of the Sparassis crispa, such as Sparassis crispa polysaccharide, for the oral absorption, the solubility of the Sparassis crispa polysaccharide is more important than the consideration of the dissociation degree, the Sparassis crispa polysaccharide comprises beta-glucan which has the antioxidation performance and can clear free radicals, and then the ergosterol is extracted and purified by the acid liquor containing ethanol to enhance the solubility of the Sparassis crispa polysaccharide.
After the alkali liquor extraction and the action of the pulse electric field, the saponification temperature is higher than 80 ℃, namely the denaturation temperature of the protein, and the pH value of the alkali liquor also makes the Sparassis crispa protein denature to be called flocculate precipitation, the Sparassis crispa protein comprises sulfur protein, the protein is polluted by heavy metal in the growth environment in the growth process of the Sparassis crispa and is used as a large fungus, the Sparassis crispa is extremely rich in heavy metal, particularly chromium, the sulfur protein is rich in short peptide of cysteine, has high affinity to various heavy metals, is a protein with low molecular weight and high content of cysteine residue and metal, forms metallothionein, the heavy metal is resolved along with the denaturation of the sulfur protein after the protein is denatured, so that the heavy metal is dissolved in the solution to be in an ionic heavy metal, the heavy metal is enriched by utilizing an electrolytic reduction method in the scheme, an electrode is inserted into the sewage containing the heavy metal, heavy metal moves directionally under the action of an electric field, heavy metal simple substances are separated out from the electrode or are enriched near the electrode, so that the heavy metal is removed, at the cathode, due to the difference of the electrode potentials of the heavy metal ions, the heavy metal ions are reduced to the heavy metal simple substances at the cathode, the mixed liquid is centrifuged, due to the high density of the heavy metal, the mixed liquid is settled at the bottommost part, the spiraea residue containing ergosterol forms flocculate and is settled at the middle part, and then the heavy metal is separated and removed.
The scheme has the following beneficial effects:
(1) in the scheme, the strong base is used as an extractant for semi-bionic extraction and also used as a saponifier for generating ergosterol, and the ergosterol is extracted under the condition of not influencing the semi-bionic extraction process;
(2) the electric field destroys the Sparassis crispa cell membrane, so that ergosterol in the cell membrane is dissolved out, the semi-bionic extraction rate is improved, and meanwhile, the electric field accelerates the diffusion rate of alkali liquor to grease, the saponification reaction is accelerated, and the reaction is more thorough;
(3) at the same time of electric field-saponification, the protein of the Sparassis crispa is deteriorated, heavy metals combined with sulfur protein are resolved, the original electric field environment is utilized again, electrolytic reduction is carried out, and the heavy metals are enriched, so that pollution-free Sparassis crispa ergosterol extract is obtained;
(4) the semi-bionic extraction is adopted, so that the loss of effective components is reduced, the active components of the mixture are considered in the extraction process, the monomer components to be extracted are also controlled, and the prepared Sparassis crispa ergosterol contains Sparassis crispa polysaccharide, so that the ergosterol can be prevented from being oxidized, and the stability of the ergosterol is enhanced.
