Preparation process for preparing instant red algae polysaccharide through compound enzyme method

Abstract

The invention relates to the technical field of red algae polysaccharide extraction, and provides a preparation technology for preparing instant red algae polysaccharide through a compound enzyme method, and the preparation technology comprises the following steps: S100, washing a red algae raw material, and sequentially carrying out mechanical crushing treatment and freeze drying treatment to obtain red algae powder; s200, sequentially performing enzymolysis treatment, centrifugal treatment and filtration treatment on the red algae powder to obtain supernatant liquid; and S300, sequentially carrying out concentration treatment, purification treatment, spray drying treatment and fluidization granulation treatment on the supernatant to obtain the red algae polysaccharide. The red algae raw material is efficiently separated and purified by adopting the compound enzyme, so that the efficient degradation of the red algae cell wall and the efficient extraction and preparation of the red algae polysaccharide are realized, a new technical way is provided for the efficient preparation and wide application of the red algae polysaccharide, and the method has important industrial application value and market prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of red algae polysaccharide extraction, and in particular to a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method. Background Art

[0002] Red algae polysaccharides are an important class of biomacromolecules extracted from red algae. Their complex and diverse structures are primarily composed of monosaccharides such as galactose and 3,6-galactose linked by glycosidic bonds, with some also containing modifying groups such as sulfate and methyl groups. The type, number, and attachment positions of these groups contribute to their unique physicochemical properties and biological activities. Red algae polysaccharides exhibit excellent viscosity and gelling properties, making them suitable for use as thickeners and stabilizers in a variety of foods. In recent years, as research on red algae polysaccharides has deepened, their rapid solubility has increasingly attracted attention. The development of rapidly soluble red algae polysaccharides not only improves product convenience but also expands their applications in the food and pharmaceutical sectors. For example, in the food industry, rapidly soluble red algae polysaccharides can be used as thickeners and stabilizers, rapidly dissolving in liquid foods and improving product texture and stability. Furthermore, their rapid solubility reduces waste caused by incomplete dissolution during processing, thereby improving production efficiency.

[0003] However, traditional extraction methods have many limitations. Although hot water extraction is simple to operate, the extraction efficiency is low and it consumes a lot of energy and water resources. At the same time, high temperature conditions can easily lead to structural damage of red algae polysaccharides, affecting their biological activity and purity. Although acid extraction and alkaline extraction can improve the extraction rate to a certain extent, the acid-base conditions are relatively harsh and will also cause irreversible damage to the polysaccharide structure. In addition, tedious neutralization treatment is required later, which increases production costs and environmental pollution risks. Therefore, how to improve the extraction efficiency of red algae polysaccharides without affecting the polysaccharide structure has always been the goal pursued by professionals in this field. Summary of the Invention

[0004] The present invention aims to solve at least one of the above technical problems.

[0005] The present invention provides a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method, comprising: S100, washing the red algae raw material and sequentially subjecting it to mechanical pulverization and freeze-drying to obtain red algae powder; S200, performing enzymatic hydrolysis, centrifugation, and filtration on the red algae powder in sequence to obtain a supernatant; S300, sequentially performing concentration treatment, purification treatment, spray drying treatment, and fluidized bed granulation treatment on the supernatant to obtain red algae polysaccharide; Among them, S200 includes: S210, performing a first enzymatic hydrolysis treatment on the red algae powder in a water bath using a first complex enzyme to obtain a first enzymatic hydrolysis intermediate; S220, performing a second enzymatic hydrolysis treatment on the first enzymatic hydrolysis intermediate using a second complex enzyme to obtain a second enzymatic hydrolysis intermediate; S230, performing a third enzymatic hydrolysis treatment on the second enzymatic hydrolysis intermediate using a third complex enzyme in a water bath, and then sequentially performing a centrifugal treatment and a filtration treatment to obtain a supernatant; The second complex enzyme and the third complex enzyme are both immobilized by nanomagnetic particles.

[0006] In the above technical features, the preparation method of the nanomagnetic particles includes: S221, dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water, introducing nitrogen, stirring thoroughly until fully dissolved, then adding NH3·H2O to adjust the pH value to obtain a precipitate, separating the precipitate with an external magnet, and sequentially washing the precipitate and drying to obtain Fe3O4; S222, dispersing Fe3O4 in DMF and performing ultrasonic treatment, adding FeCl3·6H2O and H2BDC during the ultrasonic treatment, and then performing a hydrothermal reaction. After the reaction is completed, magnetic separation, washing, and drying are performed to obtain Fe3O4 with a MOFs layer; S223. Disperse Fe3O4 with MOFs layer in Tris-HCl buffer, add dopamine solution and stir, then magnetically separate, wash to neutrality, and dry to obtain nanomagnetic particles.

[0007] In any of the above technical features, in S221, the pH is adjusted to 9.0~10; and / or in S222, the temperature of the hydrothermal reaction is 110~120℃; and / or in S222, the time of the hydrothermal reaction is 12~24h; and / or in S223, the concentration of the dopamine solution is 1~2mg / mL.

