Preparation process of high-efficiency instant red algae polysaccharide based on composite enzyme method

Abstract

The application provides a high-efficiency instant red algae polysaccharide preparation process based on a composite enzyme method, and belongs to the technical field of polysaccharide extraction. The application uses streptomyces to pre-ferment red algae raw materials, gently loosens the cell wall structure, then carries out high-pressure treatment to destroy the cell wall at normal temperature, and then sequentially carries out selective polysaccharide enzymolysis and sulfatase hydrolysis, step-by-step degrades the polysaccharide network and directionally modifies the structure, finally purifies through an ultrafiltration membrane and sprays and dries to obtain high-purity instant red algae polysaccharide powder, so that the problems of low extraction efficiency, easy damage to the polysaccharide structure and poor solubility of the product in the traditional red algae polysaccharide extraction process are solved. The polysaccharide product obtained by the red algae polysaccharide preparation process has excellent instant solubility, structural stability and functional adjustability, and can be used in the fields of food industry, medical care, cosmetics and agricultural additives.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, specifically to a highly efficient and rapidly soluble red algae polysaccharide preparation process based on a compound enzyme method. Background Technology

[0002] In the current field of biomanufacturing, the efficient extraction and structure preservation of polysaccharides is an important research direction. Traditional extraction methods generally suffer from low efficiency and poor product quality. While modern auxiliary technologies can improve efficiency, they still face challenges in systematically deciphering the complex structures of raw materials and achieving precise control over product properties. Therefore, developing green integrated processes that can combine the advantages of multiple technologies and achieve programmed, precise processing of specific raw materials has become the key to overcoming existing technological bottlenecks.

[0003] Driven by the urgent need for systematic and refined extraction processes, researchers are now focusing on integrating physical and biological technologies to seek breakthroughs. CN121086093A discloses a highly efficient method for extracting lentinan from shiitake mushrooms with antioxidant effects. By sequentially treating shiitake mushrooms with pulsed light, ultrasound, and a compound enzyme method, the polysaccharide is released by the compound enzyme after physical cell wall disruption, significantly improving the extraction rate and enhancing antioxidant activity. However, the compound enzyme used in this method acts synchronously in the same reaction system and under the same conditions. Due to the differences in the optimal temperature, pH, and action sites of different enzymes, this compromise cannot allow each enzyme to reach its optimal catalytic efficiency simultaneously, thus limiting further improvement in the overall enzymatic hydrolysis effect. Furthermore, although this process combines physical pretreatment and enzymatic hydrolysis, it does not explicitly involve real-time monitoring and precise termination control of the enzymatic hydrolysis process, making it difficult to dynamically adjust the reaction process according to different raw material batches or states. This may lead to a wide molecular weight distribution of the final product or excessive degradation of some target components. CN116396995A discloses a method for preparing red algae extract and its application. It uses a specific complex enzyme for simultaneous hydrolysis and combines it with membrane separation technology to purify the product. However, it also faces the problem of optimizing conditions when multiple enzymes act simultaneously during the enzymatic hydrolysis stage. In addition, the heat treatment step after enzymatic hydrolysis in the process is relatively simple and may not be able to completely inactivate all enzymes, which poses a potential risk of unexpected enzyme reactions in subsequent processes.

[0004] In summary, existing technologies still face challenges in achieving efficient synergy among multiple enzymes and precise control of the reaction process. Therefore, there is an urgent need to propose an extraction process that can better coordinate enzymatic hydrolysis conditions and introduce process monitoring and regulation mechanisms, thereby developing functional polysaccharide products with superior performance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a highly efficient and rapidly soluble red algae polysaccharide preparation process based on a complex enzymatic method. This invention utilizes Streptomyces to pre-ferment red algae raw materials, gently loosening the cell wall structure. Subsequently, high-pressure treatment at room temperature disrupts the cell wall, followed by selective polysaccharide hydrolysis and sulfate ester hydrolysis, stepwise degrading the polysaccharide network and directionally modifying its structure. Finally, ultrafiltration and spray drying yield high-purity, rapidly soluble red algae polysaccharide powder, which can be used in the food industry, healthcare, cosmetics, and agricultural additives. This process solves the problems of low extraction efficiency, easy destruction of polysaccharide structure, and poor product solubility in traditional red algae polysaccharide extraction processes.

[0006] This invention discloses a highly efficient and rapidly soluble red algae polysaccharide preparation process based on a compound enzyme method, such as... Figure 1 As shown, the specific technical solution is as follows: Step 1: After crushing the red algae raw material, mix it with a fermentation medium containing Streptomyces and signaling pathway initiators for fermentation. After fermentation, heat and sterilize the mixture, then cool the system and perform high-pressure homogenization.

[0007] Step 2: Adjust the pH and temperature of the mixture after high-pressure crushing, add a selective polysaccharide degrading enzyme to induce an enzymatic hydrolysis reaction in the system, and continuously monitor the viscosity of the system. When the viscosity of the system drops to less than half of the initial viscosity, raise the temperature to inactivate the enzyme.

