Method for stably extracting active ingredients of fresh lucid ganoderma and keeping activity of active ingredients of fresh lucid ganoderma

Abstract

The invention relates to the technical field of biological extraction technology, in particular to a fresh ganoderma lucidum active ingredient stable extraction and activity maintenance method which specifically comprises the following steps: a, obtaining ganoderma lucidum micro powder; b, obtaining ganoderma lucidum micro powder treated by a pulsed electric field; pre-treated lucid ganoderma micro powder is obtained; wall-broken lucid ganoderma micro powder is obtained; c, obtaining an extracting solution; d, obtaining a concentrated filtrate; e, obtaining freeze-dried powder of the active ingredients of the fresh lucid ganoderma; f, performing ultrahigh-temperature instantaneous sterilization on the freeze-dried powder of the active ingredients of the fresh lucid ganoderma, and performing vacuum sealing, filling and storing; compared with the prior art, the extraction method of the fresh ganoderma lucidum has the advantages of high efficiency, environmental protection, stability, easiness in preservation of the activity of effective components and the like. The countercurrent extraction adopted in the invention can fully utilize the concentration gradient of the solid phase and the liquid phase in the system, so that the higher-concentration balance can be achieved when the extracting solution is discharged, the extraction rate of the extracted product can be improved, the energy consumption can be saved, and the extraction time can be shortened.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction technology, and in particular to a method for stably extracting active ingredients of fresh ganoderma lucidum and maintaining their activity. Background Art

[0002] Ganoderma lucidum, also known as sedge, is a medicinal and edible fungus of the family Ganoderma, ...

[0003] Therefore, if Ganoderma lucidum is eaten directly, the absorption effect of the active ingredients will be greatly reduced, and its active ingredients cannot be effectively utilized, which greatly reduces its effect. In addition, due to the low absorption rate, a large amount of active ingredients will be wasted.

[0004] At present, most of the existing extraction methods have defects such as low polysaccharide yield, non-green extraction process, and relatively poor extraction stability.

[0005] Therefore, it is very important to develop a stable extraction technology. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for stably extracting active ingredients from fresh Ganoderma lucidum and maintaining their activity.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for stably extracting and maintaining the activity of active ingredients from fresh Ganoderma lucidum, comprising the following steps:

[0009] a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80 to -70°C for 10 to 12 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum powder;

[0010] b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field;

[0011] The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder;

[0012] Then the pretreated Ganoderma lucidum powder is subjected to enzymatic hydrolysis, inactivated at 55°C for 10 min, and dried to obtain wall-broken Ganoderma lucidum powder;

[0013] c. performing countercurrent extraction on the broken-wall Ganoderma lucidum micropowder obtained in step b to obtain an extract;

[0014] d. filtering the extract obtained in step c by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate;

[0015] e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum;

[0016] f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage;

[0017] The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s.

[0018] As a further technical solution, the distilled water cleaning in step a is performed at room temperature, and the cleaning time is 10 to 15 minutes;

[0019] The vacuum drying temperature was 50°C and the time was 10 h.

[0020] As a further technical solution, the crushed material is screened through a 60-mesh screen;

[0021] The set speed is 2200r / min and the optimal crushing time is 20min.

[0022] As a further technical solution, the pulse electric field treatment in step b is a two-stage pulse electric field treatment, and the entire pulse electric field treatment is carried out under the condition that the Ganoderma lucidum micropowder is prepared into a slurry, specifically including: an initial pulse electric field treatment, a blank stage, and a final pulse electric field treatment;

[0023] Ganoderma lucidum powder is prepared into a slurry as follows:

[0024] Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use;

[0025] Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5;

[0026] After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

[0027] As a further technical solution, the electric field intensity of the initial pulse electric field treatment is 1.2-1.6 kV / cm, the pulse width is 18-20 μs, and the number of pulses is 6-8 times;

[0028] The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment;

[0029] The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz;

[0030] The electric field intensity of the terminal pulse electric field treatment is 1.8-2.0 kV / cm, the pulse width is 22-25 μs, and the pulse number is 10-12 times.

[0031] As a further technical solution, the pressure difference expansion treatment in step b is:

[0032] The ganoderma lucidum powder treated by the pulse electric field is added to the puffing tank, and treated at a puffing temperature of 70-80°C for 1-1.5 minutes to make the puffing pressure reach 0.2-0.3MPa, and then the pressure is released, and the vacuum drying temperature is 50-55°C, maintained for 60-70 minutes to obtain the pretreated ganoderma lucidum powder.

[0033] As a further technical solution, the enzymatic treatment in step b is:

[0034] The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis;

[0035] Wherein, the mass concentration of the complex enzyme in the mixed system is 3-4%;

[0036] The complex enzyme is a mixture of cellulase, chitinase and glucosidase;

[0037] Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:1.

[0038] As a further technical solution, the countercurrent extraction in step c is carried out by a three-stage tank group dynamic countercurrent extraction method;

[0039] The three-stage tank group dynamic countercurrent extraction method specifically comprises: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, using the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and rapidly cooling the extract to 20-30° C. after filtering;

[0040] Among them, the primary extraction solvent is distilled water, the extraction temperature is 85-95°C, and the extraction time is 20-30min.

