A method for sequentially separating polysaccharide and phycoerythrin from water extract of defatted haematococcus algae residue

Abstract

This invention belongs to the field of Haematococcus pluvialis separation technology, specifically relating to a method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of degreased Haematococcus pluvialis residue. The method includes the following steps: S1. Pretreatment; S2. Deep flocculation and primary separation of polysaccharides; and S3. Specific precipitation of phycoerythrin. This invention utilizes a self-made hydrophobic flocculant and a composite precipitation inducer to construct a sequential separation process, thereby enabling the simultaneous acquisition of high-purity polysaccharide products and highly active phycoerythrin concentrate from the aqueous extract of degreased Haematococcus pluvialis residue. This invention integrates the deep flocculation of polysaccharides, the specific precipitation of phycoerythrin, and their respective subsequent purification steps into an orderly overall process, ensuring that the output of the previous step can directly enter the next processing step in the most suitable state. This not only reduces potential losses from the storage and transfer of intermediate materials but also improves the purity of the final product through condition matching and synergy between steps.

Description

Technical Field

[0001] This invention belongs to the field of Haematococcus pluvialis separation technology, specifically relating to a method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of degreased Haematococcus pluvialis residue. Background Technology

[0002] Haematococcus pluvialis is considered one of the best biological sources for producing natural astaxanthin in nature. Currently, large-scale astaxanthin extraction technologies, primarily based on organic solvent extraction or supercritical carbon dioxide fluid extraction, are quite mature and constitute a major economic pillar of the Haematococcus pluvialis industry. However, this core extraction process generates a large amount of degreasing algal residue from Haematococcus pluvialis. How to achieve high-value comprehensive utilization of this residue and avoid resource waste and secondary environmental burden has become a prominent bottleneck restricting cost reduction, efficiency improvement, and green development across the entire Haematococcus pluvialis industry chain.

[0003] After water extraction, the degreased algal residue of *Hylocereus pluvialis* yields two main types of potentially valuable natural products: one is a polysaccharide naturally synthesized during the growth of *Hylocereus pluvialis*, with a sulfate ester group on its molecular backbone, which has been proven to possess immunomodulatory and other biological activities; the other is phycoerythrin, a pigment-protein complex with characteristic red color and fluorescence properties, which has high added value in fields such as bioassay, food coloring, and cosmetics. Therefore, simultaneously recovering these two components from the degreased algal residue is widely considered a key approach to enhancing the utilization value of the raw material. However, due to the overlap in molecular size and charge distribution between the two, achieving efficient and high-purity separation from this complex colloidal system remains a major bottleneck for industrialization.

[0004] A well-known domestic microalgae processing company struggled to obtain qualified products economically and efficiently by using conventional techniques to process degreased algal residue aqueous extracts. Firstly, the aqueous extract is an extremely stable colloidal system composed of polysaccharides, proteins, pigments, and cell debris, with highly overlapping properties of the target components. The company attempted to precipitate polysaccharides using conventional cationic flocculants, but these methods exhibited poor selectivity, resulting in loose, high-water-content flocs that also contained a significant proportion of phycoerythrin. After separation, the resulting polysaccharide wet cake contained numerous impurities; without further processing, direct drying resulted in low purity and poor color, failing to meet the basic requirements for food or cosmetic raw materials.

[0005] In the subsequent phycoerythrin precipitation stage, traditional methods often rely on high-concentration ammonium sulfate salting-out. To achieve a high yield, the ammonium sulfate saturation often needs to be increased to over 70%. This harsh salting-out environment not only easily leads to the loss of the active components of phycoerythrin, but more importantly, it also precipitates residual polysaccharides and other proteins that were not completely removed in the previous step. The resulting phycoerythrin precipitate is actually a complex mixture, requiring lengthy post-processing steps such as dialysis and multiple reconstitution for initial purification. The entire process takes several days, and the purity of the final product fluctuates greatly, severely impacting the product's commercial value.

[0006] Therefore, it is necessary to design a method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of degreased algal residue from Haematococcus pluvialis. Summary of the Invention

[0007] To overcome the shortcomings of the existing technology, a method is provided for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of degreased algal residue from Haematococcus pluvialis.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue from Haematococcus pluvialis, the method comprising the following steps:

[0010] S1. Pretreatment: The defatted algal residue of *Hylocereus pluvialis* is subjected to water extraction treatment, and the pH value of the resulting water extract of defatted algal residue of *Hylocereus pluvialis* is adjusted to 5.0-5.8;

[0011] S2. Deep flocculation and primary separation of polysaccharides: A hydrophobic flocculant is added to the aqueous extract of degreased algal residue obtained in S1. After flocculation reaction at 20-30℃, solid-liquid separation is performed to obtain a flocculated wet cake containing polysaccharides and a first supernatant. The flocculated wet cake containing polysaccharides is then refined to obtain a solid polysaccharide product.

