Method and device for extracting tremella polysaccharide through combination of ultrasonic waves and high-voltage pulse electric field

Abstract

The invention relates to a method and device for extracting tremella polysaccharide through combination of ultrasonic waves and a high-voltage pulsed electric field, and the method comprises the following steps: firstly, crushing tremella to prepare a suspension, then carrying out wall breaking treatment on the suspension through combination of the ultrasonic waves and the high-voltage pulsed electric field to realize efficient disintegration of cell walls, centrifuging, taking supernate, and carrying out high-pressure homogenization to obtain a tremella polysaccharide stock solution; the stock solution can also be subjected to membrane separation treatment and vacuum freeze drying to obtain tremella polysaccharide dry powder with different molecular weights. Ultrasonic waves and a high-voltage pulse electric field are synergistically applied to white fungus wall breaking for the first time, the white fungus cell breaking rate is larger than or equal to 95%, the extraction time is shortened, the extraction rate can reach 35%, the extraction rate is improved by 50% compared with traditional white fungus polysaccharide extraction, and the defects that single physical wall breaking efficiency is low, ultrasonic treatment time is too long, solution temperature rise is caused, and extraction time is short are overcome. The absorption rate of a human body is improved, and a solution is provided for efficient utilization of the functional components of the tremella fuciformis.

Description

Technical Field

[0001] This invention relates to a method for extracting polysaccharides from Tremella fuciformis, and particularly to a method and apparatus for extracting polysaccharides from Tremella fuciformis using a combination of ultrasound and a high-voltage pulsed electric field. Background Technology

[0002] Tremellam polysaccharide is an acidic heteropolysaccharide isolated from the fruiting body of an edible fungus, Tremella fuciformis Berk. Its main chain structure consists of mannan linked by α-(1→3) glycosidic bonds, with side chains composed of glucuronic acid and xylose. A similar acidic heteropolysaccharide found in the deep-fermented spores of Tremella has a similar main chain structure, differing only in its side chains. According to literature, Tremellam polysaccharide is a polysaccharide obtained from the deep-fermentation of Tremella fuciformis, a basidiomycete. It is a white powder, soluble in water, but insoluble in organic solvents such as alcohol and ether. After hydrolysis, its composition includes xylose, mannose, fucose, glucose, and glucuronic acid, with a sugar content exceeding 75%. In addition to the polysaccharide, its products also contain spores.

[0003] Pharmacological effects of Tremella polysaccharide: Tremella polysaccharide has been reported to be an immune enhancer of basidiomycete polysaccharides. It can improve the body's immune function and increase white blood cell count. It can significantly improve the phagocytic function of reticuloendothelial cells in experimental animals, promote non-specific immunity, increase the levels of immunoglobulins and serum total complement, prevent and treat bone marrow suppression caused by radiation and anti-tumor drugs (such as CTX), and promote the synthesis of proteins in the liver.

[0004] The bioactivity of Tremella fuciformis polysaccharides (such as immunomodulation and anticancer activity) is highly dependent on their higher-order spatial structure (such as triple helix structure) and molecular weight, and is highly sensitive to temperature. Its inactivation is not a fixed "melting point" but a gradual degradation process. Safe temperature limits: It is generally recommended to control it below 80°C to maximize the preservation of its bioactivity; significant degradation temperature: When it exceeds 80°C, prolonged treatment will severely reduce its activity; complete inactivation temperature: Treatment at 121°C (the commonly used temperature for high-temperature sterilization) for 20-30 minutes is sufficient to cause the loss of the vast majority of bioactivity.

[0005] Currently, the traditional methods for extracting tremella mainly include: hot water extraction, simple ultrasonic / microwave-assisted extraction, enzymatic hydrolysis, and alkali extraction / alcohol precipitation. Although these methods have their own advantages, they also have obvious disadvantages. The existing invention patent with patent number "202011543317.1" discloses a method for preparing low molecular weight Tremella polysaccharide. However, this patent uses single ultrasonic-assisted extraction, which has a limited extraction rate. It does not design a cooling function for the ultrasonic reaction chamber. When ultrasonic treatment is performed for a long time to achieve the extraction rate, the solution temperature will rise, usually reaching 80°C. At this temperature, most of the active substances in Tremella polysaccharide are inactivated. Furthermore, the obtained low molecular weight Tremella polysaccharide is not subdivided, making it impossible to apply it to different fields.

[0006] Based on a review of the literature, we understand that the extraction of polysaccharides from Tremella fuciformis is currently in the in-depth research stage, with varying extraction rates. There are physical, chemical, and enzymatic extraction methods, but many of these technologies are still immature and in the experimental stage. The experimental products do not have a unified standard, and there are very few reports of industrialization. Summary of the Invention

[0007] The purpose of this invention is to provide a method and apparatus for extracting polysaccharides from Tremella fuciformis. The method involves first breaking up Tremella fuciformis, then pretreating the Tremella fuciformis polysaccharide aqueous solution using ultrasound to expand the micropores of the cell wall and reduce its structural strength. Next, a high-voltage pulsed electric field is used to directionally break down the cell membrane, which, combined with the microjet generated by the collapse of ultrasonic cavitation bubbles, achieves efficient cell wall disintegration. The resulting broken-cell liquid is then centrifuged to remove coarse particles, and the supernatant is subjected to high-pressure homogenization to obtain Tremella fuciformis polysaccharide stock solution. This method largely preserves the activity of Tremella fuciformis polysaccharides, improves human absorption, and can be used as a high-quality nutritional food or health product.