[0041] As a further technical solution, the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis in step d is:

[0042] Among them, the pore size of the microfiltration membrane is 0.2 μm;

[0043] The pore size of the nanofiltration membrane is 0.01 μm;

[0044] The pore size of the reverse osmosis membrane is 0.3nm;

[0045] The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz;

[0046] The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min;

[0047] The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

[0048] As a further technical solution, the specific method of freeze-drying in step e is:

[0049] The concentrated filtrate is pre-frozen at a temperature of -48 to -45°C for 20 minutes;

[0050] After pre-freezing, adjust the temperature to -25 ~ -20 ° C, turn on the vacuum at the same time, adjust the vacuum degree to 0.5Pa, maintain for 10-12 hours, then continue to adjust the temperature to -3 ~ -2 ° C, keep warm for 5-6 hours, and finally adjust the temperature to 50-52 ° C, keep warm for 6-7 hours to complete the freeze-drying process.

[0051] The storage temperature is 1-2℃.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] Compared with the prior art, the method for extracting fresh Ganoderma lucidum provided by the present invention has the advantages of high efficiency, environmental protection, stability and easy preservation of the activity of effective ingredients.

[0054] The countercurrent extraction adopted in the present invention can make full use of the concentration gradient of the solid-liquid phase in the system, so that the extract reaches a higher concentration balance when discharging, which can not only improve the extraction rate of the extract, but also save energy and shorten the extraction time.

[0055] The extraction method of the present invention can extract the active ingredients from fresh Ganoderma lucidum to a greater extent through the sequential superposition and coordination of various steps, and has a high extraction rate, which can greatly reduce the loss of active ingredients in the fresh Ganoderma lucidum during the extraction process.

[0056] The present invention can greatly improve the dissolution rate of the effective ingredients in the fresh Ganoderma lucidum through the coordination and promotion of multiple treatment steps in step b, facilitate subsequent extraction, and greatly improve the extraction stability of the fresh Ganoderma lucidum.

[0057] The present invention first freezes fresh Ganoderma lucidum and then grinds it to achieve a preliminary crushing effect on the Ganoderma lucidum for subsequent processing. Then, a high-frequency alternating current greater than a certain threshold is applied to the cell wall through a multi-stage pulse electric field treatment to generate a higher potential gradient. This strong electric field change causes the spatial orientation of charged ions in Ganoderma lucidum micropowder and polar molecules in cells to change at a high frequency with the current, and the ions and molecules rub and collide with each other, thereby destroying the cell structure. When the Ganoderma lucidum micropowder cells are exposed to a pulsed electric field, the intensity of the electric field changes rapidly to form short high-voltage pulses. The intensity of these pulses is sufficient to destroy the integrity of the cell wall, increase the permeability of the cells, and achieve a preliminary crushing effect, which can pave the way for the subsequent wall breaking of the composite enzyme, thereby improving the extraction of active ingredients from fresh Ganoderma lucidum. In combination with the pressure difference expansion treatment, the pressure difference refers to the process in which the high pressure in the cavity is changed to low pressure when the material is connected to the vacuum tank during the expansion process. This process causes the internal moisture of the Ganoderma lucidum micropowder after the pulsed electric field treatment to be instantly vaporized, so that the Ganoderma lucidum micropowder forms a fluffy structural state, which can destroy the cell wall structure of the Ganoderma lucidum cells, thereby making the active ingredients in the fresh Ganoderma lucidum cells more easily released. This is conducive to the subsequent extraction of active ingredients from fresh Ganoderma lucidum, greatly improving the extraction rate of active ingredients from fresh Ganoderma lucidum. In order to further improve the release of active ingredients from fresh Ganoderma lucidum, the present invention further cooperates with the composite enzyme treatment to further destroy the cell structure of fresh Ganoderma lucidum and further promote the release of effective ingredients in fresh Ganoderma lucidum.

[0058] In order to maintain the stability of the active ingredients, the present invention preserves the extracted active ingredients of Ganoderma lucidum in a low temperature, light-proof and vacuum environment, which can inhibit the growth of microorganisms and the activity of enzymes, thereby effectively avoiding the loss of active ingredients in Ganoderma lucidum, ensuring the high efficacy of the active ingredients in Ganoderma lucidum, and greatly extending the shelf life of fresh active ingredients of Ganoderma lucidum. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0060] Figure 1 This is the effect of countercurrent extraction at different temperatures on the yield of polysaccharides;

[0061] Figure 2 This is a graph comparing the effects of different puffing temperatures on polysaccharide yields;

[0062] Figure 3 This is an electron micrograph of the broken-wall Ganoderma lucidum powder of Example 1. DETAILED DESCRIPTION

[0063] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] The following are some of the equipment used in the present invention:

[0065] Microfiltration membrane 19778 (PVDF) Chengdu Hecheng Filtration Technology Co., Ltd.;

[0066] Nanofiltration membrane 19909 (PA, MWCO = 150-250Da) Chengdu Hecheng Filtration Technology Co., Ltd.;

[0067] Reverse osmosis membrane 19969 (PA, MWCO ≤ 150Da) Chengdu Hecheng Filtration Technology Co., Ltd.;

[0068] High pressure membrane separation equipment HC-XS18-6X Chengdu Hecheng Filtration Technology Co., Ltd.