[0012] S3. Specific precipitation of phycoerythrin: A composite precipitation inducer containing phenylboronic acid-functionalized polyethylene glycol and ammonium sulfate is added to the first supernatant obtained in S2. The mixture is allowed to stand at 2-8°C to induce precipitation, followed by solid-liquid separation to obtain phycoerythrin precipitate. The phycoerythrin precipitate is then purified to obtain phycoerythrin concentrate.

[0013] In step S1, the water extraction process includes the following steps: the astaxanthin-extracted defatted algal residue of *Hydnocarpus erythropoietin* is added to a phosphate buffer solution with a pH of 6.5-7.0 at a material-to-liquid ratio of 1g:(20-30)mL, and the mixture is stirred and extracted at 60-70℃ for 1.5-2.5 hours. The supernatant is then centrifuged and collected as the water extract of the defatted algal residue of *Hydnocarpus erythropoietin*.

[0014] In step S2, the refining step of the polysaccharide-containing flocculent wet cake is as follows: the polysaccharide-containing flocculent wet cake obtained by solid-liquid separation is stirred and washed with a dilute hydrochloric acid solution with a pH of 2.5-3.5, the solid is collected after centrifugation, and the solid is dispersed in a 60%-75% ethanol aqueous solution, stirred, centrifuged, and dried to obtain a solid polysaccharide product.

[0015] The amount of dilute hydrochloric acid solution used is 3-5 times the volume of the polysaccharide-containing flocculent wet cake, and the washing time is 10-20 minutes; the amount of ethanol aqueous solution used is 8-12 times the volume of the solid mass after washing.

[0016] In step S3, the purification of the phycoerythrin precipitate is as follows: the obtained phycoerythrin precipitate is dissolved in a buffer solution with a pH of 7.0-7.5 and containing 0.1-0.5M sorbitol; the resulting solution is subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30-50 kDa; desalting and concentration are performed to obtain a concentrated phycoerythrin solution.

[0017] In step S2, the hydrophobic flocculant is prepared by the following steps: chitosan is dissolved in an acetic acid solution with a mass fraction of 1%-2%, and glycidyltrimethylammonium chloride and dodecane oxide are added successively under nitrogen protection and at 60-70°C for 8-12 hours. The pH value is maintained at 8.5-9.5 throughout the reaction. After the reaction is completed, the product is precipitated, washed and dried to obtain the hydrophobic flocculant.

[0018] The molar ratio of glycidyltrimethylammonium chloride to the amino group in chitosan is 1.0-1.2:1, and the amount of dodecyl oxide added is 0.2 to 0.3 times the molar amount of the amino group in chitosan.

[0019] In step S3, the phenylboronic acid-functionalized polyethylene glycol is prepared by the following steps: 3-carboxyphenylboronic acid pinacol ester is dissolved in an anhydrous solvent and reacted with thionyl chloride at 0-30°C for 2-5 hours in the presence of a catalytic amount of N,N-dimethylformamide; the resulting acyl chloride intermediate solution is added to an anhydrous dichloromethane solution of monomethoxy polyethylene glycol in the presence of an ice bath and triethylamine, and the reaction is continued at 25-35°C for 6-12 hours; after the reaction is completed, the product is precipitated, washed, and dried to obtain phenylboronic acid-functionalized polyethylene glycol.

[0020] The molar ratio of the hydroxyl terminus of the 3-carboxyphenylboronic acid pinacol ester to monomethoxy polyethylene glycol is 1.10-1.25:1.