[0008] Step 3: Adjust the pH and temperature of the inactivated system, add sugar sulfatase to react the system, and raise the temperature after the reaction to inactivate the enzyme.

[0009] Step 4: The mixture after enzymatic hydrolysis and inactivation is concentrated by ultrafiltration membrane, and then the concentrate is spray-dried to obtain red algae polysaccharide powder.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. By synergistically disrupting the cell wall and sequentially enzymatically hydrolyzing, the cell wall of red algae is more thoroughly destroyed, allowing for a more complete release of polysaccharides from the intracellular matrix and cell wall matrix. This significantly improves the extraction rate of total polysaccharides and specific active polysaccharide components. Furthermore, the extraction process is carried out under mild enzymatic conditions and controllable physical conditions, avoiding the sugar chain breakage caused by prolonged high-temperature heating and the damage to sensitive groups such as sulfate ester bonds caused by strong acids and bases. This achieves efficient extraction while preserving the natural structure and key functional groups of polysaccharides to the greatest extent possible.

[0011] 2. Selective polysaccharide degrading enzymes specifically degrade the polysaccharide backbone that is prone to gel formation, fundamentally eliminating the main components that lead to high viscosity and insolubility. Under controlled conditions, sulfatases selectively cleave the key sulfate bonds that serve as cross-linking points in the polysaccharide network, rather than generally desulfurizing the polysaccharide molecules. While effectively disintegrating the structure, it retains the sulfate groups that give the polysaccharide charge and activity to the maximum extent. Combined with ultrafiltration purification to remove impurities, the target polysaccharide components with good water solubility are enriched in the final product, and the intermolecular forces are weakened and the surface negative charge is enhanced.

[0012] 3. By adjusting the pre-fermentation conditions, the degree and pattern of biodegradation of cell wall components can be affected, thereby controlling the initial state of subsequent polysaccharide release. This multi-level, multi-parameter controllable system allows key structural parameters such as the molecular weight distribution range, degree and distribution of sulfation, and branched structure characteristics of the product to be systematically adjusted according to predetermined goals, providing a customizable structural basis for different functional requirements. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the preparation process of the red algae polysaccharide of the present invention; Figure 2 This is a schematic diagram of the liquid chromatography analysis of the pretreated raw material in Example 1 of the present invention; Figure 3 This is a schematic diagram of the infrared spectrum of the red algae polysaccharide powder prepared in Example 1 of the present invention. Detailed Implementation

[0014] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0015] This invention proposes a highly efficient and rapidly soluble red algae polysaccharide preparation process based on a compound enzyme method, such as... Figure 1 As shown, the specific technical solution is as follows: 1. Combined biological and physical pretreatment Streptomyces induced by a signaling pathway initiator were used to ferment red algae feedstock. After sterilization, the red algae cell walls were ruptured by high-pressure treatment. The red algae cell wall is a rigid complex formed by crystalline cellulose microfibrils embedded in a matrix of hemicellulose, pectin, and sulfated polysaccharides. During growth, Streptomyces secretes various hydrolytic enzymes with different substrate specificities into the extracellular environment to obtain carbon sources. These enzymes form a synergistic combination. Cellulases attack the cellulose backbone, degrading crystalline and amorphous cellulose into soluble sugars. Meanwhile, another group of hemicellulases targets the hemicellulose components covalently or non-covalently cross-linked with cellulose microfibrils in the cell wall, catalyzing the hydrolysis of their main chain and side chains. Pectinases degrade the pectin substances filling the cellulose-hemicellulose network. This multi-enzyme synergistic catalytic reaction occurs in a mild, near-neutral aqueous environment, performing a precise and selective biological dismantling of the cell wall polymer network. It precisely severs the key bonds that maintain the integrity of the network, resulting in increased pore size and reduced mechanical connection points in the originally dense supramolecular structure. Signaling pathway initiators, including activity inhibitors and signaling pathway inducers, are added to the culture medium beforehand. Glucose is used as the activity inhibitor, allowing *Streptomyces* to utilize the glucokinase (GK) pathway to inhibit the transcription of the dagA gene and suppress the activity of agarase secretion, thus ensuring that *Streptomyces* will not secrete agarase. Under the action of the inducer, regulatory proteins such as CebR release the inhibition of cellulase by *Streptomyces*, thereby inducing and promoting the opening of signaling pathways for cellulase and hemicellulase secretion. During the subsequent cell wall disruption of the red algae raw material, this primarily targets cellulose and hemicellulose, specifically degrading the rigid skeleton of the red algae cell wall and protecting the integrity of the red algae polysaccharides. Simultaneously, viscosity is continuously monitored during the pre-fermentation process. As *Streptomyces* disrupts the red algae cell wall, the system viscosity initially rises slowly and then decreases significantly. When the system viscosity stabilizes, it indicates that the main cell wall of the red algae raw material has been disrupted. At this point, high-temperature sterilization can be performed to stop the pre-fermentation process, avoiding over-fermentation and damage to the structural integrity of intracellular polysaccharides. After the biological process is terminated by high-temperature sterilization, high-pressure treatment applies pressure to the cell wall structure that has been loosened by biological enzymatic hydrolysis, producing multiple physical effects. The polymer skeleton of the cell wall undergoes elastic compression under high pressure until it undergoes plastic deformation, and the stored strain energy continuously increases. When the pressure is released instantaneously or fluctuates, the gas dissolved in the liquid or the liquid itself will form microbubbles and rupture, producing a strong local cavitation effect. At the same time, it is accompanied by huge shear forces. This isotropic macroscopic compressive stress and localized microscopic cavitation shear stress form a combined force, which can easily exceed the structural strength threshold and fracture toughness of the cell wall that have been significantly reduced by biological treatment. This leads to instantaneous and complete brittle disintegration and tearing of the cell wall, causing the cell contents to be released into the liquid phase on a large scale.