[0069] LGJ-10FD ordinary vacuum freeze dryer.

[0070] ZGRVP variable temperature pressure difference expansion equipment: Zhucheng Zhonggong Machinery Co., Ltd.

[0071] Example 1

[0072] A method for stably extracting and maintaining the activity of active ingredients from fresh Ganoderma lucidum, comprising the following steps:

[0073] a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80°C for 10 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum micro powder. Wash with distilled water at room temperature for 10 minutes.

[0074] The vacuum drying temperature was 50°C and the time was 10 h.

[0075] The crushed material is sieved through a 60-mesh sieve;

[0076] The speed was set to 2200 r / min and the optimal crushing time was 20 min;

[0077] b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field; the pulse electric field treatment is performed in two stages, and the entire pulse electric field treatment is performed under the condition that the ganoderma lucidum powder is prepared into a slurry, specifically including: an initial stage of pulse electric field treatment, a blank stage, and a final stage of pulse electric field treatment;

[0078] Ganoderma lucidum powder is prepared into a slurry as follows:

[0079] Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use;

[0080] Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5;

[0081] After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

[0082] The electric field intensity of the initial pulse electric field treatment was 1.2 kV / cm, the pulse width was 18 μs, and the number of pulses was 6;

[0083] The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment;

[0084] The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz;

[0085] The electric field intensity of the final pulse electric field treatment is 1.8 kV / cm, the pulse width is 22 μs, and the pulse number is 10 times.

[0086] The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder;

[0087] The pressure differential expansion process is:

[0088] The Ganoderma lucidum micropowder treated by the pulse electric field was added to the puffing tank and treated at a puffing temperature of 75°C for 1 minute to make the puffing pressure reach 0.2MPa, and then the pressure was released. The vacuum drying temperature was 50°C and maintained for 60 minutes to obtain the pretreated Ganoderma lucidum micropowder.

[0089] The pretreated Ganoderma lucidum powder is then subjected to enzymatic hydrolysis treatment, and the enzyme is inactivated at 55°C for 10 minutes, and then dried to obtain wall-broken Ganoderma lucidum powder. The enzymatic hydrolysis treatment is as follows:

[0090] The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis;

[0091] The mass concentration of the complex enzyme in the mixed system is 3%;

[0092] The complex enzyme is a mixture of cellulase, chitinase and glucosidase;

[0093] Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:1.

[0094] c. performing countercurrent extraction on the broken-wall Ganoderma lucidum powder obtained in step b to obtain an extract; the countercurrent extraction is performed using a three-stage tank group dynamic countercurrent extraction method;

[0095] The three-stage tank group dynamic countercurrent extraction method specifically comprises: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, using the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and rapidly cooling the extract to 20° C. after filtering;

[0096] Among them, the primary extraction solvent is distilled water, the extraction temperature is 90°C, and the extraction time is 20 minutes.

[0097] d. The extract obtained in step c is filtered by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate; the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis is:

[0098] Among them, the pore size of the microfiltration membrane is 0.2 μm;

[0099] The pore size of the nanofiltration membrane is 0.01 μm;

[0100] The pore size of the reverse osmosis membrane is 0.3nm;

[0101] The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz;

[0102] The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min;

[0103] The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

[0104] e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum;

[0105] f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage;

[0106] The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s; the specific method of freeze-drying is:

[0107] The concentrated filtrate was pre-frozen at -48°C for 20 min;

[0108] After pre-freezing, the temperature was adjusted to -25°C, and the vacuum was turned on at the same time. The vacuum degree was adjusted to 0.5Pa and maintained for 10 hours. The temperature was then adjusted to -3°C and kept warm for 5 hours. Finally, the temperature was adjusted to 50°C and kept warm for 6 hours to complete the freeze-drying process.

[0109] Example 2

[0110] A method for stably extracting and maintaining the activity of active ingredients from fresh Ganoderma lucidum, comprising the following steps:

[0111] a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80°C for 10 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum micro powder. Wash with distilled water at room temperature for 10 minutes.

[0112] The vacuum drying temperature was 50°C and the time was 10 h.

[0113] The crushed material is sieved through a 60-mesh sieve;

[0114] The speed was set to 2200 r / min and the optimal crushing time was 20 min;

[0115] b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field; the pulse electric field treatment is performed in two stages, and the entire pulse electric field treatment is performed under the condition that the ganoderma lucidum powder is prepared into a slurry, specifically including: an initial stage of pulse electric field treatment, a blank stage, and a final stage of pulse electric field treatment;

[0116] Ganoderma lucidum powder is prepared into a slurry as follows:

[0117] Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use;

[0118] Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5;

[0119] After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

[0120] The electric field intensity of the initial pulse electric field treatment was 1.5 kV / cm, the pulse width was 18 μs, and the number of pulses was 8 times;

[0121] The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment;

[0122] The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz;

[0123] The electric field intensity of the final pulse electric field treatment is 1.9 kV / cm, the pulse width is 22 μs, and the pulse number is 10 times.