[0021] In step S3, the composite precipitation inducer is added as follows: first, phenylboronic acid functionalized polyethylene glycol is added to the first supernatant to make its final mass concentration reach 5%-8%, and then ammonium sulfate is added under stirring to make the saturation of ammonium sulfate in the system reach 52%-60%.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0023] 1. In achieving efficient capture of polysaccharide components, this invention employs a self-made hydrophobic flocculant instead of conventional cationic flocculants. This hydrophobic flocculant is prepared by simultaneously introducing quaternary ammonium salt groups and hydrophobic long chains onto the chitosan backbone. In the weakly acidic environment of the aqueous extract, its quaternary ammonium salt groups can electrostatically attract the negatively charged centers such as sulfate ester groups on the Haematococcus pluvialis polysaccharide molecular chains, achieving initial binding. Simultaneously, its hydrophobic segments can bind to hydrophobic microregions between or within polysaccharide molecules through hydrophobic interactions, and can act as cross-linking bridges between different polysaccharide molecules. This synergistic effect of electrostatic and hydrophobic interactions results in a stronger binding force and a broader capture capacity for polysaccharides, especially those with complex molecular structures. The resulting flocs are denser, allowing for more thorough removal of polysaccharides from the complex colloidal system in the first separation step, creating more favorable conditions for subsequent phycoerythrin purification.

[0024] 2. For the selective precipitation and activity retention of phycoerythrin, this invention uses a composite precipitation inducer comprising phenylboronic acid-functionalized polyethylene glycol (PEG). The phenylboronic acid-functionalized PEG is prepared by chemically modifying polyethylene glycol by grafting phenylboronic acid groups onto its ends. Under near-neutral conditions, the phenylboronic acid groups can form reversible covalent bonds with the cis-diol structures on the sugar chains of glycoprotein molecules such as phycoerythrin. This effect endows the precipitation process with a certain molecular recognition capability. When it acts together with ammonium sulfate, ammonium sulfate primarily provides a mild salting-out environment, moderately reducing the solubility of phycoerythrin, while the phenylboronic acid-functionalized PEG, through specific binding, guides the phycoerythrin molecules to aggregate in a directional and orderly manner. This precipitation mechanism differs from the non-selective and violent precipitation caused by traditional high-concentration salting-out, thus achieving effective precipitation of phycoerythrin at relatively low ammonium sulfate saturation and helping to maintain its natural three-dimensional conformation and fluorescent activity.

[0025] 3. To address potential impurities remaining in the primary separation products and the needs of subsequent processing, this invention designs targeted purification steps. For the polysaccharide-containing flocculent wet cake, washing with dilute hydrochloric acid solution dissolves and removes some impurities non-specifically adsorbed by the flocculant, while the moderate acidity helps maintain floc stability. Subsequent ethanol precipitation further dehydrates the polysaccharide and converts it into an easily processed solid form. For the phycoerythrin precipitate, it is dissolved in a buffer solution containing sorbitol. Sorbitol, as a competitive small molecule, can bind to phenylboronic acid groups, thereby promoting the gentle release of phycoerythrin. The subsequent tangential flow ultrafiltration step efficiently removes salts, small molecule impurities, and excess phenylboronic acid-functionalized polyethylene glycol from the solution, while concentrating phycoerythrin under mild physical conditions. These two purification steps are closely integrated with the aforementioned specific separation steps, working together to ensure the purity and bioactivity of the Haematococcus pluvialis polysaccharide and phycoerythrin products.

[0026] 4. This invention integrates the deep flocculation of polysaccharides, the specific precipitation of phycoerythrin, and their respective subsequent refining steps into an orderly whole process, ensuring that the output of the previous step can directly enter the next step in the most suitable state. This not only reduces the potential losses caused by the storage and transfer of intermediate materials, but also improves the purity of the final product through the matching and synergy of conditions between steps. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows:

[0029] The defatted residue of Haematococcus pluvialis is a byproduct of Haematococcus pluvialis processing after astaxanthin extraction, and comes from Yunnan Aierfa Biotechnology Co., Ltd.

[0030] Phosphate buffer was purchased from Shanghai Uniway Biotechnology Co., Ltd.

[0031] Dilute hydrochloric acid, purchased from Guangzhou Hewei Pharmaceutical Technology Co., Ltd.

[0032] Sodium hydroxide was purchased from Xinjiang Zhongtai Chemical Co., Ltd.

[0033] Acetic acid was purchased from Shandong Hualu Hengsheng Chemical Co., Ltd.

[0034] Acetone was purchased from Lihua Yiweiyuan Chemical Co., Ltd.

[0035] N,N-Dimethylformamide was purchased from Shandong Hengshuo Chemical Co., Ltd.

[0036] Thionyl chloride was purchased from Shandong Kaisheng New Materials Co., Ltd.

[0037] Anhydrous tetrahydrofuran, purchased from Shandong Aite Chemical Co., Ltd.

[0038] Triethylamine was purchased from Jiangsu Youlixin Chemical Co., Ltd.