[0008] The original solution can also be freeze-dried under vacuum to obtain dried tremella powder; or the original solution can be membrane separated to obtain tremella polysaccharide health products with different molecular weights and different uses.

[0009] A method for extracting polysaccharides from Tremella fuciformis using ultrasound and a combination of ultrasound and a high-voltage pulsed electric field, comprising the following extraction steps: (1) Raw material pretreatment: Fresh or dried tremella is rehydrated and then crushed to a particle size ≤2mm. Pure water is added to adjust the concentration of tremella to 0.5-5% to prepare a tremella mixed aqueous solution. (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. The temperature of the suspension before treatment is 10~30℃. The suspension is first subjected to ultrasonic treatment to expand the micropores of the tremella cell wall and reduce the structural strength. The ultrasonic frequency is 20~100kHz and the time is 5-15 minutes. The temperature is kept below 60℃ throughout the process. (3) Continue to apply a high-voltage pulsed electric field to the suspension, with an intensity of 10-80 kV / cm and a pulse width of 1-100 μs, to achieve efficient cell wall disintegration and fully dissolve the active ingredients of the tremella polysaccharide tissue for 1-5 minutes. (4) The above two treatment methods, ultrasound and high-voltage pulsed electric field, are alternated 2 to 3 times to obtain the cell wall broken liquid of tremella polysaccharide; (5) Centrifuge the above-mentioned broken tremella polysaccharide liquid, remove the coarse particles from the centrifugation, and obtain the supernatant as a tremella polysaccharide solution with a particle size D90≤10μm; (6) The above-mentioned Tremella polysaccharide stock solution is subjected to high pressure homogenization to obtain Tremella polysaccharide stock solution with a molecular weight of 5,000 to 200,000; the homogenization pressure is 20 to 30 MPa, the homogenization temperature is 40 to 70°C, and the homogenization is performed 1 to 2 times.

[0010] In the extraction steps of the method for extracting Tremella polysaccharide using a combination of ultrasound and high-voltage pulsed electric field described in this invention, after the Tremella polysaccharide stock solution obtained in step (6) above is subjected to high-pressure homogenization, it is further subjected to membrane separation to obtain Tremella polysaccharide health products with different molecular weights. The separation membrane is a micron membrane and / or a nano membrane, and 2 to 3 layers of separation membrane are used.

[0011] In the extraction steps of the method for extracting Tremella polysaccharide using a combination of ultrasound and high-voltage pulsed electric field described in this invention, the Tremella polysaccharide stock solution obtained in step (6) above is vacuum freeze-dried at a temperature of -15 to -30°C to remove the water of crystallization, and then placed in a sealed bottle to obtain a fine powder product; the vacuum degree is 40 to 100 Pa.

[0012] Furthermore, the vacuum freeze-drying process involves first pre-freezing the membrane-separated Tremella polysaccharide to lower the material temperature to -15 to -30°C until all moisture in the material chamber is completely frozen. Then, a vacuum is drawn into the material drying chamber. Once the vacuum degree in the material drying chamber reaches 40 to 100 Pa, the material is heated to above 10°C to allow the moisture in the material to sublimate and be removed under vacuum conditions. When the material temperature and the vacuum degree in the material drying chamber meet the process requirements, the vacuum freeze-drying process is completed, and the Tremella polysaccharide freeze-dried powder is obtained through packaging.

[0013] In the extraction steps of the method for extracting Tremella polysaccharides by combining ultrasound and high-voltage pulsed electric field described in this invention, there is a temperature sensor in the ultrasonic reaction chamber in step (2). When the solution temperature reaches 60°C, the ultrasonic treatment is paused, and the cooling water flow rate is intelligently controlled to continuously remove the heat generated by the ultrasound.

[0014] In the extraction steps of the method for extracting Tremella polysaccharide by combining ultrasound and high-voltage pulsed electric field described in this invention, the conductivity of the suspension in step (2) ranges from 0.1 to 5 mS / cm. The conductivity of the suspension is adjusted by adding an appropriate amount of sodium chloride or potassium chloride to the suspension.

[0015] The method for extracting Tremella polysaccharides using a combination of ultrasound and high-voltage pulsed electric field as described in this invention can be performed in the same metal container or in two separate metal containers.

[0016] Furthermore, it is preferable that the concentration of tremella is adjusted to 1-2% in step (1), the ultrasonic frequency is 30-50kHz in step (2), and the high voltage pulse electric field intensity is 10-50kV / cm and the pulse width is 20-50μs in step (3).

[0017] The device for extracting Tremella polysaccharides using a combination of ultrasound and high-voltage pulsed electric field in this invention includes a pulverizer, an extraction container cavity (containing a temperature sensor and a double-layer jacket design with circulating cooling water or chilled water in between), an ultrasonic generator, a pulsed electric field, a centrifuge, and a homogenizer; the extraction container cavity is made of stainless steel, the ultrasonic generator uses a titanium alloy ultrasonic transducer plate, and the pulsed electric field electrode plate uses platinum electrodes.

[0018] The equipment used also includes vacuum freeze dryers or membrane separation devices.

[0019] To produce products for different applications in various fields, this invention further involves high-pressure homogenization of the liquid obtained after centrifugation of the above-mentioned broken-cell-wall liquid of Tremella fuciformis polysaccharide to obtain Tremella fuciformis polysaccharide stock solution, followed by membrane separation to obtain Tremella fuciformis polysaccharides of different molecular weights. The membrane separation device employs a three-layer membrane: the first layer is a micron-sized membrane with a molecular weight cutoff of 50,000–200,000 for polysaccharides; the second layer is a micron-sized membrane with a molecular weight cutoff of 15,000–50,000 for polysaccharides; and the third layer is a nano-sized membrane with a molecular weight cutoff of 5,000–15,000 for polysaccharides. If clogging occurs after prolonged membrane separation, it can be flushed with distilled water.

