Step-by-step recovery method for active ingredients of kelp

Abstract

The invention relates to the technical field of kelp processing, in particular to a step-by-step recycling method for active ingredients of kelp. According to the method, efficient recovery of phycoxanthine, mannitol, fucoidan and cellulose contained in kelp is achieved through the following steps that the phycoxanthine is extracted from kelp fine powder through supercritical carbon dioxide, and the phycoxanthine is recovered; treating kelp residues after recovery of phycoxanthine by using high-pressure microfluidics, adjusting the pH value, and carrying out freeze drying to recover mannitol; then, carrying out enzymolysis, ultrafiltration separation, organic solvent extraction and alcohol precipitation operation on the kelp residues after mannitol recovery, so as to recover fucoidan; and finally, recovering cellulose from the kelp residues through chitosan flocculation, lactic acid bacteria fermentation decalcification, alkali treatment and enzymolysis. By adopting a step-by-step recovery mode, mutual interference among active components is effectively avoided, kelp is efficiently utilized, and the recovery rate and the purity are improved.

Description

Technical Field

[0001] The invention relates to the technical field of marine natural product extraction, in particular to a step-by-step recovery method for active components of kelp. Background Art

[0002] As an important marine resource, kelp is rich in a variety of bioactive ingredients, such as phycoxanthin, mannitol, fucoidan and cellulose. These ingredients have a wide range of biological activities and application values, such as antioxidant, anti-inflammatory, antibacterial, and use as food additives, pharmaceutical ingredients, etc. However, the effective extraction of these active ingredients from kelp is a technical challenge, which requires an efficient and environmentally friendly method.

[0003] Traditional extraction methods are often inefficient and can damage the structural integrity of active ingredients, affecting their biological activity. In addition, due to the complex composition of kelp, a single extraction technology is difficult to achieve the desired extraction effect. Therefore, it is particularly important to develop a method that can gradually recover various active ingredients in kelp.

[0004] At present, although there are some extraction technologies for specific active ingredients, most methods only focus on the extraction of one or several ingredients, lacking overall consideration and comprehensive utilization. In addition, existing technologies often involve complex process flows and high costs, which limits their large-scale application.

[0005] In summary, the problems existing in the prior art mainly include: low extraction efficiency, resulting in low recovery rate of active ingredients; the structure of the active ingredients is easily destroyed during the extraction process, affecting their biological activity; a single extraction method is difficult to fully recover the various active ingredients in kelp; the process flow is complicated and the cost is high, which is not conducive to large-scale industrial production. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a step-by-step recovery method for active ingredients of kelp, aiming to solve the above-mentioned technical problems, improve the recovery rate and purity of active ingredients, simplify the process flow and reduce production costs.

[0008] (II) Technical solution

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] A method for stepwise recovery of active ingredients of kelp is implemented according to the following steps to achieve stepwise recovery of active ingredients of kelp;

[0011] S1. Grinding the dried kelp into fine powder, wherein the particle size of the fine powder is less than 200 μm;

[0012] S2, extracting the kelp powder in step S1 in supercritical carbon dioxide, and re-dissolving the extract and eluting it by column chromatography to recover phycoxanthin;

[0013] S3, using high pressure microfluidics technology to treat the kelp residue produced in step S2 to release mannitol, then filtering the extract, adjusting the pH value, and finally freeze-drying to recover the mannitol crystals;

[0014] S4, recovering fucoidan from the kelp residue treated by high-pressure microfluidic technology in step S3 by enzymatic hydrolysis, ultrafiltration separation, organic solvent extraction and alcohol precipitation;

[0015] S5. Separate the kelp residue after ultrafiltration separation treatment in step S4, and recover cellulose through chitosan flocculation, lactic acid bacteria fermentation decalcification, alkali treatment and enzymatic hydrolysis.

[0016] In step S1, by crushing the dried kelp into fine powder with a particle size less than 200 μm, the surface area of ​​the raw material is increased, thereby improving the contact efficiency and extraction rate of the subsequent extraction step.

[0017] In step S2, supercritical carbon dioxide extraction technology is used to efficiently extract phycoxanthin under mild conditions, avoiding the problems of thermal damage and solvent residue caused by traditional solvent extraction. The purity of phycoxanthin is further improved by column chromatography elution.

[0018] In step S3, high pressure microfluidics technology is used to treat the kelp residue produced in step S2, which can effectively destroy the cell wall structure and release mannitol. Through the steps of filtration, pH adjustment and freeze drying, high-purity mannitol crystals can be efficiently recovered.

[0019] In step S4, the fucoidan can be effectively separated and purified through enzymatic hydrolysis, ultrafiltration separation, organic solvent extraction and alcohol precipitation, thereby improving its purity and recovery rate.

[0020] In step S5, high-purity cellulose can be efficiently recovered from kelp residues through chitosan flocculation, lactic acid bacteria fermentation decalcification, alkali treatment and enzymatic hydrolysis, while avoiding excessive use of chemical reagents and reducing environmental pollution.

[0021] Furthermore, in step S2, the conditions for extracting phycoxanthin in supercritical carbon dioxide are: under the conditions of 30-40°C and 180-220 standard atmospheres, using supercritical carbon dioxide as a solvent, extracting for 1-1.5 hours, and the flow rate of supercritical carbon dioxide is 50g / min-80g / min.

[0022] Under the conditions of 30-40°C temperature and 180-220 standard atmospheres, supercritical carbon dioxide can effectively dissolve phycoxanthin and increase its solubility, thereby improving the extraction efficiency of phycoxanthin. Under this condition, the extraction can be completed in a short time, further improving the extraction efficiency.

[0023] Extraction under relatively mild temperature conditions can minimize the thermal damage of phycoxanthin during the extraction process, thereby maintaining the biological activity of phycoxanthin.

[0024] Supercritical carbon dioxide, as an environmentally friendly solvent, replaces traditional organic solvents, reduces the use of harmful solvents, and reduces the impact on the environment.

[0025] Supercritical carbon dioxide is easy to recover under reduced pressure conditions. CO2 can be recovered and reused through simple reduced pressure operations, reducing production costs.

[0026] Supercritical carbon dioxide extraction can effectively separate phycoxanthin from other impurities, and the purity of phycoxanthin can be further improved through subsequent column chromatography elution.

[0027] In summary, the present invention uses supercritical carbon dioxide to extract phycoxanthin under specific conditions, which not only improves the extraction efficiency and purity of phycoxanthin, but also maintains the biological activity of phycoxanthin, while reducing production costs and environmental pollution.

[0028] Furthermore, after extracting phycoxanthin, CO2 is recovered under reduced pressure at room temperature to concentrate the extract and perform extraction. The conditions for reducing pressure to recover CO2 are: temperature range is 20-25°C, pressure is reduced from 180-220 standard atmospheres to 50-60 standard atmospheres, maintained for 30s to 60s, and then further reduced to 10-20 standard atmospheres, maintained for 3-5 minutes, and finally reduced to standard atmospheric pressure.