[0039] Anhydrous dichloromethane was purchased from Chengdu Zhong Sheng Jin Hong Chemical Co., Ltd.

[0040] Solid ammonium sulfate was purchased from Wuhan Jixin Yibang Biotechnology Co., Ltd.

[0041] Chitosan was purchased from Hubei Haijia Biotechnology Co., Ltd.

[0042] Glycidyltrimethylammonium chloride was purchased from Hubei Chushengwei Chemical Co., Ltd.

[0043] Dodecyl oxide was purchased from Hubei Jiufenglong Chemical Co., Ltd.

[0044] 3-Carboxyphenylboronic acid pinacol ester, purchased from Beijing Bailingwei Technology Co., Ltd.

[0045] Monomethoxy polyethylene glycol was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0046] Sorbitol, purchased from Beijing Solarbio Technology Co., Ltd.

[0047] The technical solution of this application is as follows:

[0048] A method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue from Haematococcus pluvialis, the method comprising the following steps:

[0049] S1. Pretreatment: The defatted algal residue of *Hylocereus pluvialis* is subjected to water extraction treatment, and the pH value of the resulting water extract of defatted algal residue of *Hylocereus pluvialis* is adjusted to 5.0-5.8;

[0050] S2. Deep flocculation and primary separation of polysaccharides: A hydrophobic flocculant is added to the aqueous extract of degreased algal residue obtained in S1. After flocculation reaction at 20-30℃, solid-liquid separation is performed to obtain a flocculated wet cake containing polysaccharides and a first supernatant. The flocculated wet cake containing polysaccharides is then refined to obtain a solid polysaccharide product.

[0051] S3. Specific precipitation of phycoerythrin: A composite precipitation inducer containing phenylboronic acid-functionalized polyethylene glycol and ammonium sulfate is added to the first supernatant obtained in S2. The mixture is allowed to stand at 2-8°C to induce precipitation, followed by solid-liquid separation to obtain phycoerythrin precipitate. The phycoerythrin precipitate is then purified to obtain phycoerythrin concentrate.

[0052] In step S1, the water extraction process includes the following steps: the astaxanthin-extracted defatted algal residue of *Hydnocarpus erythropoietin* is added to a phosphate buffer solution with a pH of 6.5-7.0 at a material-to-liquid ratio of 1g:(20-30)mL, and the mixture is stirred and extracted at 60-70℃ for 1.5-2.5 hours. The supernatant is then centrifuged and collected as the water extract of the defatted algal residue of *Hydnocarpus erythropoietin*.

[0053] The amount of dilute hydrochloric acid solution used is 3-5 times the volume of the polysaccharide-containing flocculent wet cake, and the washing time is 10-20 minutes; the amount of ethanol aqueous solution used is 8-12 times the volume of the solid mass after washing.

[0054] In step S2, the hydrophobic flocculant is prepared by the following steps: chitosan is dissolved in an acetic acid solution with a mass fraction of 1%-2%, and glycidyltrimethylammonium chloride and dodecane oxide are added successively under nitrogen protection and at 60-70°C for 8-12 hours. The pH value is maintained at 8.5-9.5 throughout the reaction. After the reaction is completed, the product is precipitated, washed and dried to obtain the hydrophobic flocculant.

[0055] The molar ratio of glycidyltrimethylammonium chloride to the amino group in chitosan is 1.0-1.2:1, and the amount of dodecyl oxide added is 0.2 to 0.3 times the molar amount of the amino group in chitosan.

[0056] The challenge in recovering water-soluble active ingredients from the defatted algal residue of *Hydrocotyle vulgaris* after astaxanthin extraction is that the aqueous extract is a stable colloidal system highly mixed with polysaccharides, phycoerythrin, and other impurities. Conventional separation methods using polyaluminum chloride or ordinary chitosan for flocculation, while capable of precipitating some substances, suffer from poor selectivity. These conventional cationic flocculants primarily rely on electrostatic interactions to bind with negatively charged components. However, the sulfate ester groups of *Hydrocotyle vulgaris* polysaccharides and the acidic amino acids on the surface of phycoerythrin are both negatively charged in this environment, leading to simultaneous flocculation of both, resulting in a loose and complex precipitate.