[0020] The specific process and connection procedure of this invention are as follows: A 0.5-5% solution of dried white fungus is prepared by adding purified water to the dried white fungus. Fresh white fungus or rehydrated dried white fungus can be used. The solution is then pulverized in a pretreatment tank until the particle size is ≤2mm, and then pumped to a physical cell-wall breaking treatment tank. The physical cell-wall breaking treatment tank uses a metal container cavity, which can be made of stainless steel. The mixture is stirred or shaken to form a suspension. Before treatment, the temperature of the suspension is approximately 10-30℃. The suspension is then subjected to ultrasonic treatment at a frequency of 20-100kHz for 5-15 minutes, maintaining the temperature below 60℃ throughout. A high-voltage pulsed electric field with an intensity of 10-80kV / cm is then applied to the suspension. The ultrasonic and high-voltage pulsed electric field treatments are applied for 1-5 minutes to achieve efficient cell wall disintegration and fully dissolve the active ingredients of Tremella fuciformis polysaccharide. The ultrasonic and high-voltage pulsed electric field treatments are alternated 2-3 times to obtain a Tremella fuciformis polysaccharide cell wall-broken liquid. The Tremella fuciformis polysaccharide cell wall-broken liquid is centrifuged to remove coarse fibers and large particles, resulting in a Tremella fuciformis liquid with a particle size D90≤10μm. The Tremella fuciformis polysaccharide stock solution is then subjected to high-pressure homogenization to obtain a Tremella fuciformis polysaccharide stock solution with a molecular weight of 5000-200000. The high-pressure homogenization is performed at a pressure of 20-30MPa, a temperature of 40-70°C, and 1-2 times to obtain a Tremella fuciformis polysaccharide stock solution with a molecular weight of 5000-200000.

[0021] The following describes the mechanism and advantages of the combined ultrasonic and high-voltage pulsed electric field extraction of Tremella fuciformis polysaccharides and the high-voltage homogenization process of this invention: 1. Ultrasonic extraction As is well known, in the physical extraction method of Tremella polysaccharides, the literature also proposes to use ultrasound alone to extract Tremella. Ultrasonic extraction utilizes the mechanical, cavitation and thermal effects of ultrasound to increase the speed of molecular motion and the penetrating power of the medium to extract bioactive components.

[0022] Mechanical Effect: The propagation of ultrasound in a medium can cause the particles in that medium to vibrate within its propagation space, thereby enhancing the diffusion and propagation of the medium. This is the mechanical effect of ultrasound. During propagation, ultrasound generates a radiation pressure that, along the direction of the sound wave, has a strong destructive effect on materials, causing cell tissue deformation and plant protein denaturation. Simultaneously, it can impart different accelerations to the medium and the suspended matter, with the medium molecules moving at a much higher speed than the suspended matter molecules. This generates friction between the two, and this frictional force can cause biomolecules to depolymerize, allowing the effective components on the cell walls to dissolve more quickly into the solvent.

[0023] Cavitation effect: Under normal circumstances, some microbubbles are dissolved inside the medium. These bubbles vibrate under the action of ultrasound. When the sound pressure reaches a certain value, the bubbles expand due to directional diffusion, forming a resonant cavity, and then suddenly close. This is the cavitation effect of ultrasound. When these bubbles close, they generate a pressure of several thousand atmospheres around them, forming micro-shock waves. These micro-shock waves can cause plant cell walls and the entire organism to rupture, and the entire rupture process is completed instantaneously, which is conducive to the dissolution of active ingredients.

[0024] Thermal effect: Like other physical waves, the propagation of ultrasound in a medium is also a process of energy propagation and diffusion. During propagation, the acoustic energy of ultrasound is continuously absorbed by the particles of the medium, which converts all or most of the absorbed energy into heat energy. This leads to an increase in the temperature of both the medium itself and the medicinal tissue, increasing the dissolution rate of the active ingredients. Because this increase in internal temperature caused by the absorption of acoustic energy is instantaneous, the biological activity of the extracted components remains unchanged.

[0025] The advantages of using ultrasound to process tremella in this invention are: (1) High extraction efficiency: The unique physical properties of ultrasound can cause plant cell tissue to break or deform, making the effective components of tremella more fully extracted, and the extraction rate is significantly higher than that of traditional hot water extraction process by 50-500%; (2) Short extraction time: Ultrasound can usually obtain the best extraction rate in 20-40 minutes, and the extraction time is shortened by more than 2 / 3 compared with traditional methods; (3) Low extraction temperature: The optimal temperature for ultrasonic extraction of Chinese medicinal materials is 40-80℃, which has a protective effect on the effective components in tremella, and at the same time saves energy; (4) Fewer impurities after extraction (no chemical interference from chemical extraction methods, and no enzymes that are difficult to remove), and the effective components are easy to separate and purify; (5) Low operating cost of extraction process and significant comprehensive economic benefits; (6) Simple and easy to operate, and convenient equipment maintenance and upkeep.