[0029] In the process of supercritical carbon dioxide extraction of phycoxanthin, phycoxanthin is dissolved in supercritical carbon dioxide. When the pressure is gradually reduced, phycoxanthin separates from carbon dioxide to form a liquid containing phycoxanthin. This liquid is the so-called "extract".

[0030] Under supercritical conditions, phycoxanthin dissolves in carbon dioxide to form a homogeneous phase. As the pressure gradually decreases, phycoxanthin separates from carbon dioxide to form a semisolid containing phycoxanthin, which is further reconstituted in ethyl acetate to form a liquid.

[0031] After the phycoxanthin in the kelp residue is dissolved by supercritical carbon dioxide, the remaining kelp residue still contains other valuable components, such as mannitol, for subsequent operations.

[0032] The temperature range is 20-25°C. Within this temperature range, phycoxanthin is not easily damaged by heat, which helps to maintain the biological activity of phycoxanthin. If the temperature is too high (over 25°C), the biological activity of phycoxanthin will be damaged; if the temperature is too low (below 20°C), the solubility of phycoxanthin will be reduced, affecting the extraction efficiency.

[0033] The pressure is reduced from 180-220 standard atmospheres to 50-60 standard atmospheres. Within this pressure range, CO2 can be effectively recovered, and the separation of phycoxanthin from CO2 is more efficient. If the initial pressure is too low (lower than 180 standard atmospheres), phycoxanthin cannot be completely dissolved; if the final pressure is too high (higher than 60 standard atmospheres), phycoxanthin cannot be completely separated from CO2.

[0034] Keep it for 30s to 60s. This period of time is long enough for the phycoxanthin to separate from CO2, but not too long to cause the loss of phycoxanthin. If the time is too short (less than 30s), the phycoxanthin cannot be completely separated; if the time is too long (more than 60s), it will lead to unnecessary energy consumption.

[0035] Further reduction to 10-20 standard atmospheres, holding time 3-5 minutes: Further reduction of pressure helps to fully recover CO2 and ensure complete separation of phycoxanthin. If the final pressure is too low (less than 10 standard atmospheres), it will lead to unnecessary energy consumption; if the holding time is too short (less than 3 minutes), phycoxanthin cannot be completely separated; if the holding time is too long (more than 5 minutes), it will increase energy consumption.

[0036] Furthermore, in step S2, the conditions for column chromatography elution to recover fucoxanthin are as follows: the extracted substance is re-dissolved with 2-4 times the mass of ethyl acetate and added to the top of the column chromatography column; the column chromatography uses a silica gel column, the elution solvent is a mixed solvent of hexane and ethyl acetate, the elution flow rate of the mixed solvent is 1-5 mL / min / cm, and the elution conditions are: initial conditions: the volume ratio of hexane: ethyl acetate is 95:5, and this high proportion of hexane is used for the preliminary elution of non-polar or low-polar impurities to ensure that fucoxanthin will not be eluted prematurely.

[0037] Using 2-4 times the mass of ethyl acetate to re-dissolve the super-carbon dioxide extract and pouring it into the chromatography column can effectively improve the purity of phycoxanthin. The amount of ethyl acetate used is moderate, which can not only improve the purity but also will not increase the cost too much.

[0038] Medium-term conditions: The volume ratio of hexane:ethyl acetate is 80:20. When the non-polar impurities are basically eluted, the proportion of ethyl acetate can be gradually increased to start eluting fucoxanthin.

[0039] Late elution conditions. Late conditions: Hexane: ethyl acetate volume ratio of 70:30. When fucoxanthin begins to elute, continue to increase the proportion of ethyl acetate to ensure that fucoxanthin is fully eluted while reducing the co-elution of highly polar impurities. Final conditions: Hexane: ethyl acetate volume ratio of 50:50. If fucoxanthin is still not completely eluted, try a higher proportion of ethyl acetate.

[0040] TLC monitoring: Use thin layer chromatography (TLC) or other detection methods to monitor the elution process, and determine whether the target compound has been eluted based on the mobility (Rf value) of each component on the TLC plate.

[0041] By using a mixed solvent of hexane and ethyl acetate as an eluent and gradually changing the solvent composition in a specific ratio, phycoxanthin can be effectively separated from other impurities and the purity of phycoxanthin can be improved. The flow rate of the elution solvent is 1-5mL / min / cm, which can not only ensure the efficiency of elution, but also avoid the rapid loss of phycoxanthin and other impurities, thereby improving the elution efficiency.

[0042] Using thin layer chromatography (TLC) or other detection methods to monitor the elution process, it can be determined whether the target compound has been eluted based on the mobility (Rf value) of each component on the TLC plate. This monitoring helps to adjust the elution conditions in time to ensure efficient purification of phycoxanthin.

[0043] Further, in step S3, the conditions for recovering mannitol by high-pressure microfluidic technology are as follows: the kelp residue in step S2 is treated by high-pressure water jet, high-pressure water 100-120MPa, high-pressure water jet, to promote the efficient release of mannitol. The treatment time is 10-30 seconds, the treatment temperature is 50-60°C, and the flow rate is 0.3-0.8mL / min. The filtered solution passes through a 0.22μm microfiltration membrane to intercept larger particles and cell debris to separate the mannitol extract.

[0044] The high-pressure water jet pressure is 100-120MPa. Within this pressure range, it can effectively destroy the cell wall structure of kelp residues and promote the efficient release of mannitol. If the pressure is too low (below 100MPa), the cell wall structure cannot be effectively destroyed, resulting in insufficient release of mannitol; if the pressure is too high (above 120MPa), it will lead to unnecessary energy consumption and cause additional wear on the equipment.

[0045] The processing time is 10-30 seconds. Within this time range, the effective release of mannitol can be ensured while avoiding the increase in energy consumption caused by too long a processing time. If the processing time is too short (less than 10 seconds), the cell wall structure cannot be fully destroyed, resulting in insufficient mannitol release; if the processing time is too long (more than 30 seconds), energy consumption will increase and other components will be lost.

[0046] The processing temperature is 50-60°C. Within this temperature range, the solubility of mannitol can be improved, which is conducive to the effective release of mannitol while maintaining the biological activity of mannitol. If the temperature is too low (below 50°C), the solubility of mannitol will be reduced and the release efficiency will be affected; if the temperature is too high (above 60°C), the biological activity of mannitol will be damaged.

[0047] The high-pressure water jet flow rate is 0.3-0.8mL / min. Within this flow rate range, the uniform distribution of the high-pressure water jet can be ensured, the release efficiency of mannitol can be improved, and energy consumption can be reduced. If the flow rate is too low (less than 0.3mL / min), the high-pressure water jet will be unevenly distributed, affecting the release efficiency of mannitol; if the flow rate is too high (more than 0.8mL / min), energy consumption will increase.