[0008] In any of the above technical features, the first complex enzyme includes at least one of cellulase, hemicellulase, xylanase and pectinase or a combination thereof; and / or the second complex enzyme includes at least one of cellulase, agarase and xylanase or a combination thereof; and / or the third complex enzyme includes at least one of papain and mannanase or a combination thereof.

[0009] In any of the above technical features, in the first complex enzyme, the amount of cellulase is 1.3~1.8%w / w, the amount of hemicellulase is 1.2~1.5%w / w, the amount of xylanase is 0.4~0.6%w / w, and the amount of pectinase is 0.8~1.2%w / w; and / or in the second complex enzyme, the amount of cellulase is 1.3~1.6%w / w, the amount of agarase is 1.0~1.5%w / w, and the amount of xylanase is 0.5~0.8%w / w; and / or in the third complex enzyme, the amount of papain is 0.5~0.8%w / w, and the amount of mannanase is 0.3~0.5%w / w.

[0010] In any of the above technical features, in S300, the concentration treatment is ultrafiltration treatment using an ultrafiltration membrane; and / or the purification treatment is adsorption treatment using COFs.

[0011] In any of the above technical features, in S210, the temperature of the first enzymatic hydrolysis treatment is 40~55°C; and / or in S210, the pH of the first enzymatic hydrolysis treatment is 5.0~5.5; and / or in S220, the temperature of the second enzymatic hydrolysis treatment is 40~60°C; and / or in S220, the pH of the second enzymatic hydrolysis treatment is 5.5~6.5; and / or in S230, the temperature of the third enzymatic hydrolysis treatment is 40~55°C; and / or in S230, the pH of the third enzymatic hydrolysis treatment is 7.0~7.5.

[0012] In any of the above technical features, in S100, after mechanical crushing, the particle size of the red algae raw material is less than 2 mm; and / or the temperature of the freeze-drying treatment is -40~-45°C; and / or the time of the freeze-drying treatment is 18~24 hours.

[0013] In any of the above technical features, in S100, the red algae raw material includes at least one of or a combination of Gracilaria, Gelidium, Porphyra, Red Hair Algae, Glucostomia, Seaweed, Sea Head Red, Polysiphonia, Centipede Algae, Partridge Algae, Seaweed and Gallinarum.

[0014] In any of the above technical features, in S300, the inlet air temperature of the spray drying process is 170°C~190°C; and / or the outlet air temperature of the spray drying process is 75°C~85°C; and / or the atomization pressure of the spray drying process is 0.1-0.2MPa.

[0015] After adopting the technical solution of the present invention, the following technical effects can be achieved: (1) By using complex enzymes to efficiently separate and purify red algae raw materials, the efficient degradation of red algae cell walls and the efficient extraction and preparation of red algae polysaccharides are achieved, providing a new technical approach for the efficient preparation and wide application of red algae polysaccharides, which has important industrial application value and market prospects; (2) The introduction of spray drying technology and granulation technology not only significantly improved the drying efficiency, but also improved the physical properties of red algae polysaccharides by controlling the morphology and particle size of the particles. It can produce particles with porous or hollow structures, which not only helps to increase the dissolution rate of red algae polysaccharides, but also can adjust the size and morphology of the particles according to needs, making it more suitable for industrial production and application. (3) The enzymatic hydrolysis treatment adopts a three-stage enzymatic hydrolysis distribution treatment, which can release the target polysaccharide components in an orderly, efficient and targeted manner, remove impurities, and improve the solubility and functionality of red algae polysaccharides. DETAILED DESCRIPTION

[0016] To make the above-mentioned objectives, features, and advantages of the present invention more clearly understood, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention.

[0017] In the related art, the extraction methods of red algae polysaccharides have many limitations, such as low extraction efficiency, the need to consume a large amount of energy and water resources, irreversible damage to the polysaccharide structure, increased production costs and environmental pollution risks, etc. In recent years, with the emphasis on environmental protection and efficient extraction technology, enzymatic extraction has gradually become a hot spot for research and application. The composite enzyme method can effectively improve the extraction rate and solubility of red algae polysaccharides through the synergistic action of multiple enzymes. However, the red algae polysaccharides after enzymatic hydrolysis usually exist in liquid or powder form, and their solubility and stability are still insufficient, which limits their wide application in food, medicine and other fields.

[0018] In view of this, the present invention provides a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method. By using a composite enzyme to perform a three-step enzymatic hydrolysis treatment on the red algae raw material, and combining spray drying and granulation technology, the extraction rate and solubility of the red algae polysaccharide are effectively improved without damaging the structure of the red algae polysaccharide, thereby preparing red algae polysaccharide particles with uniform particle size, good fluidity and excellent solubility.

[0019] Specifically, the present invention provides a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method, comprising: S100, washing the red algae raw material and sequentially subjecting it to mechanical pulverization and freeze-drying to obtain red algae powder; S200, performing enzymatic hydrolysis, centrifugation, and filtration on the red algae powder in sequence to obtain a supernatant; S300, sequentially performing concentration treatment, purification treatment, spray drying treatment, and fluidized bed granulation treatment on the supernatant to obtain red algae polysaccharide.

[0020] Preferably, the present invention uses a complex enzyme to efficiently separate and purify the red algae raw material, thereby achieving efficient degradation of the red algae cell wall and efficient extraction and preparation of red algae polysaccharides, providing a new technical approach for the efficient preparation and wide application of red algae polysaccharides, and has important industrial application value and market prospects.