[0026] Therefore, when the initial extraction of Tremella polysaccharides using chemical and biological methods proved to be complex, costly, and ineffective, we first attempted ultrasonic extraction. However, ultrasonic extraction alone has some drawbacks: (1) Due to the effect of ultrasound, the extract may contain a large number of tiny particles and impurities; (2) Ultrasonic extraction alone results in incomplete sterilization and a short shelf life, affecting the final product quality; (3) Prolonged ultrasonic treatment can lead to excessively high solution temperatures, causing the decomposition of effective substances (see...). Figure 2 If the processing time is too short, the cell breakage rate will be insufficient, and the effective substances will not be fully extracted.

[0028] 2. High-voltage pulsed electric field Current literature reports that high-voltage pulsed electric fields (PEFs) can be applied to the sterilization of liquid beverages, with power ranging from 10 to 100 kV / cm. These high-intensity electric pulses cause irreversible electroporation or electrodisintegration of cell membranes (mainly plant cell walls or microbial cell membranes), forming permanent micropores and increasing cell membrane permeability. In liquid food sterilization, the pH and protein content of milk remain stable after treatment, extending the shelf life of milk to 28 days (22℃). Peach juice treated with this technology retains 90% of its ascorbic acid, and pomegranate juice shows no significant loss of polyphenols. It can kill pathogens such as Bacillus subtilis, Lactobacillus delbrueckii, Listeria monocytogenes, Pseudomonas fluorescens, Saccharomyces cerevisiae, Staphylococcus aureus, Streptococcus thermophilus, Escherichia coli, Escherichia coli O157, molds, and yeasts. After treatment with 40 kV / cm PEF, more than 90% of the vitamins in the samples can be retained. Starch granules treated with 50 kV / cm PEF retained over 87% of vitamin C in orange juice, while those treated with 35 kV / cm PEF retained over 87%. Electroporation of the cell membrane refers to the instability of the cell membrane and the formation of pores when cells are exposed to PEF. This significantly increases cytoplasmic membrane permeability, with intracellular osmotic pressure exceeding extracellular pressure, ultimately leading to cell membrane damage.

[0029] The basic principle of a high-voltage pulsed electric field: A pulsed electric field generates a magnetic field. The alternating action of this pulsed electric and magnetic fields increases cell membrane permeability, intensifies oscillations, and weakens membrane strength, thus disrupting the membrane. This allows substances inside the membrane to easily flow out, while substances outside the membrane can easily penetrate, weakening or even eliminating the protective function of the cell membrane. Furthermore, the ionization effect of the high-voltage pulsed electric field causes the anions and cations generated by the ionization of substances near the electrodes to interact with intracellular biochemical substances, thereby blocking normal biochemical reactions and metabolic processes within the membrane. Simultaneously, the strong oxidizing effect of O3 produced by the ionization of the liquid medium can react with intracellular substances in a series of reactions. Through the combined action of these two factors, the bacteria are killed.

[0030] In this invention, the increased cell membrane permeability induced by a high-voltage pulsed electric field is the key mechanism for its application in the extraction of polysaccharides from Tremella fuciformis. This makes it easier for valuable intracellular (cytoplasmic) components to be released into the surrounding medium, thereby improving extraction efficiency, reducing extraction difficulty, and shortening extraction time.

[0031] Advantages of high voltage pulsed electric field (1) Non-thermal process: The temperature rise of the material during the processing is usually very low, which can preserve the bioactivity and sensory quality of heat-sensitive target substances (such as pigments, vitamins, flavor substances, antioxidants) to the greatest extent.

[0032] (2) Improve extraction rate: By effectively disrupting cell structure, the mass transfer rate and final yield of target substances from inside cells to extraction medium are significantly increased.

[0033] (3) Shorten extraction time: Reduce the time required for traditional extraction methods (such as hot extraction and long-term solvent soaking) and improve production efficiency.

[0034] (4) Reduced energy consumption: Compared with high-temperature extraction or mechanical crushing (such as homogenization and ball milling), PEF usually consumes less energy, especially when processing liquid materials.

[0035] 3. High-pressure homogenization High-pressure homogenization is a process that pulverizes materials through the release of pressure energy and high-speed motion. During high-pressure homogenization, severe processing conditions such as high-speed liquid impact, high shear, and extrusion may lead to changes in the macromolecular structure and properties.

[0036] The high-pressure homogenization technique described above is used to prepare low molecular weight Tremella fuciformis polysaccharides. The Tremella fuciformis polysaccharides obtained using this method have a narrow molecular weight range, a high yield of low molecular weight Tremella fuciformis polysaccharides, and improved absorption and solubility. This method also has advantages such as being purely physical, easy to operate, and leaving no organic solvents or acid / alkali residues.

[0037] The combined advantages of this invention in extracting Tremella fuciformis polysaccharides using a combination of ultrasound and high-voltage pulse: I. High extraction efficiency: The unique physical properties of ultrasound can cause plant cell tissue to break or deform, expand the micropores of the cell wall, reduce the structural strength, and then use a high-voltage pulse electric field to directionally break down the cell membrane. This, combined with the micro-jet generated by the collapse of ultrasonic cavitation bubbles, overcomes the technical bottleneck of low efficiency of single physical cell breaking and achieves efficient cell wall disintegration. Through energy field coupling and parameter optimization, the cell breakage rate of Tremella fuciformis is ≥95%.

[0038] II. Ultrasonic Enhancement Effect: The intense cavitation effect (shock waves, microjets) and mechanical vibration generated by ultrasound directly act on the cell walls / membranes that have been weakened by the electric field, efficiently tearing and breaking them apart. At the same time, ultrasound can promote the uniformity of the electric field distribution in the material.