[0048] The use of a 0.22 μm microfiltration membrane can effectively retain larger particles and cell debris, thereby improving the purity of the mannitol extract.

[0049] If a filter membrane with a pore size that is too large is used, impurities such as cell debris will be mixed into the mannitol extract; if a filter membrane with a pore size that is too small is used, the filtration resistance will increase, affecting the filtration efficiency.

[0050] Furthermore, the high-pressure water after washing the kelp residue is used as a mannitol extract, and the pH of the mannitol extract is adjusted to 6.5-7.0 to promote the crystallization of mannitol and avoid co-precipitation of other components such as protein.

[0051] The mannitol is collected by freeze drying, and the mannitol extract is cooled to below 10°C to promote the crystallization of mannitol to recover the mannitol.

[0052] The pH is adjusted to 6.5-7.0. Within this pH range, the crystallization of mannitol can be promoted, while co-precipitation of other components such as proteins can be avoided, thereby improving the purity of mannitol. If the pH value is too low (lower than 6.5), co-precipitation of other components such as proteins will occur, affecting the purity of mannitol; if the pH value is too high (higher than 7.0), the crystallization efficiency of mannitol will be affected.

[0053] Cooling to below 10°C: Low temperature can promote the crystallization of mannitol, improve the crystallization efficiency, and reduce the co-crystallization of other components, further improving the purity of mannitol. If the temperature is too low (much below 10°C), it will cause the co-crystallization of other components and affect the purity of mannitol; if the temperature is too high (close to room temperature), it will not effectively promote the crystallization of mannitol.

[0054] Mannitol is collected by freeze drying. Freeze drying can effectively remove moisture, improve the purity and stability of mannitol, and retain the biological activity of mannitol.

[0055] Further, in step S4, kelp residue treated with high-pressure water jet is used as an extraction raw material, and is added to a Tris-HCl buffer with a pH of 7.5-7.8, and maintained at 65-75°C for preliminary extraction. After the preliminary extraction, the extract is cooled to 45-55°C, and the extract is cooled to the optimal activity temperature of the enzyme.

[0056] Adjust the pH value to 7.5-7.8. Within this pH range, the solubility of fucoidan can be improved, while the activity of the enzyme can be optimized and the extraction efficiency of fucoidan can be improved. If the pH value is too low (lower than 7.5), the activity of the enzyme will be reduced, affecting the extraction efficiency of fucoidan; if the pH value is too high (higher than 7.8), the stability of fucoidan will decrease.

[0057] The initial extraction temperature is 65-75℃. Within this temperature range, the solubility of fucoidan can be improved, while the activity of the enzyme can be maintained, thereby improving the extraction efficiency of fucoidan. If the temperature is too low (below 65℃), the solubility of fucoidan and the activity of the enzyme will be reduced; if the temperature is too high (above 75℃), the stability of fucoidan and enzyme will decrease, affecting the extraction efficiency.

[0058] The extract is cooled to 45-55℃. Cooling the extract to this temperature range can ensure the optimal activity temperature of the enzyme, improve the efficiency of enzymatic hydrolysis, and avoid the damage of high temperature to the structure of fucoidan. If the temperature is too low (below 45℃), the activity of the enzyme will be reduced and the efficiency of enzymatic hydrolysis will be affected; if the temperature is too high (above 55℃), the enzyme will be inactivated and the efficiency of enzymatic hydrolysis will be reduced.

[0059] Maintain the extract at 45-55℃, add a complex enzyme preparation of cellulase and xylanase, with cellulase accounting for 80%-90%, of which the enzyme activity is at least 1000U / g substrate, and the reaction time is 4-4.5 hours; prevent the residual enzyme activity from continuing to act in subsequent treatments and affecting the structural integrity and purity of fucoidan. Heat the enzymatically hydrolyzed mixture to 80-100℃ for 10-15 minutes to ensure complete inactivation of the enzyme. After heating, quickly cool to room temperature or the temperature required for the next step to avoid degradation of fucoidan caused by long-term high temperature.

[0060] By using a cellulase and xylanase complex enzyme preparation with an enzyme activity of at least 1000U / g substrate and performing enzymatic hydrolysis at 45-55℃, the enzymatic hydrolysis efficiency can be improved, the reaction time can be shortened, and the extraction efficiency of fucoidan can be improved. Heating the enzymatically hydrolyzed mixture to 80-100℃ for 10-15 minutes can ensure the complete inactivation of the enzyme and prevent the residual enzyme activity from continuing to act in subsequent treatments, affecting the structural integrity and purity of fucoidan. After heating, rapid cooling to room temperature or the temperature required for the next step of treatment can avoid degradation of fucoidan caused by prolonged high temperature.

[0061] Maintain the temperature of the extract at 45-55°C. Within this temperature range, the optimal activity temperature of the enzyme can be ensured, the enzymatic efficiency can be improved, and the damage to the structure of fucoidan caused by high temperature can be avoided. If the temperature is too low (below 45°C), the activity of the enzyme will be reduced, affecting the enzymatic efficiency; if the temperature is too high (above 55°C), the enzyme will be inactivated, reducing the enzymatic efficiency.

[0062] The enzyme activity is at least 1000U / g substrate. Using enzyme preparations with high enzyme activity can improve enzymatic efficiency, shorten reaction time, and improve the extraction efficiency of fucoidan. If the enzyme activity is too low, the reaction time will be prolonged and the extraction efficiency of fucoidan will be reduced.

[0063] The reaction time is 4-4.5 hours. Within this time range, effective enzymatic hydrolysis of fucoidan can be ensured while avoiding degradation of fucoidan caused by too long reaction time. If the reaction time is too short (less than 4 hours), the enzymatic hydrolysis cannot be fully achieved; if the reaction time is too long (more than 4.5 hours), degradation of fucoidan will occur.

[0064] Heating to 80-100℃ for 10-15 minutes can ensure the complete inactivation of the enzyme within this temperature range, preventing the residual enzyme activity from continuing to act in subsequent processing and affecting the structural integrity and purity of fucoidan. If the temperature is too low (below 80℃), the enzyme cannot be effectively inactivated; if the temperature is too high (above 100℃), it will lead to the degradation of fucoidan.

[0065] Cool quickly after heating. Rapid cooling can avoid the degradation of fucoidan caused by long-term high temperature and reduce energy consumption. If the cooling speed is too slow, it will cause the degradation of fucoidan.

[0066] Continuous separation is performed through ultrafiltration membranes to intercept fucoidan and remove undecomposed residues and small molecular impurities. The transmembrane pressure (TMP) during ultrafiltration is usually between 0.5-2 bar, and the operating temperature should be controlled between 20-30°C to maintain the stability of fucoidan and the performance of the membrane. The flow rate is controlled by adjusting the speed of the circulation pump, and the flow rate is between 0.5-2m / s to maintain the turbulent state of the membrane surface and reduce membrane fouling. Ultrafiltration membranes include polyethersulfone (PES), polysulfone (PS), and polyamide (PA).