[0016] 2. Targeted treatment with polysaccharide-degrading enzymes The pH and temperature of the pretreated mixture were adjusted, a selective polysaccharide-degrading enzyme was added for reaction, and the enzyme was inactivated at high temperature when the viscosity decreased to a certain level. The structural polysaccharides accumulated in the cell walls and intercellular matrix of red algae are mainly agar polysaccharides and carrageenan polysaccharides. Both of these polysaccharides are long-chain linear molecules composed of galactose and its derivatives, but their precise monosaccharide composition, glycosidic bond linkage mode, and sulfation mode differ. After dissolving in hot water, these long-chain molecules form a three-dimensional hydrated network gel structure that runs through the entire solution space through their regular secondary structure and extensive hydrogen bonding and specific ionic cross-linking between molecules. This continuous network that fixes a large number of water molecules in the structure increases the viscosity of the aqueous solution and decreases its solubility. The introduction of selective polysaccharide-degrading enzymes (such as agarase and carrageenase) can specifically deconstruct the core framework of this high-viscosity gel network. The active centers of agarase and carrageenase can highly specifically recognize and bind to degradation sites composed of specific galactose derivative sequences in the corresponding polysaccharide chains. Under the corresponding pH and temperature conditions, the enzyme molecules efficiently catalyze the hydrolysis and breakage of glycosidic bonds, causing the long-chain polysaccharide polymers that make up the gel network framework to be cut one by one, generating a series of oligosaccharide fragments with significantly reduced molecular weight. This leads to a reduction in the hydrodynamic radius of individual polysaccharide chains, and the ability of short-chain oligosaccharides to form stable helical conformations and further undergo interhelical aggregation is greatly weakened or completely lost. A large number of physical cross-linking points that maintain the macroscopic gel network are removed, causing the original continuous rigid three-dimensional network structure that extends to the entire system to collapse and disintegrate from the inside out, transforming into a discrete sol state with only weak local interactions. This results in a decrease in viscosity, and the target components encapsulated in the residual cell debris and other solid phase substances can diffuse more fully into the liquid phase bulk. Simultaneously, specific polysaccharide-degrading enzymes selectively degrade and subsequently remove the dominant gel-forming polysaccharide, increasing the relative concentration and accessibility of the target active polysaccharide component in the liquid phase. This matrix-degradation effect not only enriches the target active component but also creates an ideal reaction environment for subsequent precise modification by sulfatases. In a low-viscosity, near-Newtonian fluid homogeneous medium, the diffusion coefficient between sulfatase and the target sulfated polysaccharide substrate is significantly increased, and the effective collision frequency and the probability of forming enzyme-substrate complexes are significantly increased. This ensures that the subsequent sulfated group modification reaction can proceed in a highly efficient, uniform, and controllable manner, and the reaction kinetics are no longer limited by diffusion, thus laying a solid foundation for precisely controlling the degree of sulfation and bioactivity of the final product. By monitoring viscosity changes online and immediately inactivating at high temperature when a predetermined decrease threshold is reached, the degree of degradation of such polysaccharides can be precisely controlled. This effectively breaks down the viscosity barrier while avoiding unnecessary over-hydrolysis of specific structural fragments in the target active component or gel polysaccharide that may need to be retained.