[0039] III. Synergistic Effect: The effects of the two physical fields are not simply superimposed, but rather produce a synergistic effect of "1+1>2". The electric field lowers the energy threshold required for ultrasonic cell disruption, while ultrasound amplifies the destructive effect of the electric field on cell structure. This results in a higher cell disruption rate and a shorter disruption time with lower total energy input. The introduction of a pulsed electric field reduces the ultrasonic treatment time while maintaining the highest possible extraction rate, thus avoiding the reduction in bioactivity caused by solution heating and preserving most of the bioactivity of Tremella fuciformis polysaccharides.

[0040] Fourth, the extraction time is shortened, allowing for more complete extraction of the effective components of Tremella fuciformis. This results in a 40-60% increase in the dissolution rate of polysaccharides compared to traditional Tremella fuciformis extraction methods. The extraction temperature is low (not exceeding 60°C), and the extraction time is significantly reduced by more than 2 / 3 compared to traditional methods, greatly reducing energy consumption.

[0041] Fifth, high-voltage pulses have a protective effect on the effective components in tremella, while the extraction process results in fewer impurities and easier separation and purification of the effective components; the extraction process has low operating costs and significant overall economic benefits.

[0042] VI. The equipment is simple. There are many domestic manufacturers of ultrasonic generators. The electrode materials used in the high-voltage pulse electric field are stable and can be used for a long time without damage. Cleaning and maintenance are very convenient.

[0043] VII. Environmental friendliness: The solvent used is only water, without chemical pollution, and the process generates almost no waste (the residue of tremella can be used as dietary fiber or as feed for farmed animals). The equipment is easy to clean and is more in line with the concept of green extraction.

[0044] This invention is the first to synergistically apply the ultrasonic cavitation effect and the high-voltage pulse electroporation effect to the cell wall disruption of Tremella fuciformis. Through energy field coupling and parameter optimization, the cell disruption rate of Tremella fuciformis is increased to over 96%, the extraction time is shortened, and the dissolution rate of polysaccharides extracted from Tremella fuciformis is increased by 40-60% compared with traditional methods. This invention overcomes the technical bottleneck of low efficiency of single physical cell wall disruption and provides a solution for the efficient utilization of functional components of Tremella fuciformis.

[0045] In this invention, the aqueous solution of tremella is placed in a metal container and stirred or shaken before ultrasonic cell wall breaking to form a suspension, which can also improve the efficiency of ultrasonic extraction.

[0046] This invention also introduces centrifugation, high-pressure homogenization, and membrane separation technologies. Specifically, the cell-wall-breaking liquid extracted using a combination of ultrasonic and high-voltage pulsed electric fields is centrifuged, homogenized under high pressure, and then separated using a membrane to obtain aqueous solutions of Tremella polysaccharides with different molecular weights. These solutions are then freeze-dried under vacuum to finally obtain Tremella ultrafine powders with different molecular weights (for different applications). This provides a solution for the efficient extraction and application of functional components from Tremella.

[0047] The aforementioned ultrasonic and high-voltage pulsed electric field device uses a titanium alloy ultrasonic transducer plate and a parallel electrode plate made of platinum electrode material within a metal cavity. By adjusting the ultrasonic / pulse parameters, the cell breakage rate of Tremella fuciformis is ≥95%, shortening the subsequent extraction time. This results in a 40-60% increase in the dissolution rate of polysaccharides extracted from Tremella fuciformis compared to traditional methods. With the addition of high-pressure homogenization, the resulting Tremella fuciformis polysaccharide stock solution is significantly more easily absorbed by the human body. For people with poor physical condition and immunity, drinking this solution can lead to better recovery of bodily functions. Attached Figure Description

[0048] Figure 1 This is a flowchart illustrating the specific process of extracting polysaccharides from Tremella fuciformis using a combination of ultrasound and pulse techniques according to the present invention.

[0049] Figure 2 This is a schematic diagram showing the relationship between ultrasonic treatment time and liquid temperature change. Detailed Implementation

[0050] The technical solutions of the invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The described embodiments are only a part of the invention, and not all of the embodiments.

[0051] Example 1

[0052] A method for extracting polysaccharides from Tremella fuciformis using a combination of ultrasound and high-voltage pulsed electric field, comprising the following steps: (1) Raw material pretreatment: Place freshly picked white fungus into a grinder and grind it to a particle size ≤2mm. Add purified water to adjust the white fungus concentration to 0.2-3% to make a white fungus mixed aqueous solution; (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. The temperature of the suspension before treatment is about 10-30℃. The suspension is first subjected to ultrasonic treatment to expand the micropores of the tremella cell wall and reduce the structural strength. The ultrasonic frequency is 30-80kHz and the time is 5-10 minutes. The temperature is kept below 60℃ throughout the process. (3) Continue to apply a high-voltage pulsed electric field to the suspension, with an intensity of 20-60 kV / cm and a pulse width of 20-50 μs, to achieve efficient cell wall disintegration and fully dissolve the active ingredients of the tremella polysaccharide tissue for 1-3 minutes; (4) The above two treatment methods, ultrasound and high-voltage pulsed electric field, are alternated 2 to 3 times to obtain the cell wall broken liquid of tremella polysaccharide; (5) Centrifuge the above-mentioned broken tremella polysaccharide liquid, remove the coarse particles from the centrifugation, and obtain the supernatant as a tremella polysaccharide solution with a particle size D90≤10μm; (6) The above-mentioned Tremella polysaccharide solution is subjected to high pressure homogenization to obtain Tremella polysaccharide stock solution with a molecular weight of 5,000 to 200,000; the homogenization pressure is 10 to 20 MPa, the homogenization temperature is 40 to 50°C, and the homogenization is performed 1 to 2 times to obtain Tremella polysaccharide stock solution. (7) After the separated Tremella polysaccharide is completely frozen at -15 to -20°C for 10 minutes, the water of crystallization is removed by vacuum extraction. The vacuum degree is 50 to 70 Pa to obtain an ultrafine powder product. The product is then placed in a sealed bottle and packaged to obtain a Tremella polysaccharide product with a molecular weight of 5,000 to 200,000. The product is then transported under cold storage.