[0124] The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder;

[0125] The pressure differential expansion process is:

[0126] The Ganoderma lucidum micropowder treated by the pulse electric field was added to the puffing tank and treated at a puffing temperature of 75°C for 1 minute to make the puffing pressure reach 0.25MPa, and then the pressure was released. The vacuum drying temperature was 53°C and maintained for 62 minutes to obtain the pretreated Ganoderma lucidum micropowder.

[0127] The pretreated Ganoderma lucidum powder is then subjected to enzymatic hydrolysis treatment, and the enzyme is inactivated at 55°C for 10 minutes, and then dried to obtain wall-broken Ganoderma lucidum powder. The enzymatic hydrolysis treatment is as follows:

[0128] The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis;

[0129] Among them, the mass concentration of the complex enzyme in the mixed system is 3.5%;

[0130] The complex enzyme is a mixture of cellulase, chitinase and glucosidase;

[0131] Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:1.

[0132] c. performing countercurrent extraction on the broken-wall Ganoderma lucidum powder obtained in step b to obtain an extract; the countercurrent extraction is performed using a three-stage tank group dynamic countercurrent extraction method;

[0133] The three-stage tank group dynamic countercurrent extraction method specifically includes: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, and the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and the extract is filtered out and then rapidly cooled to 25°C;

[0134] Among them, the primary extraction solvent is distilled water, the extraction temperature is 90°C, and the extraction time is 25min.

[0135] d. The extract obtained in step c is filtered by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate; the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis is:

[0136] Among them, the pore size of the microfiltration membrane is 0.2 μm;

[0137] The pore size of the nanofiltration membrane is 0.01 μm;

[0138] The pore size of the reverse osmosis membrane is 0.3nm;

[0139] The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz;

[0140] The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min;

[0141] The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

[0142] e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum;

[0143] f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage;

[0144] The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s; the specific method of freeze-drying is:

[0145] The concentrated filtrate was pre-frozen at -46°C for 20 min;

[0146] After pre-freezing, the temperature was adjusted to -22°C, and the vacuum was turned on at the same time. The vacuum degree was adjusted to 0.5Pa and maintained for 10 hours. The temperature was then adjusted to -3°C and kept warm for 5.5 hours. Finally, the temperature was adjusted to 51°C and kept warm for 6 hours to complete the freeze-drying process.

[0147] Example 3

[0148] A method for stably extracting and maintaining the activity of active ingredients from fresh Ganoderma lucidum, comprising the following steps:

[0149] a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80°C for 10 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum micro powder. Wash with distilled water at room temperature for 10 minutes.

[0150] The vacuum drying temperature was 50°C and the time was 10 h.

[0151] The crushed material is sieved through a 60-mesh sieve;

[0152] The speed was set to 2200 r / min and the optimal crushing time was 20 min;

[0153] b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field; the pulse electric field treatment is performed in two stages, and the entire pulse electric field treatment is performed under the condition that the ganoderma lucidum powder is prepared into a slurry, specifically including: an initial stage of pulse electric field treatment, a blank stage, and a final stage of pulse electric field treatment;

[0154] Ganoderma lucidum powder is prepared into a slurry as follows:

[0155] Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use;

[0156] Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5;

[0157] After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

[0158] The electric field intensity of the initial pulse electric field treatment was 1.5 kV / cm, the pulse width was 20 μs, and the number of pulses was 7 times;

[0159] The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment;

[0160] The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz;

[0161] The electric field intensity of the final pulse electric field treatment is 1.9 kV / cm, the pulse width is 24 μs, and the pulse number is 11 times.

[0162] The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder;

[0163] The pressure differential expansion process is:

[0164] The Ganoderma lucidum micropowder treated by the pulse electric field was added to the puffing tank and treated at a puffing temperature of 75°C for 1.5 minutes to make the puffing pressure reach 0.26MPa, and then the pressure was released. The vacuum drying temperature was 52°C and maintained for 65 minutes to obtain the pretreated Ganoderma lucidum micropowder.

[0165] The pretreated Ganoderma lucidum powder is then subjected to enzymatic hydrolysis treatment, and the enzyme is inactivated at 55°C for 10 minutes, and then dried to obtain wall-broken Ganoderma lucidum powder. The enzymatic hydrolysis treatment is as follows:

[0166] The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis;

[0167] Among them, the mass concentration of the complex enzyme in the mixed system is 3.3%;

[0168] The complex enzyme is a mixture of cellulase, chitinase and glucosidase;

[0169] Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:1.

[0170] c. performing countercurrent extraction on the broken-wall Ganoderma lucidum powder obtained in step b to obtain an extract; the countercurrent extraction is performed using a three-stage tank group dynamic countercurrent extraction method;

[0171] The three-stage tank group dynamic countercurrent extraction method specifically includes: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, and the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and the extract is filtered out and then rapidly cooled to 25°C;

[0172] Among them, the primary extraction solvent is distilled water, the extraction temperature is 90°C, and the extraction time is 25min.