[0057] This invention first adjusts the environment of the aqueous extract of *Hydrocotyle vulgaris* degreasing residue to a weakly acidic range of pH 5.0-5.8, creating a foundation for subsequent selective separation. A self-made hydrophobic flocculant replaces conventional reagents. This flocculant is prepared by simultaneously introducing quaternary ammonium salt groups and hydrophobic long chains onto a chitosan backbone. Within the aforementioned pH range, its quaternary ammonium salt groups effectively neutralize the negative charge of polysaccharides, while the hydrophobic segments interact with polysaccharide molecules through hydrophobic interactions, forming bridges between different polysaccharide molecules. This synergistic effect of electrostatic and hydrophobic interactions enhances the flocculant's affinity and capture efficiency for polysaccharides, especially those with larger molecular weights and more complex structures, resulting in denser flocs. Compared to conventional flocculants that rely solely on electrostatic interactions, this design improves the selectivity for polysaccharides in the first separation step, reduces the proportion of phycoerythrin entrained, and provides a supernatant with fewer impurities for subsequent steps.

[0058] In step S3, the phenylboronic acid-functionalized polyethylene glycol is prepared by the following steps: 3-carboxyphenylboronic acid pinacol ester is dissolved in an anhydrous solvent and reacted with thionyl chloride at 0-30°C for 2-5 hours in the presence of a catalytic amount of N,N-dimethylformamide; the resulting acyl chloride intermediate solution is added to an anhydrous dichloromethane solution of monomethoxy polyethylene glycol in the presence of an ice bath and triethylamine, and the reaction is continued at 25-35°C for 6-12 hours; after the reaction is completed, the product is precipitated, washed, and dried to obtain phenylboronic acid-functionalized polyethylene glycol.

[0059] The molar ratio of the hydroxyl terminus of the 3-carboxyphenylboronic acid pinacol ester to monomethoxy polyethylene glycol is 1.10-1.25:1.

[0060] In step S3, the composite precipitation inducer is added as follows: first, phenylboronic acid functionalized polyethylene glycol is added to the first supernatant to make its final mass concentration reach 5%-8%, and then ammonium sulfate is added under stirring to make the saturation of ammonium sulfate in the system reach 52%-60%.

[0061] However, even with the removal of as much polysaccharide as possible in the first step, the separation and purification of phycoerythrin in the resulting supernatant remains a challenge. Traditional methods often require salting out with high concentrations of ammonium sulfate to achieve high yields, which can easily impair the activity of phycoerythrin. Furthermore, the harsh precipitation environment can cause residual polysaccharides to co-precipitate with other proteins, affecting product purity. This invention introduces a composite precipitation inducer containing phenylboronic acid-functionalized polyethylene glycol (PEG) at this stage. PEG is synthesized by converting 3-carboxyphenylboronic acid pinacol ester to an acyl chloride, which is then coupled with monomethoxy polyethylene glycol. Under near-neutral conditions, the phenylboronic acid group can reversibly and specifically bind to the cis-diol structure of the sugar chains on the surface of glycoprotein molecules such as phycoerythrin. When synergistically acting with ammonium sulfate, the ammonium sulfate provides a mild salting-out environment, moderately reducing the solubility of phycoerythrin, while the PEG guides the phycoerythrin molecules to aggregate in a more directional and orderly manner through molecular recognition. This precipitation method reduces dependence on high salt concentrations and helps to better maintain the native conformation of phycoerythrin during precipitation, thus making it possible to obtain highly active products.

[0062] In step S2, the refining step of the polysaccharide-containing flocculent wet cake is as follows: the polysaccharide-containing flocculent wet cake obtained by solid-liquid separation is stirred and washed with a dilute hydrochloric acid solution with a pH of 2.5-3.5, the solid is collected after centrifugation, and the solid is dispersed in a 60%-75% ethanol aqueous solution, stirred, centrifuged, and dried to obtain a solid polysaccharide product.

[0063] The polysaccharide flocculent wet cake obtained in the first step also contains flocculants and impurities that may be non-specifically adsorbed, requiring further processing to transform it into a qualified product. Direct drying results in low product purity. This invention employs a purification method of acid washing followed by alcohol precipitation. Washing the flocculent wet cake with a dilute hydrochloric acid solution provides an acidic environment that helps dissolve and remove loosely bound proteins, while a certain level of acidity may also help maintain floc stability for ease of handling. The washed solid is then redispersed in an aqueous ethanol solution of a specific concentration. The ethanol alters the polarity of the solution, causing the polysaccharide to dehydrate and precipitate. This process further purifies the polysaccharide and transforms it into a solid form that is easy to store and transport. This purification step, combined with the deep flocculation step, ensures the purity of the final polysaccharide product.