[0053] Example 2

[0054] A method for extracting polysaccharides from Tremella fuciformis using a combination of ultrasound and high-voltage pulsed electric field, comprising the following steps: (1) Raw material pretreatment: After rehydrating the dried white fungus, put it into a grinder and grind it to a particle size ≤2mm. Add purified water to adjust the white fungus concentration to 0.5-2% to make a white fungus mixed aqueous solution; (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. The temperature of the suspension before treatment is about 10-30℃. The suspension is first subjected to ultrasonic treatment to expand the micropores of the tremella cell wall and reduce the structural strength. The ultrasonic frequency is 40-60kHz and the time is 5-15 minutes. (3) Continue to apply a high-voltage pulsed electric field to the suspension, with an intensity of 10-80 kV / cm and a pulse width of 1-100 μs, to achieve efficient cell wall disintegration and fully dissolve the active ingredients of the tremella polysaccharide tissue for 2-5 minutes. (4) The above two treatment methods, ultrasound and high-voltage pulsed electric field, are alternated 2 to 3 times to obtain the cell wall broken liquid of tremella polysaccharide; (5) Centrifuge the above-mentioned broken tremella polysaccharide liquid, remove the coarse particles from the centrifugation, and obtain the supernatant as a tremella polysaccharide solution with a particle size D90≤10μm; (6) The above-mentioned Tremella polysaccharide solution is subjected to high pressure homogenization to obtain Tremella polysaccharide stock solution with a molecular weight of 5,000 to 200,000; the homogenization pressure is 10 to 20 MPa, the homogenization temperature is 40 to 50°C, and the homogenization is performed 1 to 2 times to obtain Tremella polysaccharide stock solution. (7) The liquid after centrifugation of the above-mentioned broken cell wall liquid of tremella polysaccharide is further subjected to membrane separation to obtain three tremella polysaccharide solutions with different molecular weights; the membrane separation device adopts a three-layer separation membrane, the first separation membrane is a micron membrane with a molecular weight cutoff of 80,000 to 200,000 for polysaccharides; the second separation membrane is a micron membrane with a molecular weight cutoff of 20,000 to 80,000 for polysaccharides; and the third separation membrane is a nano membrane with a molecular weight cutoff of 5,000 to 20,000 for polysaccharides.

[0055] (8) The three types of Tremella polysaccharides after membrane separation are placed in a vacuum freeze-drying chamber for pre-freezing. The material temperature inside the chamber is lowered to -18 to -22°C. After the water in the chamber is completely frozen, the material drying chamber is evacuated. When the vacuum degree of the material drying chamber reaches 50 to 70 Pa, the material is heated to above 10°C so that the water in the material is sublimated and removed under vacuum conditions. When the material temperature and the vacuum degree in the material drying chamber meet the process requirements, the vacuum freeze-drying is completed, and an ultrafine powder product is obtained. It is then placed in a sealed bottle and packaged to obtain three different Tremella polysaccharide products with molecular weights of 5,000 to 200,000. The products are then transported under refrigeration.

[0056] Example 3

[0057] A method for extracting polysaccharides from Tremella fuciformis using a combination of ultrasound and high-voltage pulsed electric field, comprising the following steps: (1) Raw material pretreatment: After rehydrating the dried white fungus, put it into a grinder and grind it to a particle size ≤2mm. Add purified water to adjust the white fungus concentration to 0.5-5% to make a white fungus mixed aqueous solution; (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. The temperature of the suspension before treatment is about 10-30℃. The suspension is first subjected to ultrasonic treatment to expand the micropores of the tremella cell wall and reduce the structural strength. The ultrasonic frequency is 20-100kHz and the time is 5-15 minutes. (3) Keep the temperature constant and continue to apply a high-voltage pulse electric field to the suspension with an intensity of 10-80 kV / cm and a pulse width of 1-100 μs to achieve efficient cell wall disintegration, so that the active ingredients of the tremella polysaccharide tissue can be fully dissolved for 1-5 minutes. (4) The above two treatment methods, ultrasound and high-voltage pulsed electric field, are alternated 2 to 3 times to obtain the cell wall broken liquid of tremella polysaccharide; (5) Centrifuge the above-mentioned broken tremella polysaccharide liquid, remove the coarse particles from the centrifugation, and obtain the supernatant as a tremella polysaccharide solution with a particle size D90≤10μm; (6) The above-mentioned Tremella polysaccharide solution is subjected to high pressure homogenization to obtain Tremella polysaccharide stock solution with a molecular weight of 5,000 to 200,000; the homogenization pressure is 10 to 20 MPa, the homogenization temperature is 40 to 50°C, and the homogenization is performed 1 to 2 times to obtain Tremella polysaccharide stock solution.

[0058] (7) The liquid after centrifugation of the above-mentioned broken cell wall liquid of tremella polysaccharide is further subjected to membrane separation to obtain three tremella polysaccharide solutions with different molecular weights; the membrane separation device adopts a three-layer separation membrane, the first separation membrane is a micron membrane with a molecular weight cutoff of 80,000 to 200,000 for polysaccharides; the second separation membrane is a micron membrane with a molecular weight cutoff of 20,000 to 80,000 for polysaccharides; and the third separation membrane is a nano membrane with a molecular weight cutoff of 5,000 to 20,000 for polysaccharides.