[0017] 3. Sulfate esterase-controlled modification The pH and temperature of the system were adjusted, sulfatase was added, and the reaction was followed by high-temperature inactivation. The bioactivity of red algae polysaccharides is closely related to the content, substitution position, and distribution pattern of sulfate groups on their molecules. These negatively charged sulfate groups are covalently linked to specific hydroxyl groups on the sugar ring via sulfate bonds. They not only directly participate in electrostatic interactions with various biomacromolecules but also profoundly affect the conformation, flexibility, and intermolecular forces of the polysaccharide chains in aqueous solution. Sulfatases are a class of esterases that can hydrolyze sulfate bonds. Different sulfatases have strict substrate specificity. This specificity is reflected in the precise requirements for the type of sugar unit to which the sulfate group is attached, the position of the substituted hydroxyl group on the sugar ring, and even the stereoconfiguration of adjacent glycosidic bonds. This high degree of specificity determines that enzymatic desulfurization is an ordered and directional process, rather than an indiscriminate attack on all sulfate groups. Since the sulfate bonds on the polysaccharide molecular chains of red algae are glycosyl sulfates, glycosylsulfatases can specifically target these sulfate bonds, hydrolyzing the sulfate bond at the C-6 position of galactose residues, thereby site-specifically removing sulfate groups from the polysaccharide chains. By independently adjusting the pH and temperature of the reaction system to the optimal range for the sulfatase, the enzyme molecules diffuse and bind to the dissolved polysaccharide chains. The enzyme's active site first identifies and confirms sulfation sites on the substrate chain that meet its specific requirements, then initiates a catalytic cycle. Each successful catalysis removes a sulfate group from a specific position on the polysaccharide backbone. Reaction time is a crucial external variable controlling the final degree of desulfurization. In the initial stage of the reaction, the enzyme preferentially acts on sulfate groups with high spatial accessibility and sufficient exposure of enzyme binding sites. The removal of these sites significantly alters the local electrostatic environment and steric hindrance of the polysaccharide chain segments, further improving chain flexibility and solvent accessibility, exposing more previously hidden sites. In the early stages of the reaction, sulfatases primarily remove key sulfate groups that constitute major steric barriers to molecular solubility or cause abnormal intermolecular aggregation. While maintaining the framework of sulfation required for high bioactivity of polysaccharides, they effectively improve water solubility and dispersibility. As the reaction time increases, more sulfate groups are continuously lost, the net negative charge density of the polysaccharide chains decreases, the electrostatic repulsion between chain segments weakens, and the conformation of the polysaccharide molecule in water changes from a relatively relaxed state, revealing hydrophobic interactions. Therefore, by adjusting the reaction time, the degree of sulfation of the product can be precisely controlled, thereby controlling the product's bioactivity and physical solubility.

[0018] 4. Purification and drying to form the product The enzymatic hydrolysate is treated with an ultrafiltration membrane to remove impurities and concentrate it. A small amount of ammonium bicarbonate is then mixed in and spray-dried into powder. Under the pressure of ultrafiltration, solvent and small-sized solute molecules can pass through the membrane pores, while polysaccharide molecules, due to their larger size, are completely blocked. The process is carried out at room temperature, ensuring the thermodynamic stability of the polysaccharide conformation. This operation first achieves absolute removal of impurities based on size differences; all molecules smaller than the membrane retention threshold are irreversibly separated and discarded. Only the target component larger than the threshold is retained in the product stream. By continuously removing solvent, the product stream is concentrated proportionally, the solution volume decreases at a fixed ratio, and the solids concentration increases linearly along a predetermined trajectory. Simultaneously, a size-based narrowing effect is produced on the target polysaccharide population, and the lower limit of the molecular weight distribution is clearly defined by the membrane retention threshold, thereby improving the consistency of the product molecular weight. Spray drying atomizes the concentrated liquid into micron-sized droplets, creating a huge instantaneous evaporation interface. The droplets can quickly skip the viscous transition state of polysaccharides in the hot gas flow, preventing hydrogen bond reconstruction or tight packing between molecules that would lead to decreased solubility. After the water rapidly escapes, ammonium bicarbonate undergoes violent decomposition to produce gas. The instantaneous release of this gas forms a large number of micropores and channels on the surface and inside of the solidifying particles, giving the final powder particles a sponge-like or hollow microstructure. This fixes the polysaccharide molecules in a porous, loose solid framework. This artificially created microporous structure provides a capillary network for the instantaneous penetration of water, exposing a large internal surface area to the solvent. This allows dissolution to occur simultaneously from the inside and the surface of the particles, helping to improve the solubility of the final product.

[0019] The following are some specific embodiments of the present invention, and Table 1 shows the raw material information used in the embodiments.