[0021] Preferably, the present invention first washes the red algae raw material to remove impurities such as sea salt and mud. The red algae raw material includes at least one of or a combination of Gracilaria, Gelidium, Laver, Red Hair Algae, Glucosphaera, Sea Iris, Sea Head Red, Polycystic Algae, Centipede Algae, Partridge Algae, Sea Oophthalmia and Gallus Feather Algae. The same red algae varieties are widely distributed in various sea areas, and the seasons and yields complement each other, which helps to stabilize the supply of raw materials. The washed red algae is chopped and then mechanically crushed. After the mechanical crushing, the particle size of the red algae raw material is less than 2 mm to increase the contact area between the subsequent enzyme and the substrate. The freeze-drying process first needs to be pre-frozen at -40 ° C and then vacuum freeze-dried. The temperature is -40~-45℃ and the time is 18~24h, which can maximize the retention of heat-sensitive components in red algae such as polysaccharides and bioactive substances, and can improve the retention rate of red algae polysaccharides; the freeze-dried red algae are enzymatically hydrolyzed with a composite enzyme to the freeze-dried red algae powder. Compared with wet red algae, the enzymatic hydrolysis efficiency of the dried red algae powder is significantly improved, and the polysaccharide extraction rate is much higher than that of single enzyme or physical extraction. The molecular weight of the product after enzymatic hydrolysis is moderate, which is conducive to quick dissolution; the supernatant collected after centrifugation and filtration after enzymatic hydrolysis contains red algae polysaccharides, and the concentration and purification of the supernatant are conducive to further improving the purity of red algae polysaccharides.

[0022] Furthermore, the concentration treatment is to use an ultrafiltration membrane for ultrafiltration treatment. Ultrafiltration is usually carried out at room temperature or low temperature to avoid high temperature causing degradation or loss of activity of red algae polysaccharides. The pore size of the ultrafiltration membrane is preferably 5-10kDa, which can achieve the penetration of small molecular impurities and retention of polysaccharides, thereby quickly achieving volume concentration. In addition, the ultrafiltration treatment does not require the addition of alcohols, which can avoid the problems of protein and pigment entrainment during alcohol precipitation, and the product purity is higher; the purification treatment is to use COFs (covalent organic framework materials) for adsorption treatment. COFs have adjustable pore size and functional groups and can be designed to selectively remove proteins, polyphenols, pigments, and oligomeric impurities, while maintaining high affinity or hydrophobic separation for target polysaccharides. COFs have a high specific surface area, good structural stability and chemical resistance, not only with high processing efficiency, but also can be reused to reduce costs.

[0023] Preferably, the spray drying technology forms particles with uniform particle size, good fluidity and good solubility by atomizing the red algae polysaccharide solution into fine droplets and drying them instantly in a hot air flow. This process not only significantly improves the drying efficiency, but also improves the physical properties of the red algae polysaccharide by controlling the morphology and particle size of the particles. In addition, spray drying can produce particles with porous or hollow structures. These structures facilitate the rapid penetration of water molecules, thereby increasing the dissolution rate. The inlet air temperature of the spray drying process is 170°C to 190°C. This temperature range can ensure that the polysaccharide droplets quickly contact the hot air flow after entering the drying chamber and achieve instant evaporation of water, thereby forming solid particles, effectively achieving efficient drying, avoiding long-term heating that causes degradation of the polysaccharide structure, and quickly forming and locking the original morphology of the droplets, which helps to form a hollow or porous structure and improve solubility. The outlet air temperature reflects the actual temperature environment experienced by the particles at the end of drying. It is the key control point for the moisture content and stability of the finished product. The outlet air temperature is 75℃~85℃, which is conducive to controlling the final moisture content of the product within a reasonable range to ensure storage stability. The outlet air temperature should not be too high to avoid degradation of heat-sensitive polysaccharides or darkening of color. The physicochemical pressure determines the particle size of droplets formed by the atomizing nozzle, which in turn affects the drying rate, particle structure and fluidity. The atomizing pressure is 0.1-0.2MPa. At this time, the droplets will be finer, the drying will be more complete, and it is easy to form a porous or hollow structure, which is conducive to rapid solubility; the granulation process further enhances its rapid solubility and stability by processing the dried powder into particles with a certain particle size and shape. In addition, the granulation technology can adjust the size and shape of the particles according to needs, making it more suitable for industrial production and application; the composite enzyme method combined with spray drying and granulation technology not only improves the extraction efficiency of red algae polysaccharides, but also improves its physical properties to make it more suitable for industrial production.

[0024] Furthermore, S200 includes: S210, performing a first enzymatic hydrolysis treatment on the red algae powder in a water bath using a first complex enzyme to obtain a first enzymatic hydrolysis intermediate; S220, performing a second enzymatic hydrolysis treatment on the first enzymatic hydrolysis intermediate using a second complex enzyme to obtain a second enzymatic hydrolysis intermediate; S230, performing a third enzymatic hydrolysis treatment on the second enzymatic hydrolysis intermediate using a third complex enzyme in a water bath, and then performing a centrifugal treatment and a filtration treatment in sequence to obtain a supernatant.