Drawings
FIG. 1 shows the results of ergosterol content measurements;
FIG. 2 shows the results of ergosterol stability tests;
FIG. 3 shows the results of heavy metal content measurement.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
Taking 100g of sparassis crispa sporocarp, grinding and crushing the sporocarp, sieving the sporocarp with a 200-mesh sieve, loading the obtained sparassis crispa powder into a high-pressure pulse device, adding 2L of NaOH solution, adjusting the pH value of the solution to be 9, heating and stirring the solution for 1h at 90 ℃, adjusting the conditions of a micro-electrolysis reactor to enable the electric field intensity of an external electric field to be 20kv/cm and the pulse to be 6 microseconds, enabling a discharge electrode to act on the sparassis crispa cell membrane, and simultaneously carrying out saponification reaction;
pouring the mixed solution into a micro-electrolysis reactor, adjusting the electrodes to enable the cathode to be a stainless steel electrode plate and the anode to be a graphite electrode plate, carrying out electrolytic reduction on the mixed solution obtained in the previous step, wherein the voltage is 1.8V, the reaction temperature is 25 ℃, the reaction time is 120min, centrifuging the obtained mixed solution after slowly removing the cathode electrode, the centrifugation speed is 3000r/min, the centrifugation time is 2min, and reserving the supernatant and the first layer of precipitate, namely middle-layer flocculate;
adding 200ml ethanol and 800ml acetic acid solution into the middle layer flocculate, adjusting pH to 3, extracting in 40 deg.C water bath for 1 hr, filtering, and collecting filtrate;
mixing the filtrate and supernatant, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol, and performing data detection on the obtained powder.
Example 2
Taking 100g of sparassis crispa sporocarp, grinding and crushing the sporocarp, sieving the sporocarp with a 200-mesh sieve, loading the obtained sparassis crispa powder into a high-pressure pulse device, adding 2L of KOH solution, adjusting the pH value of the solution to be 10, heating and stirring the solution for 1h at 90 ℃, adjusting the conditions of a micro-electrolysis reactor to enable the electric field intensity of an external electric field to be 40kv/cm and the pulse to be 12 microseconds, enabling a discharge electrode to act on the sparassis crispa cell membrane and simultaneously carrying out saponification reaction;
pouring the mixed solution into a micro-electrolysis reactor, adjusting the electrodes to enable the cathode to be a stainless steel electrode plate and the anode to be a graphite electrode plate, carrying out electrolytic reduction on the mixed solution obtained in the previous step, wherein the voltage is 1.8V, the reaction temperature is 25 ℃, the reaction time is 120min, centrifuging the obtained mixed solution after slowly removing the cathode electrode, the centrifugation speed is 4000r/min, the centrifugation time is 3min, and reserving the supernatant and the first layer of precipitate, namely middle-layer flocculate;
adding 200ml ethanol and 800ml acetic acid solution into the middle layer flocculate, adjusting pH to 4, extracting in 40 deg.C water bath for 1 hr, filtering, and collecting filtrate;
mixing the filtrate and supernatant, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol, and performing data detection on the obtained powder.
Example 3
Taking 100g of sparassis crispa sporocarp, grinding and crushing the sporocarp, sieving the sporocarp with a 200-mesh sieve, loading the obtained sparassis crispa powder into a high-pressure pulse device, adding 2L of KOH solution, adjusting the pH value of the solution to be 10, heating and stirring the solution for 1h at 90 ℃, adjusting the conditions of a micro-electrolysis reactor to enable the electric field intensity of an external electric field to be 50kv/cm and the pulse to be 10 microseconds, enabling a discharge electrode to act on the sparassis crispa cell membrane, and simultaneously carrying out saponification reaction;
pouring the mixed solution into a micro-electrolysis reactor, adjusting the electrodes to enable the cathode to be a stainless steel electrode plate and the anode to be a graphite electrode plate, carrying out electrolytic reduction on the mixed solution obtained in the previous step, wherein the voltage is 1.8V, the reaction temperature is 25 ℃, the reaction time is 120min, centrifuging the obtained mixed solution after slowly removing the cathode electrode, the centrifugation speed is 3500r/min, the centrifugation time is 3min, and reserving the supernatant and the first layer of precipitate, namely middle-layer flocculate;
adding 200ml ethanol and 800ml acetic acid solution into the middle layer flocculate, adjusting pH to 3.5, extracting in 40 deg.C water bath for 1h, filtering, and collecting filtrate;
mixing the filtrate and supernatant, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol, and performing data detection on the obtained powder.