[0064] In step S3, the purification of the phycoerythrin precipitate is as follows: the obtained phycoerythrin precipitate is dissolved in a buffer solution with a pH of 7.0-7.5 and containing 0.1-0.5M sorbitol; the resulting solution is subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30-50 kDa; desalting and concentration are performed to obtain a concentrated phycoerythrin solution.

[0065] The phycoerythrin precipitate obtained through the aforementioned specific precipitation still requires further processing to remove residual salts, excess precipitant, and potentially trace impurities to obtain a high-purity product. Traditional methods such as dialysis are time-consuming and may cause activity loss. This invention designs a targeted purification step, dissolving the precipitate in a weakly alkaline buffer containing sorbitol. Sorbitol molecules contain multiple hydroxyl groups, which can act as a competitive agent, binding to phenylboronic acid groups, thereby promoting the gentle release and dissolution of phycoerythrin. Subsequently, the solution is treated using tangential flow ultrafiltration. The ultrafiltration membrane, based on molecular size sieving, efficiently retains large phycoerythrin molecules while allowing small molecules such as ammonium sulfate, sorbitol, and unbound phenylboronic acid-functionalized polyethylene glycol to permeate into the filtrate. This process is carried out at room temperature, simultaneously desalting and removing small molecule impurities while concentrating the phycoerythrin solution, ultimately yielding a high-purity phycoerythrin concentrate. This purification step is closely integrated with the aforementioned specific precipitation step, forming a complete purification unit.

[0066] The method provided by this invention constructs a complete process for sequentially separating two target products from a complex colloidal system through a series of interconnected and complementary steps. The application of pretreatment and a self-made hydrophobic flocculant improves the selectivity and efficiency of the polysaccharide separation stage; the design of a composite precipitation inducer achieves high selectivity and gentle precipitation of phycoerythrin; and subsequent purification steps, designed separately for the polysaccharide and phycoerythrin precipitates, remove various impurities, resulting in a high-purity final product. This overcomes the problems of poor separation selectivity, low product purity, difficulty in maintaining activity, and discontinuous process flow in existing technologies.

[0067] The present invention will be described in detail below through examples and comparative examples, but the scope of protection of the present invention is not limited to these examples. Unless otherwise specified, the chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products.

[0068] Example 1

[0069] A method for sequentially separating polysaccharides and phycoerythrin from an aqueous extract of defatted Haematococcus pluvialis residue includes the following steps: First, pretreatment is performed by weighing 100 g of defatted Haematococcus pluvialis residue after astaxanthin extraction and adding it to a phosphate buffer solution with a pH of 7.0 at a material-to-liquid ratio of 1 g to 30 mL. The mixture is then stirred and extracted at 70°C for 1.5 hours. After centrifugation, the supernatant is collected to obtain the aqueous extract of defatted Haematococcus pluvialis residue. The pH of this aqueous extract is adjusted to 5.8 using dilute hydrochloric acid. Next, deep flocculation and primary separation of the polysaccharides are performed by adding a self-made hydrophobic flocculant to the pretreated aqueous extract.

[0070] The preparation method of this hydrophobic flocculant is as follows: 10 grams of chitosan is dissolved in a 2% (w / w) acetic acid solution. Under nitrogen protection and at 70°C, glycidyltrimethylammonium chloride and dodecyl oxide are added sequentially and reacted for 8 hours. The molar ratio of glycidyltrimethylammonium chloride to amino groups in chitosan is 1.2:1. The amount of dodecyl oxide added is 0.3 times the molar amount of amino groups in chitosan. The pH value is maintained at 9.5 using sodium hydroxide solution throughout the reaction. After the reaction is completed, the product is precipitated with acetone, washed, and dried to obtain the final product.

[0071] The hydrophobic flocculant was prepared into an aqueous solution with a mass fraction of 0.5%. The flocculant solids were added at a ratio of 5% of the total solids in the aqueous extract. The mixture was stirred at 100 rpm for 40 minutes at 30°C, and then allowed to stand for 1 hour to mature. The polysaccharide-containing flocculent wet cake and the first supernatant were obtained by centrifugation.

[0072] The flocculated wet cake was refined by washing it with a dilute hydrochloric acid solution with a pH of 3.5, which was 5 times the volume of the wet cake, for 10 minutes. After centrifugation, the solid was collected. The solid was then dispersed in a 75% ethanol aqueous solution, which was 12 times the volume of the washed solid. After stirring and centrifugation, the solid was dried to obtain a solid Haematococcus pluvialis polysaccharide product.