[0173] d. The extract obtained in step c is filtered by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate; the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis is:

[0174] Among them, the pore size of the microfiltration membrane is 0.2 μm;

[0175] The pore size of the nanofiltration membrane is 0.01 μm;

[0176] The pore size of the reverse osmosis membrane is 0.3nm;

[0177] The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz;

[0178] The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min;

[0179] The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

[0180] e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum;

[0181] f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage;

[0182] The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s; the specific method of freeze-drying is:

[0183] The concentrated filtrate was pre-frozen at -46°C for 20 min;

[0184] After pre-freezing, the temperature was adjusted to -24°C, and the vacuum was turned on at the same time. The vacuum degree was adjusted to 0.5Pa and maintained for 11 hours. The temperature was then adjusted to -3°C and kept warm for 5 hours. Finally, the temperature was adjusted to 51°C and kept warm for 6 hours to complete the freeze-drying process.

[0185] Example 4

[0186] A method for stably extracting and maintaining the activity of active ingredients from fresh Ganoderma lucidum, comprising the following steps:

[0187] a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80°C for 10 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum micro powder. Wash with distilled water at room temperature for 10 minutes.

[0188] The vacuum drying temperature was 50°C and the time was 10 h.

[0189] The crushed material is sieved through a 60-mesh sieve;

[0190] The speed was set to 2200 r / min and the optimal crushing time was 20 min;

[0191] b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field; the pulse electric field treatment is performed in two stages, and the entire pulse electric field treatment is performed under the condition that the ganoderma lucidum powder is prepared into a slurry, specifically including: an initial stage of pulse electric field treatment, a blank stage, and a final stage of pulse electric field treatment;

[0192] Ganoderma lucidum powder is prepared into a slurry as follows:

[0193] Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use;

[0194] Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5;

[0195] After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

[0196] The electric field intensity of the initial pulse electric field treatment was 1.4 kV / cm, the pulse width was 18 μs, and the number of pulses was 6;

[0197] The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment;

[0198] The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz;

[0199] The electric field intensity of the final pulse electric field treatment is 1.8 kV / cm, the pulse width is 22 μs, and the pulse number is 12 times.

[0200] The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder;

[0201] The pressure differential expansion process is:

[0202] The Ganoderma lucidum micropowder treated by the pulse electric field was added to the puffing tank and treated at a puffing temperature of 75°C for 1.2 minutes to make the puffing pressure reach 0.28MPa, and then the pressure was released. The vacuum drying temperature was 53°C and maintained for 65 minutes to obtain the pretreated Ganoderma lucidum micropowder.

[0203] The pretreated Ganoderma lucidum powder is then subjected to enzymatic hydrolysis treatment, and the enzyme is inactivated at 55°C for 10 minutes, and then dried to obtain wall-broken Ganoderma lucidum powder. The enzymatic hydrolysis treatment is as follows:

[0204] The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis;

[0205] Among them, the mass concentration of the complex enzyme in the mixed system is 3.8%;

[0206] The complex enzyme is a mixture of cellulase, chitinase and glucosidase;

[0207] Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:1.

[0208] c. performing countercurrent extraction on the broken-wall Ganoderma lucidum powder obtained in step b to obtain an extract; the countercurrent extraction is performed using a three-stage tank group dynamic countercurrent extraction method;

[0209] The three-stage tank group dynamic countercurrent extraction method specifically includes: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, and the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and the extract is filtered out and then rapidly cooled to 26°C;

[0210] Among them, the primary extraction solvent is distilled water, the extraction temperature is 90°C, and the extraction time is 25min.

[0211] d. The extract obtained in step c is filtered by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate; the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis is:

[0212] Among them, the pore size of the microfiltration membrane is 0.2 μm;

[0213] The pore size of the nanofiltration membrane is 0.01 μm;

[0214] The pore size of the reverse osmosis membrane is 0.3nm;

[0215] The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz;

[0216] The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min;

[0217] The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

[0218] e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum;

[0219] f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage;

[0220] The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s; the specific method of freeze-drying is:

[0221] The concentrated filtrate was pre-frozen at -47°C for 20 min;

[0222] After pre-freezing, the temperature was adjusted to -23°C, and the vacuum was turned on at the same time. The vacuum degree was adjusted to 0.5Pa and maintained for 12 hours. The temperature was then adjusted to -3°C and kept warm for 5 hours. Finally, the temperature was adjusted to 51°C and kept warm for 6.5 hours to complete the freeze-drying process.

[0223] Example 5

[0224] A method for stably extracting and maintaining the activity of active ingredients from fresh Ganoderma lucidum, comprising the following steps:

[0225] a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80°C for 10 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum micro powder. Wash with distilled water at room temperature for 10 minutes.

[0226] The vacuum drying temperature was 50°C and the time was 10 h.