[0025] Preferably, the enzymatic hydrolysis treatment adopts a three-stage enzymatic hydrolysis distribution treatment, which can release the target polysaccharide components in an orderly, efficient and directionally manner, remove impurities, and improve the solubility and functionality of red algae polysaccharides; the first complex enzyme used in the first enzymatic hydrolysis treatment includes cellulase, hemicellulase, xylanase and pectinase, which can destroy the cell wall and intercellular mucilage, loosen the tissue structure, and lay the foundation for the second enzymatic hydrolysis treatment to release the main polysaccharide, wherein the amount of cellulase is 1.3~1.8%w / w, which is mainly used to degrade the cellulose main chain and destroy the cell wall skeleton, and the amount of hemicellulase is 1.2~1.5%w / w, which is mainly used to degrade the cellulose main chain and destroy the cell wall skeleton. Used to decompose hemicellulose side chains and reduce cell wall density, the dosage of xylanase is 0.4~0.6%w / w, which is mainly used to degrade xylan and cooperate with cellulase to enhance the wall breaking effect. The dosage of pectinase is 0.8~1.2%w / w, which hydrolyzes pectin and releases polysaccharide-pectin complexes; the temperature of the first enzymatic hydrolysis treatment is 40~55℃, and the pH is 5.0~5.5. At this temperature and pH, the activities of cellulase, hemicellulase, xylanase and pectinase are all good, which is conducive to enhancing the efficiency of the first enzymatic hydrolysis treatment. The time of the first enzymatic hydrolysis treatment is more suitable for 1.5-2h.

[0026] Furthermore, the first complex enzyme used in the first enzymatic hydrolysis treatment does not need to be fixed with magnetic particles. This is because the main purpose of the first enzymatic hydrolysis treatment is to break the cell wall of red algae and loosen the polysaccharide skeleton to create better substrate accessibility for the subsequent main enzymatic hydrolysis. The reaction time of the first enzymatic hydrolysis treatment is short. After the treatment is completed, the enzyme becomes ineffective or enters the next step. The first complex enzyme used includes cellulase, hemicellulase, xylanase and pectinase. If they are fixed, not only will there be problems with carrier adaptation, but also increased costs. Moreover, the immobilized enzyme will delay the diffusion of some enzyme release products, which may affect the continuity of substrate treatment. The free enzyme can quickly act on the surface and microstructure, and is suitable for primary lysis. In summary, the first complex enzyme does not need to be immobilized.

[0027] Preferably, the second enzymatic hydrolysis treatment is mainly to specifically degrade the main structural polysaccharides in red algae, such as agar, algin, and red algae cell wall polysaccharides, break the polymer chains, generate soluble oligosaccharides or oligosaccharides, improve solubility, reduce viscosity, and facilitate subsequent filtration, centrifugation, and drying. The second complex enzyme used includes cellulase, agarase, and xylanase, wherein the amount of cellulase is 1.3-1.6% w / w to further degrade residual cellulose and improve the purity of polysaccharides, and the amount of agarase is 1. 0~1.5%w / w, which can specifically cut the β-1,3 glycosidic bonds of agar polysaccharides, and the dosage of xylanase is 0.5~0.8%w / w, which supplements the degradation of hemicellulose-lignin complexes; the temperature of the second enzymatic hydrolysis treatment is 40~60℃, the pH is 5.5~6.5, and the time of the second enzymatic hydrolysis treatment is preferably 2-3h. After the second enzymatic hydrolysis treatment, the average molecular weight of the polysaccharide decreases and the dissolution rate increases, which is conducive to the formation of instant products, stable viscosity control, and convenient standardized production.

[0028] Preferably, in order to further improve the effect of the second enzymatic hydrolysis treatment, the second enzymatic hydrolysis treatment is carried out under ultrasonic or microwave-assisted conditions, the microwave power is preferably 300 W, and intermittent irradiation is adopted. After the addition of the second complex enzyme, microwave irradiation is performed for 10 seconds every 5 minutes to promote the breakage of the polysaccharide chain; the ultrasonic frequency is preferably 20 kHz. After the addition of the second complex enzyme, ultrasound is performed for 10 minutes every 30 minutes to improve the permeability of the second complex enzyme.

[0029] Preferably, the third enzymatic hydrolysis treatment is mainly to degrade non-target components such as residual protein and mannan gum, reduce protein residue, prevent precipitation and flocculation, and improve the purity and clarity of polysaccharides. The third complex enzyme used includes papain and mannanase. The dosage of papain is 0.5~0.8%w / w, which is mainly used to degrade protein impurities and improve the purity of polysaccharides. The dosage of mannanase is 0.3~0.5%w / w, which is mainly used to degrade protein impurities and improve the purity of polysaccharides. The temperature of the third enzymatic hydrolysis treatment is 40~55°C, the pH is 7.0~7.5, and the time is 1-1.5h; after the third enzymatic hydrolysis treatment, the subsequent COFs load pressure can be reduced and the purification efficiency can be improved.