[0067] Within this pressure range, fucoidan can be effectively pushed through the ultrafiltration membrane while avoiding damage to the membrane or degradation of fucoidan due to excessive pressure. If the pressure is too low (below 0.5 bar), the separation efficiency will decrease; if the pressure is too high (above 2 bar), energy consumption will increase and the membrane will be damaged.

[0068] The operating temperature is 20-30℃. Within this temperature range, the stability of fucoidan and the performance of the membrane can be maintained, and the separation efficiency can be improved. If the temperature is too low (below 20℃), the fluidity of fucoidan will be reduced, affecting the separation efficiency; if the temperature is too high (above 30℃), it will cause the degradation of fucoidan.

[0069] The flow rate is 0.5-2m / s. Within this flow rate range, the turbulent state of the membrane surface can be maintained, membrane fouling can be reduced, and separation efficiency can be improved. If the flow rate is too low (lower than 0.5m / s), membrane fouling will be aggravated; if the flow rate is too high (higher than 2m / s), energy consumption will increase and the membrane will be damaged.

[0070] Further, the fucoidan concentrate is extracted with an organic solvent, wherein the organic solvent is a mixed solution of water and ethanol in a volume ratio of 9:1-8:2, the volume ratio of the organic solvent to the fucoidan concentrate is 3-8:1, the extraction temperature is 20-30°C, and the extraction time is 0.5-1.5 hours to ensure that the fucoidan is fully dissolved without over-extraction; the extracted solution containing fucoidan is cooled to 3-5°C to reduce side reactions during precipitation and improve purity. Slowly add ethanol to it until the volume of ethanol is 70-80% of the total volume, stir evenly and leave for 16-24 hours to allow the fucoidan to completely precipitate.

[0071] The volume ratio of water to ethanol is 9:1-8:2. Within this volume ratio range, fucoidan can be effectively dissolved, while reducing the co-dissolution of other impurities and improving the purity of fucoidan. If the ethanol ratio is too low (lower than 1:9), fucoidan cannot be effectively dissolved; if the ethanol ratio is too high (higher than 2:8), other impurities will be co-dissolved, affecting the purity of fucoidan.

[0072] The volume ratio of the organic solvent to the fucoidan concentrate is 3-8:1. Within this volume ratio range, the fucoidan can be effectively dissolved while avoiding the increase in energy consumption caused by excessive solvent.

[0073] The extraction temperature is 20-30℃. Within this temperature range, the stability of fucoidan can be maintained while the extraction efficiency can be improved. If the temperature is too low (below 20℃), the extraction efficiency will be reduced; if the temperature is too high (above 30℃), the fucoidan will be degraded.

[0074] The extraction time is 0.5-1.5 hours. Within this time range, the fucoidan can be fully dissolved while avoiding degradation caused by over-extraction.

[0075] Cooling to 3-5°C, within this temperature range, can reduce side reactions during precipitation and improve the purity of fucoidan.

[0076] The volume of ethanol is 70-80% of the total volume. Within this ethanol ratio range, the precipitation of fucoidan can be effectively promoted and the purity can be improved. If the ethanol ratio is too low (less than 70%), fucoidan cannot be effectively precipitated; if the ethanol ratio is too high (more than 80%), other impurities will be co-precipitated.

[0077] Leave it for 16-24 hours. Within this time frame, the fucoidan can be completely precipitated and the purity can be improved.

[0078] By using the organic solvent extraction conditions of a water to ethanol volume ratio of 9:1-8:2, an organic solvent to fucoidan concentrate volume ratio of 3-8:1, an extraction temperature of 20-30°C, and an extraction time of 0.5-1.5 hours, fucoidan can be effectively dissolved and the purity of fucoidan can be improved. Cooling to 3-5°C, ethanol volume of 70-80% of the total volume, and leaving for 16-24 hours can effectively promote the precipitation of fucoidan and improve the purity.

[0079] Separate the precipitate and the supernatant by centrifugation or filtration. During centrifugation, the speed is controlled at 3000-5000 rpm for 10-15 minutes. Wash with a low concentration buffer (such as Tris-HCl buffer at pH 7.5) and freeze-dry at a temperature of -50°C to -20°C.

[0080] The centrifugal speed is 3000-5000rpm. Within this speed range, the precipitate and supernatant can be effectively separated, while avoiding the loss of precipitate caused by high-speed centrifugation. If the centrifugal speed is too low (lower than 3000rpm), the precipitate cannot be effectively separated; if the centrifugal speed is too high (higher than 5000rpm), the precipitate will be lost.

[0081] The centrifugation time is 10-15 minutes. Within this time range, the effective separation of the precipitate can be ensured, while avoiding the loss of the precipitate caused by too long centrifugation. If the centrifugation time is too short (less than 10 minutes), the precipitate cannot be completely separated; if the centrifugation time is too long (more than 15 minutes), the precipitate will be lost.

[0082] Use a low concentration buffer (such as Tris-HCl buffer at pH 7.5). Using Tris-HCl buffer at pH 7.5 can effectively wash the precipitate and remove impurities while maintaining the stability of fucoidan. If the pH value of the buffer is too high or too low, the stability of fucoidan will decrease.

[0083] The freeze drying condition controls the temperature between -50℃ and -20℃. Within this temperature range, the precipitate can be effectively freeze dried while maintaining the biological activity of fucoidan. If the temperature is too low (below -50℃), the energy consumption of the equipment will increase; if the temperature is too high (above -20℃), the efficiency of freeze drying will be reduced.

[0084] The precipitate and supernatant can be effectively separated by centrifugation conditions of 3000-5000 rpm and 10-15 minutes. The precipitate can be effectively washed and impurities can be removed by using Tris-HCl buffer at pH 7.5. The freeze-drying temperature at -50°C to -20°C can effectively freeze-dry the precipitate while maintaining the biological activity of fucoidan.

[0085] The kelp residue separated by ultrafiltration membrane is dried at 50-60°C to constant weight. A chitosan aqueous solution with a concentration of 0.5-0.6% w / v is added, and the pH is adjusted to 5.5-5.7 with acetic acid to optimize the flocculation effect. After stirring at 3000-4000 rpm for 5-7 minutes, the mixture is allowed to stand for 30-40 minutes to collect the flocculants. The mass ratio of kelp residue to chitosan aqueous solution is 1:20-30.

[0086] The drying temperature is 50-60℃. Within this temperature range, the kelp residue can be effectively dried while avoiding the degradation of components such as fucoidan caused by excessively high temperatures. If the temperature is too low (below 50℃), the drying time will be too long; if the temperature is too high (above 60℃), the degradation of components such as fucoidan will occur.

[0087] The concentration of chitosan aqueous solution is 0.5-0.6% w / v. Within this concentration range, flocculation can be effectively promoted and the flocculation efficiency can be improved. If the concentration is too low (lower than 0.5% w / v), flocculation cannot be effectively achieved; if the concentration is too high (higher than 0.6% w / v), the floccules will be too dense and difficult to separate.