[0073] Finally, specific precipitation of phycoerythrin was performed by adding a composite precipitation inducer to the first supernatant obtained in the previous step. First, phenylboronic acid-functionalized polyethylene glycol was prepared by reacting the hydroxyl terminus of 3-carboxyphenylboronic acid pinacol ester with that of monomethoxy polyethylene glycol at a molar ratio of 1.25:1. Specifically, 3-carboxyphenylboronic acid pinacol ester was dissolved in anhydrous tetrahydrofuran and reacted with thionyl chloride at 30°C for 2 hours in the presence of a catalytic amount of N,N-dimethylformamide. The resulting acyl chloride intermediate solution was added to an anhydrous dichloromethane solution of monomethoxy polyethylene glycol in the presence of an ice bath and triethylamine, and the reaction was continued at 35°C for 6 hours. After the reaction was completed, the precipitated product was washed and dried to obtain phenylboronic acid-functionalized polyethylene glycol. First, phenylboronic acid-functionalized polyethylene glycol was added to the first supernatant to achieve a final mass concentration of 8%. Then, solid ammonium sulfate was added under stirring to achieve an ammonium sulfate saturation of 60%. The mixture was placed at 2°C and allowed to stand for 36 hours to induce precipitation. The phycoerythrin precipitate was obtained by centrifugation. This precipitate was dissolved in Tris-HCl buffer solution with a pH of 7.5 and containing 0.5 M sorbitol. The resulting solution was subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa at an operating temperature of 25°C until the permeate conductivity stabilized, yielding a phycoerythrin concentrate concentrated approximately 10 times.

[0074] Example 2

[0075] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0076] A method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue from *Haematococcus pluvialis* is disclosed, with the following specific steps: In the pretreatment stage, 100 g of defatted algal residue from *Haematococcus pluvialis* is weighed and added to a phosphate buffer solution with a pH of 6.5 at a material-to-liquid ratio of 1 g to 20 mL. The mixture is then stirred and extracted at 60°C for 2.5 hours. After centrifugation, the supernatant is collected, and the pH of the aqueous extract is adjusted to 5.0.

[0077] In the deep flocculation and separation of polysaccharides, the hydrophobic flocculant was prepared under the following conditions: chitosan was dissolved in a 1% (w / w) acetic acid solution at 65°C; the molar ratio of glycidyltrimethylammonium chloride to chitosan amino group was 1.0:1; the amount of dodecane oxide was 0.2 times the molar amount of amino group; the reaction time was 12 hours; and the reaction pH was maintained at 8.5. This flocculant was added at a ratio of 1% of the total solids in the aqueous extract, stirred at 20°C for 80 minutes, allowed to stand for 3 hours, and then centrifuged. The resulting flocculent wet cake was washed for 20 minutes with a dilute hydrochloric acid solution (pH 2.5), at a volume three times the volume of the wet cake; after centrifugation, the solid was dispersed in a 60% ethanol aqueous solution (ethanol volume eight times the solid mass), and then dried to produce the polysaccharide product.

[0078] During the phycoerythrin precipitation stage, in the preparation of phenylboronic acid-functionalized polyethylene glycol, the molar ratio of the hydroxyl terminus of 3-carboxyphenylboronic acid pinacol ester to monomethoxy polyethylene glycol was 1.10:1. The acyl chloride reaction was carried out at 0°C for 5 hours, and the esterification reaction was carried out at 25°C for 12 hours. The product was added to the first supernatant to achieve a final mass concentration of 5%, and then ammonium sulfate was added to a saturation of 52%, and the mixture was allowed to stand at 8°C for 12 hours. The precipitate was dissolved in a buffer solution containing 0.1M sorbitol at pH 7.0, and then subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a concentrated phycoerythrin solution.

[0079] Example 3

[0080] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0081] A method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of *Hylocereus pluvialis* is disclosed, with the following specific steps: In the pretreatment, defatted algal residue of *Hylocereus pluvialis* is added to a phosphate buffer solution with a pH of 6.8 at a material-to-liquid ratio of 1 g to 25 mL. The mixture is then stirred and extracted at 65°C for 2.0 hours. After centrifugation, the aqueous extract is obtained, and its pH is adjusted to 5.4.