[0030] Furthermore, the second and third complex enzymes are both immobilized with nanomagnetic particles. The types selected for the second and third complex enzymes are functional high-value enzymes. If used in free form, the enzymes are difficult to recover after the enzymatic hydrolysis is completed, resulting in cost waste. Therefore, these enzymes are immobilized with nanomagnetic particles. After the reaction is completed, they can be quickly separated by the magnetic particles and reused multiple times. In addition, the immobilized structure provides certain protection, reducing the risk of enzyme inactivation at high temperature and pH fluctuation. The contact between the immobilized enzyme and the substrate is more controllable, avoiding excessive degradation or non-target site cleavage, which is particularly important for controlling the retention of oligosaccharide structure in the second enzymatic hydrolysis treatment and avoiding the destruction of functional components in the third enzymatic hydrolysis treatment.

[0031] Preferably, when immobilizing the second or third complex enzyme, the nanomagnetic particles are first dispersed in phosphate buffer, an appropriate amount of the second or third complex enzyme is added, and the mixture is gently stirred at 4° C. for 4-6 hours to complete the immobilization of the second or third complex enzyme.

[0032] Furthermore, the preparation method of the nanomagnetic particles includes: S221, dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water, introducing nitrogen, stirring thoroughly until fully dissolved, then adding NH3·H2O to adjust the pH value to obtain a precipitate, separating the precipitate with an external magnet, and sequentially washing the precipitate and drying to obtain Fe3O4; S222, dispersing Fe3O4 in DMF and performing ultrasonic treatment, adding FeCl3·6H2O and H2BDC during the ultrasonic treatment, and then performing a hydrothermal reaction. After the reaction is completed, magnetic separation, washing, and drying are performed to obtain Fe3O4 with a MOFs layer; S223. Disperse Fe3O4 with MOFs layer in Tris-HCl buffer, add dopamine solution and stir, then magnetically separate, wash to neutrality, and dry to obtain nanomagnetic particles.

[0033] Preferably, FeCl3·6H2O and FeCl2·4H2O are used to prepare Fe3O4. Both FeCl3·6H2O and FeCl2·4H2O are easily soluble in water at room temperature and can quickly provide sufficient Fe 3+ and Fe 2+ ions, ensuring the uniformity of the reaction system, quickly precipitating Fe3O4, easy post-processing without the need for high-temperature calcination and other processes, and the by-products are non-toxic and environmentally friendly; if only FeCl3 is used, it will tend to generate γ-Fe2O3, while using only FeCl2 will be unstable and easily oxidized by air; when preparing Fe3O4, the pH is preferably adjusted to 10.

[0034] Preferably, in order to further increase the loading capacity of the nanomagnetic particles for the complex enzyme, the Fe3O4 needs to be further surface modified. First, MOFs (metal organic framework compounds) are used to embed the Fe3O4 surface. The present invention specifically adopts the method of dispersing Fe3O4 in DMF (N, N-dimethylformamide) and then adding FeCl3·6H2O and H2BDC for hydrothermal reaction, thereby embedding MOFs on the Fe3O4. Since MOFs have a porous structure with a high specific surface area and a large pore size and strong hydrophilicity, they facilitate the entry of enzyme molecules. In addition, MOFs can buffer external temperature / acid-base fluctuations to a certain extent, further improving the stability of the enzyme. On the Fe3O4 with the MOFs layer, the Fe3O4 with the MOFs layer is dispersed in Tris-HCl buffer and a dopamine solution is added to form a polydopamine layer. Polydopamine contains a large number of phenolic hydroxyl groups and can form chemical bonds with enzyme molecules, further increasing the amount of enzyme immobilization and simultaneously improving the oxidation resistance, acid and alkali resistance and mechanical stability of the magnetic particles.

[0035] First embodiment This embodiment provides a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method, comprising: S100, washing the red algae raw material and mechanically crushing it, wherein the particle size of the red algae raw material is less than 2 mm after the mechanical crushing, pre-freezing the red algae raw material at −40° C., and then vacuum freeze-drying the red algae raw material at −40° C. for 24 hours to obtain red algae powder; S200, performing enzymatic hydrolysis, centrifugation, and filtration on the red algae powder in sequence to obtain a supernatant; S300, the supernatant is subjected to ultrafiltration treatment using an ultrafiltration membrane, and then subjected to adsorption treatment using COFS, and then subjected to spray drying treatment at an inlet air temperature of 170°C, an outlet air temperature of 85°C, and a physicochemical pressure of 0.1 MPa, and finally subjected to fluidized bed granulation treatment to obtain red algae polysaccharide; Among them, S200 includes: S210, performing a first enzymatic hydrolysis treatment on the red algae powder using a first complex enzyme in a 40° C. water bath at a pH of 5.0 for 1.5 hours to obtain a first enzymatic hydrolysis intermediate; S220, performing a second enzymatic hydrolysis treatment on the first enzymatic hydrolysis intermediate using a second complex enzyme at 40° C., pH 5.5, and 300 W microwave irradiation with an intermittent 10 s every 5 min for 2 h to obtain a second enzymatic hydrolysis intermediate; S230, performing a third enzymatic hydrolysis treatment on the second enzymatic intermediate using a third complex enzyme in a 40° C. water bath at pH 7.0 for 1 hour, and then sequentially performing centrifugation and filtration to obtain a supernatant; The first complex enzyme is not immobilized by nano-magnetic particles, and the second and third complex enzymes are both immobilized by nano-magnetic particles, and the nano-magnetic particles are Fe3O4; The first complex enzyme is 1.3% w / w cellulase, 1.2% w / w hemicellulase, 0.4% w / w xylanase, and 0.8% w / w pectinase, the second complex enzyme is 1.3% w / w cellulase, 1.0% w / w agarase, and 0.5% w / w xylanase, and the third complex enzyme is 0.5% w / w papain and 0.3% w / w mannanase.