[0020] Table 1 Raw Material Information Table Example 1 S1: Weigh 1.5g glucose, 0.5g peptone, 0.1g yeast extract powder and 0.05g magnesium sulfate heptahydrate, dissolve in 90mL deionized water and bring the volume to 100mL to obtain a liquid culture medium. Adjust the pH of the culture medium to 7.0 with 1mol / L NaOH. Then, inoculate 5mL of activated Streptomyces white into the culture medium and stir well. Then, add 1g microcrystalline cellulose and 6.67g red algae powder derived from Carrageenan to the culture medium. Ferment at 30℃ and 150rpm in a constant temperature shaker for 24h. Then, transfer the system to an autoclave and sterilize at 121℃ and 0.1MPa for 15min. After sterilization, cool the mixture to room temperature and transfer it to a high pressure homogenizer. Circulate the mixture twice under a hydrostatic pressure of 400MPa, holding the pressure for 2min each time to obtain a suspension. S2: Adjust the pH of the suspension obtained in S1 to 6.5 with 1 mol / L NaOH and heat it to 45°C in a constant temperature water bath. Then add 33.3 mg of β-agarase and react at 45°C and 100 rpm for 2 h. During the reaction, continuously monitor the viscosity of the system with a rotational viscometer. When the viscosity drops to 45% of the initial viscosity of the system, immediately transfer the reaction system to a 90°C water bath and maintain it for 15 min to inactivate the β-agarase. S3: Cool the inactivated system obtained in S2 to 40℃, adjust the pH to 5.5 with 1mol / L hydrochloric acid, then add 13.3mg of glycosulfatase, react at 40℃ and 100rpm for 4h, then heat the system to 85℃ and hold for 15min to inactivate the sulfatase, and obtain a clear polysaccharide hydrolysate. S4: The polysaccharide hydrolysate obtained in S3 is passed through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 30 kDa. Under conditions of 0.3 MPa pressure and 25°C, deionized water is continuously added until the conductivity of the filtrate is lower than 50 μS / cm. 0.5% sodium bicarbonate, 0.1% maltodextrin, and 0.5% β-cyclodextrin by mass are added to the concentrate in the ultrafiltration membrane. After mixing evenly, the mixture is sent to a spray dryer. The inlet air temperature is set to 180°C, the outlet air temperature to 95°C, the feed flow rate to 5 mL / min, and the atomizer speed to 20,000 rpm to obtain a dried powder, namely, high-efficiency fast-soluble red algae polysaccharide powder.

[0021] Example 2 The preparation method according to Example 1 differs in that: S1: The red algae powder was derived from Euphorbia milii. White Streptomyces was replaced with gray Streptomyces. The amount of glucose added was 1g. Microcrystalline cellulose was replaced with coarse red algae powder and added at a rate of 0.5g. The pH of the culture medium was adjusted to 6.5. The fermentation temperature was 28℃. The fermentation time was 12h. The pressure of the high-pressure homogenization treatment was 200MPa. The number of cycles was 1. S2: Adjust the pH of the system to 6.0, the reaction temperature to 40℃, replace β-agarase with α-agarase, and add 6.67mg. When the reaction is terminated, the viscosity of the system is 30% of the initial viscosity. S3: Adjust the pH of the system to 5.0, the reaction temperature to 35℃, the amount of sugar sulfatase added to be 3.3mg, and the reaction time to be 2h; S4: The amount of sodium bicarbonate added is 0.2% of the mass of the concentrate, the inlet air temperature for spray drying is 160℃, and the remaining steps are the same.

[0022] Example 3 The preparation method according to Example 1 differs in that: S1: The red algae powder was derived from Gracilaria. White Streptomyces was replaced with purple Streptomyces. The amount of glucose added was 2g, the amount of microcrystalline cellulose added was 1.5g, the pH of the culture medium was adjusted to 7.5, the fermentation temperature was 32℃, the fermentation time was 48h, the pressure of the high-pressure homogenization treatment was 600MPa, and the number of cycles was 4. S2: Adjust the pH of the system to 7.0, the reaction temperature to 50℃, replace β-agarase with carrageenase at an addition amount of 100mg, and the viscosity of the system at the end of the reaction is 50% of the initial viscosity; S3: Adjust the pH of the system to 6.0, the reaction temperature to 45℃, the amount of sugar sulfatase added to be 33mg, and the reaction time to be 8h; S4: The amount of sodium bicarbonate added is 0.8% of the mass of the concentrate, the inlet air temperature for spray drying is 200℃, and the remaining steps are the same.

[0023] Example 4 The preparation method according to Example 1 differs in that: S1: Adjust the pH of the culture medium to 7.2, the fermentation temperature to 31℃, the fermentation time to 36h, the pressure of the high-pressure homogenization treatment to 500MPa, and the number of cycles to 3; S2: The pH of the system was adjusted to 6.8, the reaction temperature was 48℃, the amount of β-agarase added was 66.7 mg, and the viscosity of the system at the end of the reaction was 40% of the initial viscosity. S3: Adjust the pH of the system to 5.2, the reaction temperature to 42℃, the amount of sugar sulfatase added to be 6.67mg, and the reaction time to be 6h; S4: The amount of sodium bicarbonate added is 0.4% of the mass of the concentrate, the inlet air temperature for spray drying is 190℃, and the remaining steps are the same.

[0024] Comparative Example 1 The preparation method according to Example 1 differs in that: S1: The Streptomyces fermentation and high-pressure homogenization processes were omitted. 6.67g of red algae powder was directly added to 100mL of deionized water and treated at 121℃ for 15min as the starting material. All other steps were the same.

[0025] This comparative preparation lacks the red algae polysaccharide extracted after bio-physical synergistic cell wall disruption pretreatment.