[0227] The crushed material is sieved through a 60-mesh sieve;

[0228] The speed was set to 2200 r / min and the optimal crushing time was 20 min;

[0229] b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field; the pulse electric field treatment is performed in two stages, and the entire pulse electric field treatment is performed under the condition that the ganoderma lucidum powder is prepared into a slurry, specifically including: an initial stage of pulse electric field treatment, a blank stage, and a final stage of pulse electric field treatment;

[0230] Ganoderma lucidum powder is prepared into a slurry as follows:

[0231] Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use;

[0232] Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5;

[0233] After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

[0234] The electric field intensity of the initial pulse electric field treatment was 1.6 kV / cm, the pulse width was 20 μs, and the number of pulses was 8 times;

[0235] The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment;

[0236] The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz;

[0237] The electric field intensity of the final pulse electric field treatment is 2.0 kV / cm, the pulse width is 25 μs, and the pulse number is 12 times.

[0238] The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder;

[0239] The pressure differential expansion process is:

[0240] The Ganoderma lucidum micropowder treated by the pulse electric field was added to the puffing tank and treated at a puffing temperature of 75°C for 1.5 minutes to make the puffing pressure reach 0.3MPa, and then the pressure was released. The vacuum drying temperature was 55°C and maintained for 60 minutes to obtain the pretreated Ganoderma lucidum micropowder.

[0241] The pretreated Ganoderma lucidum powder is then subjected to enzymatic hydrolysis treatment, and the enzyme is inactivated at 55°C for 10 minutes, and then dried to obtain wall-broken Ganoderma lucidum powder. The enzymatic hydrolysis treatment is as follows:

[0242] The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis;

[0243] Among them, the mass concentration of the complex enzyme in the mixed system is 4%;

[0244] The complex enzyme is a mixture of cellulase, chitinase and glucosidase;

[0245] Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:1.

[0246] c. performing countercurrent extraction on the broken-wall Ganoderma lucidum powder obtained in step b to obtain an extract; the countercurrent extraction is performed using a three-stage tank group dynamic countercurrent extraction method;

[0247] The three-stage tank group dynamic countercurrent extraction method specifically includes: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, and the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and the extract is filtered out and then rapidly cooled to 30°C;

[0248] Among them, the primary extraction solvent is distilled water, the extraction temperature is 90°C, and the extraction time is 30min.

[0249] d. The extract obtained in step c is filtered by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate; the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis is:

[0250] Among them, the pore size of the microfiltration membrane is 0.2 μm;

[0251] The pore size of the nanofiltration membrane is 0.01 μm;

[0252] The pore size of the reverse osmosis membrane is 0.3nm;

[0253] The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz;

[0254] The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min;

[0255] The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

[0256] e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum;

[0257] f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage;

[0258] The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s; the specific method of freeze-drying is:

[0259] The concentrated filtrate was pre-frozen at -45°C for 20 min;

[0260] After pre-freezing, the temperature was adjusted to -20°C, and the vacuum was turned on at the same time. The vacuum degree was adjusted to 0.5Pa and maintained for 12 hours. The temperature was then adjusted to -2°C and kept warm for 5-6 hours. Finally, the temperature was adjusted to 52°C and kept warm for 7 hours to complete the freeze-drying process.

[0261] Comparative Example 1: This comparative example is basically the same as Example 1, except that it is not subjected to enzymatic treatment.

[0262] Comparative Example 2: This comparative example is basically the same as Example 1, except that it does not undergo pressure differential expansion treatment.

[0263] Comparative Example 3: This comparative example is basically the same as Example 1, except that it is not subjected to pulse electric field treatment.

[0264] Comparative Example 4: This comparative example is basically the same as Example 1, except that the three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis is replaced by microfiltration only.

[0265] Test experiment:

[0266] Polysaccharide content detection:

[0267] Ganoderma lucidum was accurately weighed and divided into 5 groups, 3.0 g in each group. The active ingredients were extracted by the methods of the embodiment and the comparative example respectively, and the weights were weighed and recorded. The active ingredients were dissolved in distilled water and the volume was fixed to 100 mL to obtain a solution. The solution was then determined by phenol-concentrated sulfuric acid method. The mass concentration of polysaccharides in the solution was calculated from the absorbance according to the regression equation of the standard curve, and the polysaccharide yield of each treatment was calculated according to the following formula:

[0268]

[0269] wPolysaccharide yield, %;

[0270] cPolysaccharide mass concentration, mg / mL;

[0271] v constant volume, mL;

[0272] m total mass of Ganoderma lucidum, g;

[0273] Table 1

[0274]

[0275]

[0276] It can be seen from Table 1 that the polysaccharide content extracted by the method of the present invention is relatively high.

[0277] Ganoderma lucidum brass content detection:

[0278] The total flavonoid content in the samples extracted from the examples and comparative examples was determined by NaNO2-Al(NO3)3-NaOH colorimetry, and the yield of total flavonoids in each example was calculated:

[0279] Table 2

[0280] Total flavonoids yield% Example 1 0.355 Example 2 0.373 Example 3 0.335 Example 4 0.330 Example 5 0.346 Comparative Example 1 0.294 Comparative Example 2 0.307 Comparative Example 3 0.312 Comparative Example 4 0.270

[0281] It can be seen from Table 2 that the extraction method of the present invention can extract more total flavonoids from Ganoderma lucidum, and can increase the content of the extracted active ingredients of Ganoderma lucidum.