[0036] Second embodiment The embodiment provides a preparation process of red algae polysaccharide prepared by a complex enzyme method, which comprises the following steps: S100, washing and mechanically crushing red algae raw materials, and then performing vacuum freeze-drying at-45 ℃ for 18 h after pre-freezing at-40 ℃, to obtain red algae powder with a particle size of less than 2 mm; S200, sequentially performing enzymatic hydrolysis, centrifugal treatment, and filtration treatment on the red algae powder to obtain supernatant; S300, performing ultrafiltration treatment on the supernatant by using an ultrafiltration membrane, then performing adsorption treatment by using COFs, then performing spray drying treatment at an inlet air temperature of 190 ℃, an outlet air temperature of 75 ℃, and a physical and chemical pressure of 0.2 MPa, and finally performing fluidized granulation treatment, to obtain red algae polysaccharide; S200 comprises the following steps: S210, performing first enzymatic hydrolysis treatment on the red algae powder by using a first complex enzyme at 55 ℃ water bath and pH 5.5 for 1.5 h to obtain a first enzymatic hydrolysis intermediate; S220, performing second enzymatic hydrolysis treatment on the first enzymatic hydrolysis intermediate by using a second complex enzyme at 60 ℃, pH 6.5, and an ultrasonic frequency of 20 Hz, and intermittently ultrasonicizing for 10 min every 30 min for 2 h to obtain a second enzymatic hydrolysis intermediate; S230, performing third enzymatic hydrolysis treatment on the second enzymatic hydrolysis intermediate by using a third complex enzyme at 55 ℃ water bath and pH 7.5 for 1.5 h, and then sequentially performing centrifugal treatment and filtration treatment to obtain supernatant; The first complex enzyme is not immobilized by nano-magnetic particles, and the second and third complex enzymes are both immobilized by nano-magnetic particles, and the nano-magnetic particles are Fe3O4; The first complex enzyme is 1.8% w / w cellulase, 1.5% w / w hemicellulase, 0.6% w / w xylanase and 1.2% w / w pectinase in an amount of 0.8% w / w, the second complex enzyme is 1.6% w / w cellulase, 1.5% w / w agarase and 0.8% w / w xylanase, and the third complex enzyme is 0.8% w / w papain and 0.5% w / w mannanase.

[0037] Third embodiment This embodiment provides a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method, comprising: S100, washing the red algae raw material and mechanically crushing it, wherein the particle size of the red algae raw material is less than 2 mm after the mechanical crushing, pre-freezing the red algae raw material at −40° C., and then vacuum freeze-drying the red algae raw material at −40° C. for 18 hours to obtain red algae powder; S200, performing enzymatic hydrolysis, centrifugation, and filtration on the red algae powder in sequence to obtain a supernatant; S300, the supernatant is subjected to ultrafiltration treatment using an ultrafiltration membrane, and then subjected to adsorption treatment using COFs, and then subjected to spray drying treatment at an inlet air temperature of 180°C, an outlet air temperature of 80°C, and a physicochemical pressure of 0.2 MPa, and finally subjected to fluidized bed granulation treatment to obtain red algae polysaccharide; Among them, S200 includes: S210, performing a first enzymatic hydrolysis treatment on the red algae powder using a first complex enzyme in a 50° C. water bath at a pH of 5.0 for 1.5 hours to obtain a first enzymatic hydrolysis intermediate; S220, performing a second enzymatic hydrolysis treatment on the first enzymatic intermediate using a second complex enzyme at 55° C., pH 6.0, and 300 W microwave irradiation with an intermittent 10 s every 5 min for 2 h to obtain a second enzymatic hydrolysis intermediate; S230, performing a third enzymatic hydrolysis treatment on the second enzymatic intermediate using a third complex enzyme in a 50° C. water bath at pH 7.0 for 1.5 h, then sequentially performing centrifugation and filtration to obtain a supernatant; The first complex enzyme has not been immobilized with nanomagnetic particles, and the second complex enzyme and the third complex enzyme have been immobilized with nanomagnetic particles. The preparation method of the nanomagnetic particles includes: S221, dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water, introducing nitrogen, and stirring thoroughly until fully dissolved, then adding NH3·H2O to adjust the pH to 10 to obtain a precipitate, separating the precipitate with an external magnet, and sequentially washing the precipitate and drying to obtain Fe3O4; S222, dispersing Fe3O4 in DMF and performing ultrasonic treatment, adding FeCl3·6H2O and H2BDC during the ultrasonic treatment, and then performing a hydrothermal reaction. After the reaction is completed, magnetic separation, washing, and drying are performed to obtain Fe3O4 with a MOFs layer; S223, dispersing Fe3O4 with MOFs layer in Tris-HCl buffer, adding dopamine solution and stirring, magnetic separation, washing to neutrality, and drying to obtain nanomagnetic particles. The first complex enzyme is 1.5% w / w cellulase, 1.4% w / w hemicellulase, 0.5% w / w xylanase and 1.0% w / w pectinase in an amount of 0.8% w / w, the second complex enzyme is 1.3% w / w cellulase, 1.0% w / w agarase and 0.6% w / w xylanase, and the third complex enzyme is 0.8% w / w papain and 0.5% w / w mannanase.