[0026] Comparative Example 2 The preparation method according to Example 1 differs in that: S2 and S3: Adjust the pH of the suspension obtained in S1 to 6.0, add β-agarase and sulfatase simultaneously at 42℃, react for 6 hours, and then inactivate at 90℃. The remaining steps are the same.

[0027] This comparative preparation used red algae polysaccharides obtained by simultaneous enzymatic hydrolysis rather than sequential enzymatic hydrolysis.

[0028] Comparative Example 3 The preparation method according to Example 1 differs in that: S1: The pretreatment of Streptomyces fermentation and high pressure homogenization was cancelled. The red algae powder was added to 100 mL of 1 mol / L NaOH solution and stirred in an 80℃ water bath for 2 h. Then the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid and boiled for 10 min. The remaining steps were the same.

[0029] This comparative preparation used red algae polysaccharides obtained through an uncontrolled, single, and drastic chemical pretreatment.

[0030] Example 1 The red algae polysaccharide powders prepared in Examples 1-4 and Comparative Examples 1-3 were dried to constant weight in a vacuum drying oven at 60°C and -0.1 MPa. Then, they were transferred to a desiccator to cool to room temperature. 0.5 g of the dried powder sample was weighed and added to 100 mL of deionized water. The time required from addition to complete dissolution was measured at 60°C and 500 rpm. The state of the solution at 1 min was recorded. The results are shown in Table 2.

[0031] Table 2 Solubility test results for the examples and comparative samples As can be seen from Table 2, Examples 1 to 4 all formed a homogeneous solution in about 1 minute, indicating that the products all have good solubility. Comparative Example 1 lacked a bio-physical synergistic cell wall disruption process during preparation, resulting in incomplete disintegration of the red algae cell wall. The released polysaccharides tightly bound to cell wall residues and impurities such as proteins, forming a large number of insoluble complexes, leading to incomplete dissolution. Comparative Example 2 did not place β-agarase and sulfatase under their respective optimal conditions during enzymatic hydrolysis. Instead, the two enzymes were mixed and subjected to compromised hydrolysis conditions. This resulted in both β-agarase and sulfatase operating under suboptimal conditions, leading to low efficiency in the hydrolysis reaction. Consequently, the agar network within the red algae cells and the sulfate groups on the polysaccharide molecular chains were insufficiently degraded, resulting in excessively high system viscosity and the formation of an irreversible gel network, hindering further dissolution. Comparative Example 3 used strong alkali pretreatment, which severely disrupted the cell wall and degraded polysaccharide molecules into small fragments. However, strong alkali pretreatment also caused Maillard reactions and carbonization of the polysaccharide structure, resulting in the formation of dark-colored and insoluble flocculent impurities. Therefore, although the solution was transparent, its color was abnormal, and the product was inactivated and denatured.

[0032] Experiment Example 2 The red algae polysaccharide powders prepared in Examples 1-4 and Comparative Examples 1-3 were dried to constant weight in a vacuum drying oven at 60°C and -0.1 MPa. They were then transferred to a desiccator and cooled to room temperature. Subsequently, they were placed into 50 mL centrifuge tubes, and 20 mL of anhydrous ethanol was added to each tube. The tubes were vortexed for 10 min and then centrifuged at 4000 rpm for 10 min. The supernatant was discarded and the process was repeated once. The precipitate was dried in ethanol at 60°C. The solid was then extracted in 50 mL of 90°C deionized water for 1.5 h and centrifuged at 4000 rpm while hot for 15 min. The supernatant was collected, and the residue was extracted again with 20 mL of hot water. The two supernatants were then combined. Add Sevage reagent to the supernatant at a solid-liquid ratio of 4:1, shake at 500 rpm for 30 min, then aspirate the upper aqueous phase. Repeat this operation until no white protein precipitate is found at the interface. The collected aqueous phase is placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with flowing deionized water at 4 °C for 48 h. The solution in the dialysis bag is then transferred to a beaker and concentrated to a volume of 10–15 mL in a 60 °C water bath. Four times the volume of anhydrous ethanol is added, and the mixture is allowed to stand at 4 °C for 12 h. The mixture is then centrifuged at 8000 rpm for 20 min and the precipitate is collected. The precipitate is washed twice each with anhydrous ethanol and acetone, then transferred to a 60 °C, -0.1 MPa vacuum drying oven and dried to constant weight. The solid is then cooled to room temperature in a desiccator. The mass m of the final dried solid is weighed, and the polysaccharide yield Y is calculated using the formula: The results are shown in Table 3.