[0088] Acetic acid adjusts the pH to 5.5-5.7. At this pH value, the flocculation effect can be optimized and the flocculation efficiency can be improved.

[0089] The stirring condition is 3000-4000rpm, stirring for 5-7 minutes. Under this stirring condition, the chitosan aqueous solution and the kelp residue can be fully mixed to improve the flocculation efficiency. If the stirring speed is too low (lower than 3000rpm), it will cause uneven mixing; if the stirring speed is too high (higher than 4000rpm), it will cause the floccules to break.

[0090] The mass ratio of kelp residue and chitosan aqueous solution is 1:20-30. Within this mass ratio range, the flocculation effect can be ensured and the flocculation efficiency can be improved. If the mass ratio is too low (lower than 1:20), flocculation cannot be effectively achieved; if the mass ratio is too high (higher than 1:30), the floccules will be too dispersed and difficult to separate.

[0091] By using the drying and pulverizing conditions of drying temperature of 50-60°C and crushing through an 80-mesh sieve, the kelp residue can be effectively dried and the surface area can be increased. The flocculation conditions of chitosan aqueous solution concentration of 0.5-0.6% w / v, acetic acid to adjust the pH to 5.5, stirring conditions of 3000-4000 rpm stirring for 5-7 minutes, standing time of 30-40 minutes, and the mass ratio of kelp residue to chitosan aqueous solution of 1:20-30 can effectively promote flocculation and improve flocculation efficiency.

[0092] Further, the collected flocculants were transferred to a medium containing a concentration of 10 -7 -10 -8 CFU / mL lactic acid bacteria fermentation broth, fermentation at 37-40 ℃ for 18-24 hours, biological removal of calcium, reduce damage to cellulose structure. Use 0.5-0.6% w / v potassium carbonate aqueous solution, soak floccules at 45-48 ℃ for 2-2.5 hours to neutralize the acidic substances produced during the fermentation process and soften the fiber structure; add 0.5-0.6% v / v hydrogen peroxide aqueous solution and 0.5mM-1mM photosensitizer ethanol solution in equal volumes to the floccules solution after potassium carbonate aqueous solution treatment, and treat under ultraviolet irradiation for 1-1.2 hours, wavelength 254nm, intensity 10mW / cm 2 ; Add cellulase to the floc solution irradiated by ultraviolet light, at 45-55℃, the enzyme activity is at least 20U / g, hydrolyze for 16-24 hours; combine with ultrasonic assistance, power 100W, intermittent: open for 10 seconds, stop for 10 seconds, ultrasonic for 10 minutes to accelerate the enzymatic hydrolysis process. Finally, vacuum dry at -50--55℃ for 20-24 hours to obtain a dry cellulose product.

[0093] Lactobacillus concentration 10 -7 -10 -8 CFU / mL: Within this concentration range, biological calcium removal can be effectively performed to reduce damage to the cellulose structure. -7 CFU / mL), it is impossible to effectively remove calcium; if the lactic acid bacteria concentration is too high (higher than 10 -8 CFU / mL), which will lead to excessive acidity of the fermentation liquid and affect the stability of cellulose.

[0094] The fermentation temperature is 37-40℃. Within this temperature range, the activity of lactic acid bacteria can be maintained and the fermentation efficiency can be improved. If the temperature is too low (below 37℃), the fermentation efficiency will be reduced; if the temperature is too high (above 40℃), the lactic acid bacteria will be inactivated.

[0095] The fermentation time is 18-24 hours. Within this time range, calcium can be effectively removed while avoiding damage to the cellulose structure caused by too long fermentation. If the fermentation time is too short (less than 18 hours), calcium cannot be completely removed; if the fermentation time is too long (more than 24 hours), it will cause damage to the cellulose structure.

[0096] The concentration of potassium carbonate aqueous solution is 0.5-0.6% w / v. Within this concentration range, it can effectively neutralize the acidic substances produced during the fermentation process and soften the fiber structure. If the potassium carbonate concentration is too low (lower than 0.5% w / v), it cannot effectively neutralize the acidic substances; if the potassium carbonate concentration is too high (higher than 0.6% w / v), it will cause damage to the cellulose structure.

[0097] The soaking temperature is 45-48℃. Within this temperature range, the fiber structure can be effectively softened and the efficiency of subsequent treatment can be improved. If the temperature is too low (below 45℃), the softening efficiency will be reduced; if the temperature is too high (above 48℃), the cellulose structure will be damaged.

[0098] The soaking time is 2-2.5 hours. Within this time range, the fiber structure can be fully softened and the efficiency of subsequent treatment can be improved. If the soaking time is too short (less than 2 hours), the fiber structure cannot be fully softened; if the soaking time is too long (more than 2.5 hours), the cellulose structure will be damaged.

[0099] The concentration of hydrogen peroxide solution is 0.5-0.6% v / v. Within this concentration range, the fiber structure can be effectively oxidized, thereby improving the efficiency of subsequent treatment. If the concentration of hydrogen peroxide is too low (lower than 0.5% v / v), the fiber structure cannot be effectively oxidized; if the concentration of hydrogen peroxide is too high (higher than 0.6% v / v), the cellulose structure will be damaged.

[0100] The photosensitizer concentration is 0.5mM-1mM. Within this concentration range, the photocatalytic reaction can be effectively promoted and the efficiency of subsequent treatment can be improved. If the photosensitizer concentration is too low (lower than 0.5mM), the photocatalytic reaction cannot be effectively promoted; if the photosensitizer concentration is too high (higher than 1mM), it will cause damage to the cellulose structure.

[0101] The enzyme activity is at least 20U / g. Within this range, cellulose can be effectively hydrolyzed, which improves the efficiency of subsequent treatment. If the enzyme activity is too low (less than 20U / g), cellulose cannot be effectively hydrolyzed; if the enzyme activity is too high, the cost will increase.

[0102] The enzymatic hydrolysis temperature is 45-55℃. Within this temperature range, the activity of the enzyme can be maintained and the efficiency of enzymatic hydrolysis can be improved. If the temperature is too low (below 45℃), the activity of the enzyme will be reduced; if the temperature is too high (above 55℃), the enzyme will be inactivated.

[0103] The enzymatic hydrolysis time is 16-24 hours. Within this time range, the effective enzymatic hydrolysis of cellulose can be ensured, and the efficiency of subsequent treatment can be improved. If the enzymatic hydrolysis time is too short (less than 16 hours), the cellulose cannot be completely hydrolyzed; if the enzymatic hydrolysis time is too long (more than 24 hours), the cellulose structure will be damaged.

[0104] The ultrasonic auxiliary condition is a power of 100 W. Within this power range, the enzymatic hydrolysis process can be effectively accelerated and the efficiency of subsequent treatment can be improved. If the power is too low (less than 100 W), the enzymatic hydrolysis process cannot be effectively accelerated; if the power is too high, it will cause damage to the cellulose structure.