[0282] Stability test:

[0283] Tested using the extraction method of Example 1:

[0284] Equal amounts of fresh Ganoderma lucidum samples were extracted 8 times, and the changes in polysaccharide yields were compared each time:

[0285] Table 3

[0286] Polysaccharide yield% 1st time 2.093 2nd 2.088 3rd 2.079 4th 2.083 5th 2.081 6th 2.087 7th 2.084 8th 2.082

[0287] It can be seen from Table 3 that the method of the present invention has a high stable extraction performance, and multiple extractions can maintain a small extraction error.

[0288] DPPH free radical scavenging assay

[0289] The active ingredient sample solution of Ganoderma lucidum (1 mL, concentration 100 μg / mL) was extracted and uniformly mixed with 0.2 mmol of LDPPH anhydrous ethanol solution (1 mL), and then reacted for 30 minutes under light-proof conditions. The absorbance value (517 nm) was measured in a preheated visible light spectrophotometer as Ai;

[0290] The active ingredient sample solution of Ganoderma lucidum (1 mL, concentration 100 μg / mL) and anhydrous ethanol solution (1 mL) were extracted and mixed evenly. The mixture was reacted for 30 min in a light-proof condition. The absorbance value (517 nm) was measured in a preheated visible light spectrophotometer as Aj.

[0291] 0.2mmol / LDPPH anhydrous ethanol solution (1mL) and anhydrous ethanol solution (1mL) were mixed evenly and reacted for 30min in a light-proof condition. The absorbance value (517nm) was measured as A0 in a preheated visible light spectrophotometer.

[0292] The formula for DPPH free radical scavenging rate (I%) is:

[0293] Hydroxyl radical scavenging rate (I%) = [1-(Ai-Aj) / A0] × 100%;

[0294] Where: A0 is the absorbance of the blank control group; Ai is the absorbance after adding the polysaccharide solution; Aj is the absorbance of the background of the test solution;

[0295] Table 4

[0296] Hydroxyl free radical scavenging rate% Example 1 94.6 Example 2 95.8 Example 3 95.1 Example 4 95.3 Example 5 94.2 Comparative Example 1 88.6 Comparative Example 2 89.2 Comparative Example 3 92.7 Comparative Example 4 85.7

[0297] It can be seen from Table 4 that the active ingredients extracted by the method of the present invention have strong antioxidant activity and a high hydroxyl radical scavenging rate.

[0298] Taking Example 1 as the basic sample, the samples were stored at different temperatures for 60 days and then tested to see the effects on the scavenging rate of hydroxyl radicals in each group of samples:

[0299] Table 5

[0300] Temperature ℃ Hydroxyl free radical scavenging rate% 1 92.1 2 91.9 3 88.5 4 86.7 5 84.1 6 81.2 7 78.8

[0301] It can be seen from Table 5 that with the increase of storage temperature, the hydroxyl radical scavenging rate decreases continuously after long-term storage, indicating that storage at 1-2°C can better ensure the activity stability of Ganoderma lucidum active ingredients.

[0302] Based on Example 1, the effects of different countercurrent extraction temperatures on polysaccharide yields were compared. Figure 1 .

[0303] Based on Example 1, the effects of different expansion temperatures on polysaccharide yields were compared. Figure 2 .

[0304] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for stable extraction and activity maintenance of active ingredients of fresh Ganoderma lucidum, characterized in that: The specific steps include: a. After picking fresh Ganoderma lucidum, wash it with distilled water, and then vacuum dry it. After drying, freeze it in an ultra-low temperature freezer at -80 to -70°C for 10 to 12 hours. Put the frozen fresh Ganoderma lucidum into a ball mill for crushing. Sieve the crushed material to obtain Ganoderma lucidum powder; b. treating the ganoderma lucidum powder obtained in step a with a pulse electric field to obtain ganoderma lucidum powder treated with a pulse electric field; The Ganoderma lucidum treated by the pulse electric field is subjected to pressure difference expansion treatment to obtain pre-treated Ganoderma lucidum micro powder; Then the pretreated Ganoderma lucidum powder is subjected to enzymatic hydrolysis, inactivated at 55°C for 10 min, and dried to obtain wall-broken Ganoderma lucidum powder; c. performing countercurrent extraction on the broken-wall Ganoderma lucidum micropowder obtained in step b to obtain an extract; d. filtering the extract obtained in step c by a three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis to obtain a concentrated filtrate; e. freeze-drying the concentrated filtrate to obtain freeze-dried powder of active ingredients of fresh Ganoderma lucidum; f. subjecting the fresh Ganoderma lucidum active ingredient freeze-dried powder obtained in step e to ultra-high temperature instantaneous sterilization, and vacuum-sealing and filling for storage; The heating temperature of ultra-high temperature instantaneous sterilization is 136°C and the heating time is 4s.

2. The method for stable extraction and activity preservation of active ingredients of fresh Ganoderma lucidum according to claim 1, characterized in that: The distilled water cleaning in step a is performed at room temperature for 10 to 15 minutes; The vacuum drying temperature was 50°C and the time was 10 h.