[0038] Fourth embodiment This embodiment provides a preparation process for fast-soluble red algae polysaccharide by a composite enzyme method, comprising: S100, washing the red algae raw material and mechanically crushing it, wherein the particle size of the red algae raw material is less than 2 mm after the mechanical crushing, pre-freezing the red algae raw material at −40° C., and then vacuum freeze-drying the red algae raw material at −40° C. for 18 hours to obtain red algae powder; S200, performing enzymatic hydrolysis, centrifugation, and filtration on the red algae powder in sequence to obtain a supernatant; S300, the supernatant is subjected to ultrafiltration treatment using an ultrafiltration membrane, and then subjected to adsorption treatment using COFs, and then subjected to spray drying treatment at an inlet air temperature of 180°C, an outlet air temperature of 80°C, and a physicochemical pressure of 0.2 MPa, and finally subjected to fluidized bed granulation treatment to obtain red algae polysaccharide; Among them, S200 includes: S210, performing a first enzymatic hydrolysis treatment on the red algae powder using a first complex enzyme in a 50° C. water bath at a pH of 5.0 for 1.5 hours to obtain a first enzymatic hydrolysis intermediate; S220, performing a second enzymatic hydrolysis treatment on the first enzymatic intermediate using a second complex enzyme at 60° C., pH 6.5, and an ultrasonic frequency of 20 Hz with intermittent ultrasonication for 10 minutes every 30 minutes for 2 hours to obtain a second enzymatic hydrolysis intermediate; S230, performing a third enzymatic hydrolysis treatment on the second enzymatic intermediate using a third complex enzyme in a 50° C. water bath at pH 7.0 for 1.5 h, then sequentially performing centrifugation and filtration to obtain a supernatant; The first complex enzyme has not been immobilized with nanomagnetic particles, and the second complex enzyme and the third complex enzyme have been immobilized with nanomagnetic particles. The preparation method of the nanomagnetic particles includes: S221, dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water, introducing nitrogen, and stirring thoroughly until fully dissolved, then adding NH3·H2O to adjust the pH to 10 to obtain a precipitate, separating the precipitate with an external magnet, and sequentially washing the precipitate and drying to obtain Fe3O4; S222, dispersing Fe3O4 in DMF and performing ultrasonic treatment, adding FeCl3·6H2O and H2BDC during the ultrasonic treatment, and then performing a hydrothermal reaction. After the reaction is completed, magnetic separation, washing, and drying are performed to obtain Fe3O4 with a MOFs layer; S223, dispersing Fe3O4 with MOFs layer in Tris-HCl buffer, adding dopamine solution and stirring, magnetic separation, washing to neutrality, and drying to obtain nanomagnetic particles. The first complex enzyme is 1.5% w / w cellulase, 1.4% w / w hemicellulase, 0.5% w / w xylanase and 1.0% w / w pectinase in an amount of 0.8% w / w, the second complex enzyme is 1.3% w / w cellulase, 1.0% w / w agarase and 0.6% w / w xylanase, and the third complex enzyme is 0.8% w / w papain and 0.5% w / w mannanase.

[0039] Performance Testing The immobilized enzymes of Examples 1 to 4 were subjected to multiple enzymolysis cycles, and the enzyme activity retention rate of each round was recorded. The blank control group was free enzyme that was not immobilized. The immobilized enzymes of Examples 1 and 2 were inactivated after 5 repeated uses, and the immobilized enzymes of Examples 3 and 4 were inactivated after 10 repeated uses, while the free enzyme was inactivated after only 2 repeated uses, indicating that the enzymes can be reused multiple times after immobilization. The immobilized enzymes of Examples 3 and 4 were immobilized using the nanomagnetic particles described in the present invention, indicating that the use of the magnetic particles described in the present invention can improve the enzyme activity retention rate. Solubility test of red algae polysaccharide: take the same amount of red algae polysaccharide prepared in the first to fourth embodiments and place it in the same volume of deionized water. Observe whether it dissolves within 30 seconds, and measure the residual mass after 1 minute. Extraction rate of red algae polysaccharide: The total mass of the red algae polysaccharide prepared in the first to fourth embodiments was measured by dry weight method, and the yield based on the dry weight of the red algae was calculated; The test results are shown in the following table: As can be seen from the above table, the red algae polysaccharides prepared in the first to fourth embodiments can all be dissolved within 30 seconds, and the 1-minute dissolution rate is all above 91%, indicating that the red algae polysaccharides prepared by the method described in the present invention are fast-soluble; the yield of red algae polysaccharides is all above 20%, which is higher than that of the traditional method, indicating that the preparation of red algae polysaccharides by the method described in the present invention has better results.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A process for preparing fast-soluble red algae polysaccharide by a composite enzyme method, characterized in that: include: S100, washing the red algae raw material and sequentially subjecting it to mechanical pulverization and freeze-drying to obtain red algae powder; S200, sequentially performing enzymatic hydrolysis, centrifugation, and filtration on the red algae powder to obtain a supernatant; S300, sequentially performing concentration treatment, purification treatment, spray drying treatment, and fluidized bed granulation treatment on the supernatant to obtain red algae polysaccharide; Wherein, the S200 includes: S210, performing a first enzymatic hydrolysis treatment on the red algae powder in a water bath using a first complex enzyme to obtain a first enzymatic hydrolysis intermediate; S220, performing a second enzymatic hydrolysis treatment on the first enzymatic hydrolysis intermediate using a second complex enzyme under ultrasound / microwave-assisted conditions to obtain a second enzymatic hydrolysis intermediate; S230, performing a third enzymatic hydrolysis treatment on the second enzymatic intermediate using a third complex enzyme in a water bath, and then sequentially performing a centrifugal treatment and a filtration treatment to obtain the supernatant; The second complex enzyme and the third complex enzyme are both immobilized by nanomagnetic particles.