[0059] (8) Place the three types of Tremella polysaccharides after membrane separation in a vacuum freeze-drying chamber for pre-freezing, so that the material temperature inside the material drops to -16 to -25°C. After the moisture in the material chamber is completely frozen, vacuum the material drying chamber. When the vacuum degree of the material drying chamber reaches 50 to 80 Pa, heat the material to above 12°C so that the moisture in the material sublimates and is removed under vacuum conditions. When the material temperature and the vacuum degree in the material drying chamber meet the process requirements, the vacuum freeze-drying is completed, and an ultrafine powder product is obtained. It is then placed in a sealed bottle and packaged to obtain three different Tremella polysaccharide products with molecular weights ranging from 5,000 to 200,000. The products are then transported under refrigeration.

[0060] Example 4

[0061] A method for extracting polysaccharides from Tremella fuciformis using a combination of ultrasound and high-voltage pulsed electric field, comprising the following steps: (1) Raw material pretreatment: After rehydrating the dried white fungus, put it into a grinder and grind it to a particle size ≤2mm. Add purified water to adjust the white fungus concentration to 0.5-4% to make a white fungus mixed aqueous solution; (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. The temperature of the suspension before treatment is 10~30℃. The suspension is first subjected to ultrasonic treatment to expand the micropores of the tremella cell wall and reduce the structural strength. The ultrasonic frequency is 20~100kHz and the time is 20~30 minutes. (3) Keep the temperature constant and continue to apply a high-voltage pulse electric field to the suspension with an intensity of 30-90 kV / cm and a pulse width of 10-90 μs to achieve efficient cell wall disintegration, so that the active ingredients of the tremella polysaccharide tissue can be fully dissolved for 3-4 minutes. (4) The above two treatment methods, ultrasound and high-voltage pulsed electric field, were alternated twice to obtain the cell wall broken liquid of tremella polysaccharide; (5) Centrifuge the above-mentioned broken tremella polysaccharide liquid, remove the coarse particles from the centrifugation, and obtain the supernatant as a tremella polysaccharide solution with a particle size D90≤10μm; (6) The above-mentioned Tremella polysaccharide stock solution is subjected to high pressure homogenization to obtain Tremella polysaccharide stock solution with a molecular weight of 5,000 to 200,000; the homogenization pressure is 50 to 80 MPa, the homogenization temperature is 50 to 65°C, and the homogenization is performed 1 to 2 times.

[0062] Comparative Example 1 (Ultrasonic Treatment Only) A method for ultrasonic extraction of polysaccharides from Tremella fuciformis, comprising the following steps: (1) Raw material pretreatment: After rehydrating the dried white fungus, put it into a grinder and grind it until the particle size is ≤2mm. Add purified water to adjust the white fungus concentration to 1-5%. After fully rehydrating, put it into a grinder and grind it until the particle size is ≤2mm to make a white fungus mixed aqueous solution. (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. The temperature of the suspension before treatment is about 10-30℃. The suspension is subjected to ultrasonic treatment to expand the micropores of the tremella cell wall and reduce the structural strength. The ultrasonic frequency is 20-100kHz and the time is 10-30 minutes. (3) The above-mentioned mixed aqueous solution of tremella was circulated by ultrasound twice to obtain a tremella polysaccharide cell wall-breaking liquid; (4) Centrifuge the above-mentioned tremella polysaccharide cell wall-breaking liquid to remove coarse fibers and large particles, and obtain tremella liquid with particle size D90≤10μm; (5) The separated Tremella polysaccharide is vacuum freeze-dried at a temperature of -15 to -30°C to remove the water of crystallization and obtain an ultrafine powder product. The product is then placed in a sealed bottle with a vacuum degree of 40 to 70 Pa to obtain an ultrasonic Tremella polysaccharide solution.

[0063] Comparative Example 2 (Simple High-Voltage Pulsed Electric Field) A method for extracting polysaccharides from Tremella fuciformis using a high-voltage pulsed electric field, comprising the following steps: (1) Raw material pretreatment: After rehydrating the dried white fungus, put it into a grinder and grind it until the particle size is ≤2mm. Add purified water to adjust the white fungus concentration to 1-5%. After fully rehydrating, put it into a grinder and grind it until the particle size is ≤2mm to make a white fungus mixed aqueous solution. (2) Place the mixed aqueous solution of tremella in a metal container and stir or shake it to form a suspension. Heat it to 50-60°C and apply a high voltage pulse electric field to the suspension with an intensity of 3-60 kV / cm, a pulse width of 20-60 μs, and a duration of 3-5 minutes. (3) The above-mentioned mixed aqueous solution of Tremella fuciformis was circulated twice under a high-voltage pulsed electric field to obtain Tremella fuciformis polysaccharide liquid; (4) Centrifuge the above-mentioned tremella polysaccharide cell wall-breaking liquid to remove coarse fibers and large particles, and obtain tremella liquid with particle size D90≤30μm; (5) The separated Tremella polysaccharide is vacuum freeze-dried at a temperature of -15 to -30°C to remove the water of crystallization and obtain an ultrafine powder product. The product is then placed in a sealed bottle with a vacuum degree of 40 to 70 Pa to obtain an ultrasonic Tremella polysaccharide solution.