3. The method for stable extraction and activity preservation of active ingredients of fresh Ganoderma lucidum according to claim 2, characterized in that: The crushed material is sieved through a 60-mesh sieve; The set speed is 2200r / min and the optimal crushing time is 20min.

4. The method for stable extraction and activity preservation of active ingredients of fresh Ganoderma lucidum according to claim 1, characterized in that: The pulse electric field treatment in step b is a two-stage pulse electric field treatment, and the entire pulse electric field treatment is carried out under the condition that the Ganoderma lucidum micropowder is prepared into a slurry, specifically including: an initial pulse electric field treatment, a blank stage, and a final pulse electric field treatment; Ganoderma lucidum powder is prepared into a slurry as follows: Preheat distilled water to 50°C, and then keep it warm for 10 minutes to obtain preheated distilled water, then add Ganoderma lucidum powder to the preheated distilled water, stir at a speed of 500r / min for 20 minutes to obtain Ganoderma lucidum powder slurry, and keep it warm for later use; Ganoderma lucidum powder and distilled water were mixed in a mass ratio of 1:5; After being treated by two pulse electric fields, the Ganoderma lucidum micropowder slurry is spray-dried to obtain Ganoderma lucidum micropowder treated by the pulse electric field.

5. The method for stable extraction and activity preservation of active ingredients of fresh Ganoderma lucidum according to claim 4, characterized in that: The electric field intensity of the initial pulse electric field treatment is 1.2-1.6 kV / cm, the pulse width is 18-20 μs, and the number of pulses is 6-8 times; The blank period is the time between the end of the initial pulse electric field treatment and the beginning of the final pulse electric field treatment; The blank period is 5 minutes, and the blank period is treated with ultrasound at a frequency of 40 kHz; The electric field intensity of the terminal pulse electric field treatment is 1.8-2.0 kV / cm, the pulse width is 22-25 μs, and the pulse number is 10-12 times.

6. The method for stable extraction and activity preservation of active ingredients from fresh Ganoderma lucidum according to claim 1, characterized in that: The pressure difference expansion process in step b is: The ganoderma lucidum powder treated by the pulse electric field is added to the puffing tank, and treated at a puffing temperature of 70-80°C for 1-1.5 minutes to make the puffing pressure reach 0.2-0.3MPa, and then the pressure is released, and the vacuum drying temperature is 50-55°C, maintained for 60-70 minutes to obtain the pretreated ganoderma lucidum powder.

7. The method for stable extraction and activity preservation of active ingredients from fresh Ganoderma lucidum according to claim 1, characterized in that: The enzymatic treatment in step b is: The pretreated Ganoderma lucidum powder was mixed with distilled water at a mass ratio of 1:5, and then the complex enzyme was added, the temperature was adjusted to 38°C, and the water bath was kept warm for 4 hours to complete the enzymatic hydrolysis; Wherein, the mass concentration of the complex enzyme in the mixed system is 3-4%; The complex enzyme is a mixture of cellulase, chitinase and glucosidase; Among them, the mixed mass ratio of cellulase, chitinase and glucosidase is 3:1:

1.

8. The method for stable extraction and activity preservation of active ingredients from fresh Ganoderma lucidum according to claim 1, characterized in that: In step c, the countercurrent extraction is performed using a three-stage tank group dynamic countercurrent extraction method; The three-stage tank group dynamic countercurrent extraction method specifically comprises: using the primary extract obtained from the primary extraction as the extraction solvent for the secondary extraction, using the secondary extract obtained from the secondary extraction as the extraction solvent for the tertiary extraction, and rapidly cooling the extract to 20-30° C. after filtering; Among them, the primary extraction solvent is distilled water, the extraction temperature is 85-95°C, and the extraction time is 20-30min.

9. The method for stable extraction and activity preservation of active ingredients from fresh Ganoderma lucidum according to claim 1, characterized in that: The three-in-one filtration method of microfiltration, nanofiltration and reverse osmosis in step d is: Among them, the pore size of the microfiltration membrane is 0.2 μm; The pore size of the nanofiltration membrane is 0.01 μm; The pore size of the reverse osmosis membrane is 0.3nm; The microfiltration parameters were as follows: microfiltration treatment for 40 min at a temperature of 36 °C, an operating pressure of 0.15 MPa, and an operating frequency of 30 Hz; The nanofiltration parameters are: temperature 36°C, operating pressure 0.52MPa, operating frequency 35Hz, and membrane pass time 40min; The reverse osmosis parameters are: feed liquid temperature 36°C, operating pressure 2.1MPa, operating frequency 35Hz, and membrane time 40min.

10. The method for stable extraction and activity preservation of active ingredients from fresh Ganoderma lucidum according to claim 1, characterized in that: The specific method of freeze-drying in step e is: The concentrated filtrate is pre-frozen at a temperature of -48 to -45°C for 20 minutes; After pre-freezing, adjust the temperature to -25 ~ -20 ° C, turn on the vacuum at the same time, adjust the vacuum degree to 0.5Pa, maintain for 10-12 hours, then continue to adjust the temperature to -3 ~ -2 ° C, keep warm for 5-6 hours, and finally adjust the temperature to 50-52 ° C, keep warm for 6-7 hours to complete the freeze-drying process.

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