[0033] Table 3. Polysaccharide yield in the examples and comparative samples As shown in Table 3, the samples in the examples all had high polysaccharide yields, indicating that the bio-physical synergistic cell wall disruption pretreatment can maximize polysaccharide release, while the subsequent sequential enzymatic hydrolysis process can maximize polysaccharide preservation. Comparative Example 1 sample did not undergo the mild biodegradation of the cell wall network by cellulase and hemicellulase produced by Streptomyces fermentation, nor the instantaneous physical disintegration provided by subsequent high-pressure homogenization. The structural integrity of the red algae cell wall was not effectively destroyed, resulting in most of the target polysaccharides in the intracellular and cell wall matrix failing to be released into the solution phase. Comparative Example 2 sample mixed two enzymes with different optimal working environments for simultaneous enzymatic hydrolysis, forcing them to operate at a compromise pH and temperature, leading to a significant decrease in catalytic efficiency and failure to preferentially and fully degrade the polysaccharide network. Therefore, the system viscosity was high, severely hindering the contact and reaction between sulfatase and the substrate. Simultaneously, the chaotic enzymatic hydrolysis environment also led to the deterioration of the sulfatase... Esterases cause non-specific cleavage, which not only affects the yield but also destroys the active structure. The comparative sample used strong alkaline chemical pretreatment instead of controllable biological pretreatment. Although the alkaline properties severely damage the cell wall, the process is uncontrollable and highly destructive. Due to the strong alkaline conditions, the β-elimination reaction of the polysaccharide chain is triggered, which leads to the breakage of glycosidic bonds and a sharp decrease in molecular weight. At the same time, the sulfate ester bonds are easily hydrolyzed and detached in the alkaline environment, resulting in a large amount of irreversible loss of active groups. It can also lead to side reactions such as Maillard reaction of polysaccharides, generating pigments and by-products. Therefore, although the cell wall is opened, the target polysaccharide structure has been severely damaged and degraded, and the content of intact active polysaccharides that can be recovered through purification is very low.

[0034] Experimental Example 3 Take 5 mL of the suspension obtained in step S1 of Example 1, centrifuge at 10000 rpm for 10 min, take 1 mL of the supernatant, add 1 mL of 4 mol / L trifluoroacetic acid, hydrolyze at 110 °C for 2 h, remove the trifluoroacetic acid, and redissolve to obtain the treated solution. Take 200 μL of the treated solution, add 200 μL of 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution and 200 μL of 0.3 mol / L NaOH solution, mix well, react in a 70 °C water bath for 60 min, then cool to room temperature, add 200 μL of 0.3 mol / L HCl to neutralize, then extract three times with chloroform shaking, discard the lower organic phase, filter the upper aqueous phase through a 0.45 μm microporous membrane and analyze its composition by HPLC. The HPLC column used was a C18 reversed-phase column, the detection wavelength was 250 nm, and the mobile phase was 0.1 mol / L pH 6.8 phosphate buffer and acetonitrile, with a volume ratio of 82:18. The analytical results are as follows: Figure 2 As shown.

[0035] Take the red algae raw material powder from Example 1, the product after pretreatment and β-agar hydrolysis, and the final product powder, respectively. Prepare the pretreated and β-agar hydrolyzed product into a powder by freeze-drying as an intermediate. Mix the three powders with spectral grade KBr at a mass ratio of 1:100, grind to below 2 μm, and press into transparent sheets. [The last part, "4000~4000 cm," appears to be a separate, unrelated sentence fragment and is left untranslated.] -1 Infrared spectral scanning was performed within the range, and the scanning results are as follows: Figure 3 As shown.

[0036] from Figure 2 As can be seen, the impurity peaks appearing at 5-10 min are caused by residual PMP reagent. Strong peaks representing xylose and glucose appear at 13 min and 16 min, respectively, with the glucose peak being the strongest. This is partly due to the added activity inhibitor and partly due to the degradation products of microcrystalline cellulose and cellulose in the red algal cell wall skeleton by cellulase secreted by Streptomyces. The xylose peak is a product released by the specific degradation of hemicellulose components in the red algal cell wall by hemicellulase secreted by Streptomyces. Weaker peaks representing arabinose and mannose appear around 12 min and 14.5 min, respectively, originating from the degradation products of hemicellulose side chains or pectin substances in the red algal cell wall. An extremely weak galactose peak appears after 20 min. It can be seen that after Streptomyces pretreatment and high-pressure cell wall disruption, the dense cell wall of red algae was successfully dismantled, and the agarase and carrageenanase secretion signaling pathways of Streptomyces were successfully inhibited in the presence of the activity inhibitor, thus the red algal polysaccharide structure in the cells was basically preserved intact.