[0105] In this intermittent mode, overheating can be effectively avoided and the efficiency of ultrasonic treatment can be improved.

[0106] Vacuum drying at a temperature of -50°C to -55°C. Within this temperature range, cellulose products can be effectively dried while avoiding damage to the cellulose structure caused by high temperature. If the temperature is too low (below -55°C), the energy consumption of the equipment will increase; if the temperature is too high (above -50°C), the drying efficiency will be reduced.

[0107] By using lactic acid bacteria concentration 10 -7 -10 -8 CFU / mL, fermentation temperature 37-40°C, fermentation time 18-24 hours, potassium carbonate aqueous solution concentration 0.5-0.6% w / v, immersion temperature 45-48°C, immersion time 2-2.5 hours, hydrogen peroxide aqueous solution concentration 0.5-0.6% v / v, photosensitizer concentration 0.5mM-1mM, ultraviolet irradiation conditions (wavelength 254nm, intensity 10mW / cm 2 , irradiation time 1-1.2 hours), enzyme activity of at least 20U / g, enzymatic hydrolysis temperature 45-55℃, enzymatic hydrolysis time 16-24 hours, ultrasonic assisted conditions (power 100W, intermittent: on 10 seconds, off 10 seconds, ultrasonic time 10 minutes) and vacuum drying conditions (temperature -50℃ to -55℃, drying time 20-24 hours) can effectively recover cellulose and improve the efficiency of subsequent treatment.

[0108] (III) Beneficial effects

[0109] The beneficial effects of the present invention are as follows: the present invention adopts a step-by-step recovery method, and can efficiently extract multiple active ingredients such as phycoxanthin, mannitol, fucoidan and cellulose from kelp, thereby improving the recovery rate of these ingredients.

[0110] Through the designed process conditions, such as temperature, pressure, solvent ratio, etc., the damage to the structure of the active ingredients is minimized to the greatest extent, and the biological activity of the extracted product is guaranteed. The present invention makes full use of the characteristics of kelp, realizes the maximum utilization of kelp resources, and avoids the waste of resources caused by a single extraction method.

[0111] The use of modern green technologies such as supercritical carbon dioxide extraction, high-pressure microfluidics and enzymatic hydrolysis reduces the use of organic solvents and reduces the impact on the environment. By optimizing the process flow, the extraction steps are simplified, energy consumption and raw material consumption are reduced, thus achieving effective cost control. The extraction technology and conditions used help to improve the purity and stability of the extracted products, making the final product of higher quality. DETAILED DESCRIPTION

[0112] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods.

[0113] In order to better understand the above technical scheme, exemplary embodiments of the present invention will be described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a clearer and more thorough understanding of the present invention, and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0114] Unless otherwise specified, "parts" in the following examples refer to parts by mass.

[0115] Example 1: Recovery of Phycoxanthin

[0116] The dried kelp was crushed into fine powder with a particle size of less than 200 μm, and 100 portions of kelp powder were weighed and dried to constant weight for standby use. 100 portions of kelp powder were added to a supercritical carbon dioxide extraction device; the extraction conditions were set as follows: temperature 35 ° C, pressure 200 standard atmospheres, flow rate 65 g / min, and time 1.5 hours. The extraction was started, and the pressure and temperature changes during the extraction process were monitored and recorded. CO2 was recovered under reduced pressure at 25 ° C, and the pressure dropped from 200 standard atmospheres to 50 standard atmospheres, maintained for 45 seconds, further reduced to 15 standard atmospheres, maintained for 4 minutes, and finally reduced to standard standard atmospheres. After releasing the pressure, an extract was obtained, and the extract was redissolved with 3 times the mass of ethyl acetate and injected into the top of the column of the chromatography column.

[0117] A silica gel column was used as the stationary phase, and the elution solvent was a mixed solvent of hexane and ethyl acetate. Initial conditions: the volume ratio of hexane: ethyl acetate = 95:5, mid-term conditions: the volume ratio of hexane: ethyl acetate = 80:20, late conditions: the volume ratio of hexane: ethyl acetate = 70:30, and the final conditions: the volume ratio of hexane: ethyl acetate = 50:50, and the elution flow rate was 3mL / min / cm. Thin layer chromatography (TLC) was used to monitor the elution process, and the elution solvent ratio was adjusted as needed to ensure that phycoxanthin was fully eluted. The eluate was concentrated to 20% of the initial volume and cooled to 4°C to promote the crystallization of phycoxanthin. Ethanol was used for alcohol precipitation, and the volume of ethanol was 75% of the total volume, and it was placed for 20 hours to allow phycoxanthin to precipitate completely. The precipitate and the supernatant were separated by centrifugation at 3500rpm for 12 minutes. The crystalline precipitate was washed with Tris-HCl buffer at pH 7.5. Freeze-dried at -45°C for 22 hours. The obtained product was tested for recovery and purity. After testing, 100 parts of kelp residues contained 5 parts of phycoxanthin. In this embodiment, 4.0 parts of phycoxanthin were recovered, the recovery rate of phycoxanthin was 80%, and the purity of phycoxanthin was 85%.

[0118] Embodiment 2, mannitol recovery

[0119] Take the kelp powder extracted by supercritical carbon dioxide in Example 1, dry and weigh 90 parts of kelp residues, prepare the equipment for high-pressure microfluidic technology treatment, high-pressure water jet pressure 110MPa, processing time 20 seconds, processing temperature 50 ° C, flow rate 0.5mL / min. Use a 0.22 μm microfiltration membrane to filter the treated liquid to remove larger particles and cell debris. Adjust the pH of the filtered solution to 6.7, and cool the solution to below 9 ° C to promote mannitol crystallization. Minus 40 ° C, cool and dry for 22 hours to obtain mannitol. After testing, kelp residues in 90 parts contain 13.5 parts of mannitol, and the mannitol recovered by this application is 10.4 parts, and the mannitol recovery rate is 77%, and the mannitol purity is 88%.

[0120] Example 3: Recovery of Fucoidan

[0121] Take the kelp residue treated by high-pressure microfluidics in Example 2, dry it to constant weight, the mass of the kelp residue is 70 parts, add it to a pH equal to 7.5 Tris-HCl buffer, and keep it at 65°C for preliminary extraction. The extract is cooled to 50°C, and a composite enzyme preparation of cellulase and xylanase is added, with cellulase accounting for 80%, enzyme activity of 1000U / g substrate, and reaction time of 4 hours. The extract is cooled to 50°C, and a composite enzyme preparation of cellulase and xylanase is added, with an enzyme activity of 1000U / g substrate, and the reaction time is 4 hours. The mixture after enzymolysis is heated to 80°C for 10 minutes. The mixture after enzymolysis is heated to 90°C for 12 minutes. After heating, it is quickly cooled to room temperature. Use a cold water bath or an ice water bath to quickly cool the mixture to room temperature. Use an ultrafiltration membrane for continuous separation, with a transmembrane pressure of 1.25bar, an operating temperature of 25°C, and a flow rate of 1.2m / s. The volume ratio of water to ethanol was 9:1, the volume ratio of organic solvent to fucoidan concentrate was 5.5:1, the extraction temperature was 25°C, and the extraction time was 1 hour. The extract was cooled to 4°C, and ethanol was added until the volume of ethanol was 75% of the total volume. After stirring evenly, it was placed for 20 hours. The precipitate and the supernatant were separated by a centrifuge at a centrifugal speed of 4000rpm for 12 minutes. The precipitate was washed with Tris-HCl buffer at pH 7.5. The precipitate was freeze-dried at -35°C to obtain the product.