Example 4
Taking 100g of Sparassis crispa fruiting body, grinding and crushing, sieving with a 200-mesh sieve, adding 2L of KOH solution, adjusting the pH value of the solution to 10, heating and stirring at 90 ℃ for 1h, centrifuging the obtained mixed solution at 3500r/min for 3min, taking supernatant, adding 200ml of ethanol and 800ml of acetic acid solution into the precipitate, adjusting the pH value to 3.5, extracting in water bath at 40 ℃ for 1h, filtering, and taking filtrate;
mixing the filtrate and supernatant, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol, and performing data detection on the obtained powder.
Example 5
Taking 100g of sparassis crispa sporocarp, grinding and crushing, sieving with a 200-mesh sieve, adding 2L of KOH solution into the obtained sparassis crispa powder, adjusting the pH value of the solution to 10, heating and stirring at 90 ℃ for 1h, extracting with petroleum ether, performing rotary evaporation to obtain a concentrated solution, performing vacuum freeze drying on the concentrated solution to obtain powder, namely antioxidant sparassis crispa ergosterol, and performing data detection on the obtained powder.
Example 6
The powder obtained in the above examples 1 to 5 was subjected to data detection according to a known method, and the powder was dissolved in 1L of solution for detection, and the results of ergosterol content detection are shown in FIG. 1, the results of ergosterol stability detection by treating the powder with light and an oxidizing agent are shown in FIG. 2, and the results of heavy metal content detection are shown in FIG. 3, and it was found that the ergosterol obtained by the present invention has a high extraction rate, a high content, stable properties, and no heavy metal residue.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (8)
1. A method for preparing Sparassis crispa ergosterol by electric field-semi-bionic technology is characterized by comprising the following steps:
(1) taking sparassis crispa sporocarp, grinding and sieving to obtain sparassis crispa powder;
(2) under the condition of an external electric field, taking a strong alkaline solution with the pH of 9-10 as a raw material, and performing saponification reaction at 90 ℃ to obtain free ergosterol;
(3) electrolytically reducing the heavy metal ions desorbed from the denatured proteins in the step (2);
(4) taking the mixed solution obtained in the step (3), and performing centrifugal separation to obtain bottom sediment, middle layer flocculate and supernatant;
(5) taking the middle layer flocculate, taking an ethanol water solution as an extracting solution, adjusting the pH to 3-4 with weak acid, extracting in a water bath, and filtering to obtain a filtrate;
(6) mixing the supernatant and the filtrate, adjusting pH to neutral, rotary evaporating to obtain concentrated solution, and vacuum freeze drying the concentrated solution to obtain powder, i.e. antioxidant Sparassis crispa ergosterol.
2. The method for preparing Sparassis crispa ergosterol by electric field-semi-bionic technology as claimed in claim 1, wherein in step (3), the cathode of the electrolytic reduction is stainless steel electrode plate, and the anode is graphite electrode plate.
3. The method for preparing Sparassis crispa ergosterol by using an electric field-semi-bionic technology according to claim 1, wherein in the step (2), the electric field intensity of an external electric field is 20-40kv/cm, the pulse is 6-12 μ s, and the material-liquid ratio is 1: 20.
4. The method for preparing Sparassis Crispa ergosterol by electric field-semi-bionic technology as claimed in claim 1, wherein the strong base in step (2) is one of NaOH or KOH.
5. The method for preparing Sparassis Crispa ergosterol by electric field-semi-bionic technology as claimed in claim 1, wherein the weak acid in step (5) is acetic acid.
6. The method for preparing Sparassis Crispa ergosterol by electric field-semi-bionic technology as claimed in claim 1, wherein in step (4), the centrifugation speed is 3000-.
7. The method for preparing Sparassis Crispa ergosterol by electric field-semi-bionic technology as claimed in claim 1, wherein the heavy metal ion in step (3) is one or more of chromium, lead and arsenic.
8. The method for preparing Sparassis Crispa ergosterol by electric field-semi-bionic technology as claimed in claim 1, wherein the volume ratio of ethanol to water in the extractive solution in step (5) is 2: 8.
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