[0037] from Figure 3 As can be seen, the basic structures of raw materials, intermediates, and final products are similar, ranging from 1240 to 1260 cm. -1 The S=O stretching vibration peak appears at this point, representing the sulfate ester group. The raw material has a strong absorption peak at this point, indicating that it contains a high density of sulfate ester groups. The peak intensity of the intermediate at this point is basically the same as that of the raw material. This is because the intermediate only involves the treatment of glycosidic bonds by β-agarase and does not involve desulfurization. However, the relative intensity of the peak in the final product at this point is significantly lower, indicating that the added sulfatase successfully achieved a specific desulfurization effect, thereby improving the water solubility of the molecule. However, due to the control of enzyme dosage and reaction time, the final product did not completely remove the sulfate ester bonds, thus ensuring a certain level of biological activity in the product. At 930 cm⁻¹ -1 The presence of a COC vibration peak nearby, exhibiting clear and strong peaks in the raw materials, intermediates, and final product, indicates that β-agarase and sulfatase only modified the side chains or cleaved the long chains, without destroying the core helical structural unit that determines the activity of the red algae polysaccharide gel, thus preserving the integrity of the polysaccharide backbone; at 850 cm⁻¹ -1 and 745cm-1 The peaks at the point are fingerprint peaks of some sulfate-specific sites. Compared with the peak intensities of the raw materials and intermediates, the intensity of these fingerprint peaks in the finished product is weakened to varying degrees, indicating that the sulfatase used only removes sulfur at specific sites that hinder dissolution or affect activity.

Claims

1. A highly efficient and rapidly soluble red algae polysaccharide preparation process based on a compound enzymatic method, comprising cell wall disruption pretreatment, compound enzymatic hydrolysis, ultrafiltration purification, and spray drying posttreatment, characterized in that: The cell wall disruption pretreatment includes a Streptomyces fermentation pretreatment stage and a high-pressure cell wall disruption stage; the compound enzymatic hydrolysis involves sequentially using selective polysaccharide degrading enzymes and sugar sulfatases to enzymatically hydrolyze the pretreated raw materials; the Streptomyces fermentation pretreatment stage includes using a signaling pathway initiator to inhibit the activity of agarase secreted by Streptomyces, inducing the secretion of cellulase and hemicellulase signaling pathways, and then prompting Streptomyces to specifically disrupt the red algal cell wall skeleton along the signaling pathway.

2. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 1, characterized in that: The signaling pathway initiator includes an activity inhibitor and a signaling pathway inducer; the activity inhibitor is glucose; the signaling pathway inducer is one or more of microcrystalline cellulose and crude red algae powder.

3. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to any one of claims 1 to 2, characterized in that, It is prepared through the following steps: S1: After crushing the red algae raw material, mix it with a fermentation medium containing Streptomyces and signaling pathway initiators for fermentation. After fermentation, heat and sterilize the system, then cool the system and perform high-pressure homogenization. S2: Adjust the pH and temperature of the mixture after high-pressure crushing, add selective polysaccharide degrading enzyme to induce enzymatic hydrolysis, and continuously monitor the viscosity of the system. When the viscosity of the system drops to less than half of the initial viscosity, raise the temperature to inactivate the enzyme. S3: Adjust the pH and temperature of the inactivated system, add sulfatase to carry out the enzymatic hydrolysis reaction, and raise the temperature after the reaction to inactivate the enzyme. S4: The mixture after enzymatic hydrolysis and inactivation is concentrated by ultrafiltration membrane. Then, functional additives are added to the concentrate and spray-dried to obtain red algae polysaccharide powder.

4. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 3, characterized in that: The Streptomyces mentioned in S1 is one or more of Streptomyces white, Streptomyces gray, and Streptomyces purplish-red; the red algae raw material is derived from one of Carrageenan, Euphorbia lactea, and Gracilaria.

5. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 3, characterized in that: The amount of active inhibitor added to the signaling pathway initiator in S1 is 1%~2% w / v of the culture medium; the amount of signaling pathway inducer added to the signaling pathway initiator is 0.5%~1.5% w / v of the culture medium; the pH of the culture medium is 6.5~7.5; the fermentation temperature is 28~32℃, and the time is 12~48h; the pressure of the high-pressure homogenization treatment is 200~400MPa, and the number of treatments is 1~4 times.

6. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 3, characterized in that: The selective polysaccharide degrading enzyme mentioned in S2 is one of β-agarase, α-agarase, and carrageenase.

7. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 3, characterized in that: The pH of the mixture in S2 is 6.0~7.0, and the temperature is 40~50℃; the amount of selective polysaccharide degrading enzyme added is 0.1%~1.5% of the mass of red algae powder; the viscosity of the system decreases to 30%~50% of the initial viscosity.

8. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 3, characterized in that: The pH of S3 is 5.0~6.0; the temperature is 35~45℃; the amount of sulfatase added is 0.05%~0.5% of the mass of red algae powder; and the enzymatic hydrolysis reaction time is 2~8h.

9. The efficient and fast-dissolving red algae polysaccharide preparation process based on a compound enzyme method according to claim 3, characterized in that: The functional additives in S4 are sodium bicarbonate, maltodextrin, and β-cyclodextrin; the amount of sodium bicarbonate added is 0.2% to 0.8% of the mass of the concentrate; and the inlet air temperature of the spray dryer is 160 to 200°C.

10. A highly efficient, fast-dissolving red algae polysaccharide based on a compound enzymatic method as described in any one of claims 1 to 9, characterized in that: The yield of the red algae polysaccharide is ≥25%, and the time required for its 0.5% aqueous solution to completely dissolve at 60℃ is <120s.

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