[0122] After testing, 70 parts of kelp residues contained 2.82 parts of fucoidan, and the fucoidan recovered in the present application was 2.03 parts, the fucoidan recovery rate was 72%, and the fucoidan purity was 87%.

[0123] Example 4: Cellulose recovery

[0124] Take the kelp residue after ultrafiltration membrane separation in Example 3 and dry it to constant weight to 65 parts, and dry it at 55°C to constant weight. Use 0.55% w / v chitosan aqueous solution, adjust the pH to 5.5 with acetic acid, stir at 3500rpm, stir for 6 minutes, and let stand for 35 minutes. The mass ratio of kelp residue to chitosan aqueous solution is 1:25. Transfer the floccules to a 10% concentration -7 CFU / mL lactic acid bacteria fermentation broth, fermentation temperature 38.5 ℃, fermentation for 21 hours. Use 0.55% w / v potassium carbonate aqueous solution, soak the floccules at 46 ℃ for 2 hours. Add equal volumes of 0.55% v / v hydrogen peroxide aqueous solution and 0.75mM photosensitizer ethanol solution to the floccules solution treated with potassium carbonate aqueous solution, and treat under ultraviolet light for 1.1 hours, wavelength 254nm, intensity 10mW / cm 2. Add cellulase to the floc solution irradiated with ultraviolet light, at 50°C, the enzyme activity is 20U / g, and hydrolysis is carried out for 20 hours. Combined with ultrasonic assistance, the power is 100W, intermittent: turn on for 10 seconds, stop for 10 seconds, and ultrasonic for 10 minutes. Vacuum dry at -52.5°C for 22 hours to obtain the product. After testing, 65 parts of kelp residues contain 3.92 parts of cellulose, and the cellulose recovered in this application is 2.74 parts, the cellulose recovery rate is 70%, and the cellulose purity is 88%.

[0125] Comparative Example 1

[0126] The dried kelp was crushed into a fine powder with a particle size of less than 200 μm, and 100 portions of kelp fine powder were weighed for use. Hexane was used as the extraction solvent, the extraction temperature was 25°C, and the extraction was performed for 3 hours. The extract was concentrated at 40°C using a rotary evaporator. A silica gel column was used as the stationary phase, and the elution solvent was a mixed solvent of hexane and ethyl acetate, with gradient elution in different proportions, and the specific proportions were the same as in Example 1. Thin layer chromatography (TLC) was used to monitor the elution process to ensure that phycoxanthin was fully eluted. Ethyl acetate was used to further extract phycoxanthin, the volume ratio of ethyl acetate to concentrate was 5:1, the extraction temperature was 25°C, and the time was 1 hour. Cool to 4°C, add ethanol until the volume of ethanol is 75% of the total volume, stir evenly and place for 20 hours. Centrifugal speed 3500rpm, time is 12 minutes, and the precipitate is washed with Tris-HCl buffer at pH 7.5. Freeze-dry at -45°C for 22 hours. After testing, 100 parts of kelp residues contained 5 parts of phycoxanthin. The present application recovered 3.9 parts of phycoxanthin, with a recovery rate of 78% and a purity of 83%.

[0127] It can be seen that the use of supercritical carbon dioxide extraction technology can more effectively extract phycoxanthin, with a recovery rate of 80%, which is higher than 78% in Comparative Example 1. The purity of phycoxanthin is 85%, which is higher than 83% in Comparative Example 1.

[0128] Supercritical carbon dioxide extraction technology is a green extraction method. Compared with traditional organic solvent extraction methods, it reduces the use of toxic organic solvents and is more environmentally friendly. The use of supercritical carbon dioxide extraction technology can reduce the impurities extracted from kelp, which is conducive to the subsequent recovery and purification of substances.

[0129] In Comparative Example 1, n-hexane was directly added to the kelp powder to extract phycoxanthin. N-hexane is a highly volatile organic solvent, and even after the concentration and washing steps, there are still some residual solvents. These residual solvents affect the effect of the subsequent extraction steps.

[0130] In order to verify the effect of n-hexane on subsequent operations, the kelp residue powder of comparative example 1 was verified according to the processes of Example 2, Example 3, and Example 4, and the data obtained were:

[0131] The recovery rate of mannitol decreased to 73% and the purity decreased to 82%. The recovery rate of cellulose decreased to 64% and the purity decreased to 81%. The recovery rate of fucoidan decreased to 68% and the purity decreased to 82%.

[0132] It can be seen that the residual n-hexane will affect the crystallization process of mannitol, resulting in incomplete crystallization or decreased crystal purity.

[0133] The presence of n-hexane will interfere with the enzymatic hydrolysis of cellulose, reduce enzyme activity or change the structure of cellulose, thereby affecting the recovery rate and purity of cellulose.

[0134] The residual n-hexane will affect the enzymatic hydrolysis and ultrafiltration process of fucoidan, resulting in a decrease in the recovery rate and purity of fucoidan.

[0135] Comparative Example 2

[0136] The dried kelp was crushed into fine powder with a particle size of less than 200 μm and dried to a constant weight, and mannitol was directly recovered according to the recovery method of Example 2.

[0137] The recovery rate of mannitol was 78% and the purity of mannitol was 88%.

[0138] Comparative Example 3

[0139] The dried kelp was crushed into fine powder with a particle size of less than 200 μm and dried to a constant weight, and the fucoidan was directly recovered according to the recovery method of Example 3.

[0140] The recovery rate of fucoidan was 72% and the purity of fucoidan was 87%.

[0141] Comparative Example 4

[0142] The dried kelp was crushed into fine powder with a particle size of less than 200 μm and dried to a constant weight, and the cellulose was directly recovered according to the recovery method of Example 3.

[0143] The recovery rate of cellulose was 71%, and the purity of fucoidan was 89%.

[0144] In summary, the present application ensures that the recovery of each step will not affect the recovery rate and purity of the subsequent steps by gradually recovering the active ingredients in the kelp, thereby maximizing the utilization of resources. In this way, a variety of valuable active ingredients can be extracted from the kelp, improving the overall recovery efficiency. The recovery process of each step is independent, and the components extracted in the previous step will not interfere with the recovery of the active ingredients in the subsequent steps. Such a design helps to maintain the purity of each active ingredient and reduce the impact of impurities on the subsequent extraction process.