[0064] The experimental data of the above embodiments are shown in the table below. The extraction rate was determined by the phenol-sulfuric acid method to measure the total sugar content, and the purity was determined by HPLC to measure the polysaccharide content.

[0065]

[0066] The above description is not intended to limit the present invention, nor is the present invention limited to the above examples. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should be protected by the present invention.

Claims

1. A method for combined ultrasonic and high-voltage pulsed electric field extraction of tremella polysaccharide, characterized in that, The steps are as follows: (1) raw material pretreatment: the fresh or dried tremella is crushed to a particle size of ≤2 mm after rehydration, and pure water is added to adjust the concentration of tremella to 0.5-5% to prepare a tremella mixed aqueous solution; (2) the tremella mixed aqueous solution is placed in a metal container and stirred or shaken to form a suspension, and the temperature of the suspension before treatment is about 10-30°C. The suspension is first subjected to ultrasonic treatment to expand the cell wall micropores of tremella and reduce the structural strength. The ultrasonic frequency is 20-100 kHz, and the time is 5-15 minutes, and the temperature is kept below 60°C throughout the process; (3) continue to apply high-voltage pulsed electric field to the suspension, with a strength of 10-80 kV / cm and a pulse width of 1-100 μs, to achieve efficient cell wall disintegration and fully dissolve the active components of tremella polysaccharide, and the time is 1-5 minutes; (4) the above two treatment methods of ultrasonic and high-voltage pulsed electric field are alternately cycled 2-3 times to obtain a tremella polysaccharide broken wall liquid; (5) the above tremella polysaccharide broken wall liquid is centrifuged to remove the coarse particles, and the supernatant is obtained as a tremella polysaccharide solution with a particle size D90 of ≤10 μm; (6) the above tremella polysaccharide stock solution is subjected to high-pressure homogenization treatment to obtain a tremella polysaccharide stock solution with a molecular weight of 5000-200000; the homogenization pressure is 20-30 MPa, the homogenization temperature is 40-70°C, and the homogenization times is 1-2 times.

2. The method according to claim 1, wherein the ultrasonic and high-voltage pulsed electric field combined extraction of tremella polysaccharides is characterized by, There is a temperature sensor in the ultrasonic reaction chamber of step (2) above. When the solution temperature reaches 60°C, the ultrasonic treatment is paused, and the flow of cooling water is intelligently controlled to continuously remove the heat generated by the ultrasonic waves. 3.The method of claim 1, wherein the ultrasonic and high-voltage pulsed electric field combined extraction of tremella polysaccharides is characterized by, The tremella polysaccharide stock solution obtained in step (6) above is vacuum freeze-dried at a temperature of -15 to -30°C to remove the crystal water, and is packed in a sealed bottle to obtain a fine powder product; the vacuum degree is 40-100 Pa.

4. The method according to claim 1, wherein the ultrasonic and high-voltage pulsed electric field combined extraction method of tremella polysaccharides is characterized by, After the tremella polysaccharide stock solution obtained in step (6) above is subjected to high-pressure homogenization treatment, it is further subjected to membrane separation to obtain different molecular weight tremella polysaccharide health products; vacuum freeze-drying is carried out at a temperature of -15 to -30°C to remove the crystal water, and is packed in a sealed bottle, the separation membrane is a micron membrane and / or a nanometer membrane, and 2-3 layers of separation membranes are used.

5. The method according to claim 4, wherein the ultrasonic and high-voltage pulsed electric field combined extraction method of tremella polysaccharides is characterized by, The first layer of separation membrane in the membrane separation is a micron membrane with a polysaccharide molecular weight cutoff of 80000-200000; the second layer of separation membrane is a micron membrane with a polysaccharide molecular weight cutoff of 20000-80000; and the third layer of separation membrane is a nanometer membrane with a polysaccharide molecular weight cutoff of 5000-20000.

6. The method according to claim 3-4, wherein the method is characterized in that, The vacuum freeze-drying is a pre-freezing of the liquid after high-pressure homogenization or membrane separation, which reduces the temperature of the material in the material to -15 to -30°C. After the water in the material chamber is completely frozen and frozen, the material drying chamber is vacuumed, and when the vacuum degree of the material drying chamber reaches 40-100 Pa, the material is heated to above 10°C, and the water in the material is sublimed and removed under vacuum conditions. When the material temperature and the vacuum degree in the material drying chamber meet the process requirements, the vacuum freeze-drying is completed, and the tremella polysaccharide freeze-dried powder is packaged.

7. The method according to claim 1, wherein the ultrasonic and high-voltage pulsed electric field combined extraction method of tremella polysaccharides is characterized by, The temperature of the suspension is 45-65 DEG C, the conductivity of the suspension ranges from 0.1 to 5 mS / cm, and the conductivity of the suspension is adjusted by adding proper amount of sodium chloride or potassium chloride into the suspension. 8.The method of claim 1, wherein the ultrasonic and high-voltage pulsed electric field combined extraction of tremella polysaccharides is characterized by, The ultrasonic wave and pulse combined extraction is carried out in the same metal container or in two separate metal containers. 9.The method of claim 1, wherein the ultrasonic and high-voltage pulsed electric field combined extraction of tremella polysaccharides is characterized by, The adopted tremella polysaccharide extraction device comprises a pulverizer, an extraction container cavity, an ultrasonic generator, a high-voltage pulse device, a centrifuge and a homogenizer; the extraction container cavity is made of stainless steel, the ultrasonic generator adopts a titanium alloy ultrasonic vibration plate, and the pulse electric field electrode plate adopts a platinum electrode.

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