2. The preparation process according to claim 1, characterized in that The preparation method of the nanomagnetic particles comprises: S221, dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water, introducing nitrogen, stirring thoroughly until fully dissolved, then adding NH3·H2O to adjust the pH value to obtain a precipitate, separating the precipitate with an external magnet, and sequentially washing the precipitate and drying to obtain Fe3O4; S222, dispersing Fe3O4 in DMF and performing ultrasonic treatment, adding FeCl3·6H2O and H2BDC during the ultrasonic treatment, and then performing a hydrothermal reaction. After the reaction is completed, magnetic separation, washing, and drying are performed to obtain Fe3O4 with a MOFs layer; S223, dispersing the Fe3O4 with the MOFs layer in a Tris-HCl buffer solution, adding a dopamine solution and stirring, and then magnetically separating, washing to neutrality, and drying to obtain the nanomagnetic particles.

3. The preparation process according to claim 1, characterized in that In the S221, the pH is adjusted to 9.0-10; and / or In S222, the temperature of the hydrothermal reaction is 110-120° C.; and / or In S222, the hydrothermal reaction time is 12 to 24 hours; and / or In the S223, the concentration of the dopamine solution is 1-2 mg / mL.

4. The preparation process according to claim 1, characterized in that The first complex enzyme comprises at least one of cellulase, hemicellulase, xylanase and pectinase or a combination thereof; and / or The second complex enzyme comprises at least one of cellulase, agarase and xylanase or a combination thereof; and / or The third complex enzyme includes at least one of papain and mannanase or a combination thereof.

5. The preparation process according to claim 4, characterized in that: In the first complex enzyme, the amount of cellulose is 1.3-1.8% w / w, the amount of hemicellulase is 1.2-1.5% w / w, the amount of xylanase is 0.4-0.6% w / w, and the amount of pectinase is 0.8-1.2% w / w; and / or In the second complex enzyme, the amount of the cellulase is 1.3-1.6% w / w, the amount of the agarase is 1.0-1.5% w / w, and the amount of the xylanase is 0.5-0.8% w / w; and / or In the third complex enzyme, the amount of papain is 0.5-0.8% w / w, and the amount of mannanase is 0.3-0.5% w / w.

6. The preparation process according to claim 1, characterized in that In the above S300, The concentration treatment is ultrafiltration treatment using an ultrafiltration membrane; and / or The purification process is to use COFs for adsorption treatment.

7. The preparation process according to claim 1, characterized in that In said S210, the temperature of said first enzymatic hydrolysis treatment is 40-55° C.; and / or In said S210, the pH of said first enzymatic hydrolysis treatment is 5.0-5.5; and / or In said S220, the temperature of said first enzymatic hydrolysis treatment is 40-60° C.; and / or In said S220, the pH of said first enzymatic hydrolysis treatment is 5.5-6.5; and / or In said S230, the temperature of said third enzymatic hydrolysis treatment is 40-55° C.; and / or In the S230, the pH of the third enzymatic hydrolysis treatment is 7.0-7.

5.

8. The preparation process according to claim 1, characterized in that In the S100, After the mechanical crushing process, the particle size of the red algae raw material is less than 2 mm; and / or The freeze-drying temperature is -40 to -45°C; and / or The freeze-drying process lasts for 18 to 24 hours.

9. The preparation process according to claim 1, characterized in that: In the S100, The red algae raw material includes at least one of Gracilaria, Gelidium, Porphyra, Red Hair Algae, Glucostomia, Seaweed, Sea Head Red, Polysiphonia, Centipede Algae, Partridge Algae, Hyssop Noodles and Gallinae or a combination thereof.

10. The preparation process according to claim 1, characterized in that: In the above S300, The inlet air temperature of the spray drying process is 170°C to 190°C; and / or The outlet air temperature of the spray drying process is 75°C to 85°C; and / or The atomization pressure of the spray drying process is 0.1-0.2 MPa.

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