[0145] This application uses a series of green technologies, such as supercritical carbon dioxide extraction, high-pressure microfluidics technology and enzymolysis, which reduce the use of organic solvents and reduce the impact on the environment. These technologies not only help to improve recovery efficiency, but also reduce waste generation, meeting the requirements of sustainable development. The adopted technology helps to improve the purity and stability of the extracted product, so that the final product has a higher quality. Through continuous recovery, it can be ensured that the active ingredients extracted in each step have stable performance, which is convenient for subsequent applications.

[0146] 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A step-by-step recovery method for active ingredients of kelp, characterized in that: The following steps are implemented to achieve the step-by-step recovery of the active ingredients of kelp; S1, drying the kelp and crushing the dried kelp into fine powder, wherein the particle size of the fine powder is less than 200 μm; S2, extracting the kelp powder in step S1 in supercritical carbon dioxide, and re-dissolving the extract and eluting it by column chromatography to recover phycoxanthin; S3, using high-pressure microfluidics technology to treat the kelp residue produced in step S2 to release mannitol, then filtering the extract, adjusting the pH value, and finally freeze-drying to recover mannitol crystals; S4, recovering fucoidan from the kelp residue treated by high-pressure microfluidic technology in step S3 by enzymatic hydrolysis, ultrafiltration separation, organic solvent extraction and alcohol precipitation; S5. The kelp residue separated after the ultrafiltration separation treatment in step S4 is subjected to chitosan flocculation, lactic acid bacteria fermentation decalcification, alkali treatment and enzymatic hydrolysis to recover cellulose.

2. The method for recovering active ingredients from kelp step by step according to claim 1, characterized in that: In step S2, the conditions for extracting phycoxanthin in supercritical carbon dioxide are: under the conditions of 30-40° C. and 180-220 standard atmospheric pressures, using supercritical carbon dioxide as a solvent, and extracting for 1-1.5 hours.

3. The method for recovering active ingredients from kelp step by step according to claim 2, characterized in that: After extracting phycoxanthin, CO2 is recovered under reduced pressure at room temperature, the extract is concentrated and extracted, and the conditions for reducing pressure to recover CO2 are: temperature range is 20-25°C, pressure is reduced from 180-220 standard atmospheres to 50-60 standard atmospheres, maintained for 30s to 60s, and then further reduced to 10-20 standard atmospheres, maintained for 3-5 minutes, and finally reduced to standard atmospheric pressure.

4. The method for recovering active ingredients from kelp step by step according to claim 3, characterized in that: In step S2, the conditions for recovering phycoxanthin by column chromatography elution are as follows: the extracted substance is re-dissolved with 2-4 times the mass of ethyl acetate and added to the top of the column chromatography column; the elution solvent is a mixed solvent of hexane and ethyl acetate, the elution flow rate of the mixed solvent is 1-5 mL / min / cm, and the elution conditions are: Initial conditions: volume ratio of hexane:ethyl acetate was 95:5; Medium-term conditions: the volume ratio of hexane:ethyl acetate is 80:20; Late stage conditions: volume ratio of hexane:ethyl acetate was 70:30; Final conditions: hexane:ethyl acetate volume ratio of 50:

50.

5. The method for recovering active ingredients from kelp step by step according to claim 1, characterized in that: In step S3, the conditions for recovering mannitol using high pressure microfluidic technology are: The kelp residue produced in step S2 is treated by high-pressure water jet, the high-pressure water pressure is 100-120 MPa, the treatment time is 10-30 seconds, the treatment temperature is 50-60° C., and the flow rate is 0.3-0.8 mL / min.

6. The method for recovering active ingredients from kelp step by step according to claim 5, characterized in that: In step S3, the high-pressure water after washing the kelp residue is used as a mannitol extract, the pH of the mannitol extract is adjusted to 6.5-7.0, and the mannitol extract is cooled to below 10° C. to promote mannitol crystallization to recover mannitol.

7. The method for recovering active ingredients from kelp step by step according to claim 5, characterized in that: In step S4, the kelp residue produced in S3 is used as an extraction raw material, and is added to a Tris-HCl buffer with a pH of 7.5-7.8, maintained at 65-75° C. for preliminary extraction, and after the preliminary extraction, the extract is cooled to 45-55° C.; Maintaining the extract at 45-55°C, adding a composite enzyme preparation of cellulase and xylanase, wherein the enzyme activity is at least 1000U / g substrate, and the reaction time is 4-4.5 hours; The extract after enzymatic hydrolysis is subjected to ultrafiltration separation to recover the fucoidan concentrate and remove the undecomposed kelp residues and small molecular impurities.

8. The method for stepwise recovery of active ingredients from kelp according to claim 7, characterized in that: The fucoidan concentrate is extracted with an organic solvent, wherein the organic solvent is a mixed solution of water and ethanol in a volume ratio of 9:1-8:2, the volume ratio of the organic solvent to the fucoidan concentrate is 3-8:1, the extraction temperature is 20-30°C, and the extraction time is 0.5-1.5 hours; The extracted solution containing fucoidan is cooled to 3-5°C, ethanol is slowly added thereto until the volume of ethanol is 70-80% of the total volume, and then stirred evenly and allowed to stand for 16-24 hours to allow the fucoidan to completely precipitate.

9. The method for recovering active ingredients from kelp step by step according to claim 7, characterized in that: The kelp residue separated by ultrafiltration membrane is dried at 50-60°C to constant weight, a chitosan aqueous solution with a concentration of 0.5-0.6% w / v is added, and the mixture is stirred at 3000-4000 rpm for 5-7 minutes and then allowed to stand for 30-40 minutes to collect floccules. The mass ratio of the kelp residue to the chitosan aqueous solution is 1:20-30.

10. The method for stepwise recovery of active ingredients from kelp according to claim 8, characterized in that: The collected flocs were transferred to a -7 -10 -8 CFU / mL lactic acid bacteria fermentation broth, fermentation at 37-40℃ for 18-24 hours; Using 0.5-0.6% w / v potassium carbonate aqueous solution, immerse the flocculent for 2-2.5 hours at 45-48°C to neutralize the acidic substances produced during the fermentation process and soften the fiber structure; The method comprises the following steps: adding a 0.5-0.6% v / v hydrogen peroxide aqueous solution and a 0.5 mM-1 mM photosensitizer ethanol solution in equal volumes to the flocculent solution after the potassium carbonate aqueous solution treatment, and treating the flocculent solution under ultraviolet irradiation for 1-1.2 hours; adding cellulase to the flocculent solution irradiated with ultraviolet light, and hydrolyzing the solution at 45-55° C. with an enzyme activity of at least 20 U / g for 16-24 hours; and finally vacuum drying the solution at -50 to -55° C. for 20-24 hours to obtain a dry cellulose product.