[0082] The preparation conditions for the hydrophobic flocculant used in polysaccharide flocculation were as follows: chitosan was dissolved in a 1.5% (w / w) acetic acid solution; the reaction temperature was 65℃; the molar ratio of glycidyltrimethylammonium chloride to chitosan amino group was 1.1:1; the amount of dodecane oxide was 0.25 times the molar amount of amino group; the reaction time was 10 hours; and the reaction pH was maintained at 9.0. The flocculant was added at a ratio of 3% of the total solids in the aqueous extract, stirred at 25℃ for 60 minutes, allowed to stand for 2 hours, and then centrifuged. When refining the flocculated wet cake, a dilute hydrochloric acid solution with a pH of 3.0 was used, with a volume four times the volume of the wet cake, and the cake was washed for 15 minutes. Subsequently, it was treated with a 70% ethanol aqueous solution, with the ethanol volume being 10 times the solid mass. After drying, the polysaccharide product was obtained.

[0083] In the phycoerythrin precipitation, the preparation of phenylboronic acid-functionalized polyethylene glycol involved a molar ratio of 3-carboxyphenylboronic acid pinacol ester to monomethoxy polyethylene glycol hydroxyl-terminated ester at 1.18:1. Acyl chloride was reacted at 15°C for 3.5 hours, and esterification was carried out at 30°C for 9 hours. The product was added to the first supernatant to a final mass concentration of 6.5%, followed by the addition of ammonium sulfate to a saturation of 56%, and allowed to stand at 5°C for 24 hours. The precipitate was dissolved in a buffer solution containing 0.3 M sorbitol at pH 7.3, and tangential flow ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 40 kDa to obtain a concentrated phycoerythrin solution.

[0084] Comparative Example 1

[0085] This comparative example is used to illustrate the role of the hydrophobic flocculant. The steps are basically the same as those in Example 3, except that in step S2, the self-made hydrophobic flocculant is not used, but instead an equal mass of ordinary chitosan is used, that is, chitosan that has not been modified by glycidyltrimethylammonium chloride and dodecane oxide.

[0086] Comparative Example 2

[0087] This comparative example is used to illustrate the role of phenylboronic acid-functionalized polyethylene glycol. The steps are basically the same as those in Example 3, except that in step S3, when preparing the composite precipitation inducer, phenylboronic acid-functionalized polyethylene glycol is not used, but instead an equal mass of ordinary polyethylene glycol 6000 is used.

[0088] Comparative Example 3

[0089] This comparative example is used to illustrate the role of the polysaccharide refining step. The steps are basically the same as those in Example 3, except that in step S2, after obtaining the flocculated wet cake containing polysaccharides, it is not washed with dilute hydrochloric acid and purified by ethanol precipitation. Instead, the wet cake is directly dried at 60°C to obtain crude polysaccharide.

[0090] Comparative Example 4

[0091] This comparative example is used to illustrate the role of the phycoerythrin purification step. The steps are basically the same as those in Example 3, except that in step S3, after obtaining the phycoerythrin precipitate, it is not dissolved or subjected to tangential flow ultrafiltration. Instead, the precipitate is directly washed twice with water and then freeze-dried to obtain crude phycoerythrin.

[0092] Comparative Example 5

[0093] This comparative example illustrates the synergy of the overall process sequence. Its steps are essentially the same as in Example 3, except that the order of steps is changed: phycoerythrin is precipitated first, followed by deep flocculation of polysaccharides. Specifically, the composite precipitation inducer from Example 3 is first added to the pretreated aqueous extract to precipitate phycoerythrin. The supernatant after separation is then added to the hydrophobic flocculant from Example 3 for polysaccharide flocculation. The purification methods for each of these two steps are the same as in Example 3.

[0094] Performance Test Results and Analysis

[0095] To evaluate the effectiveness of the present invention, the products obtained from the three embodiments and five comparative examples were tested. The yield of the polysaccharide product was determined by the phenol-sulfuric acid method and calculated as a percentage of the total soluble polysaccharides in the algal residue; the protein residue in the polysaccharide product was determined by the Coomassie brilliant blue method. The yield of the phycoerythrin product was calculated by measuring the absorbance of the extract and the final product at 565 nm; the purity of phycoerythrin was determined by a UV-Vis spectrophotometer and expressed as the A565 / A280 ratio; the activity of phycoerythrin was determined by measuring its fluorescence intensity by a fluorescence spectrophotometer, and the fluorescence intensity retention rate was expressed as the ratio of the fluorescence intensity of phycoerythrin in the original aqueous extract without any precipitation treatment. The specific test results are shown in Table 1.

[0096] As shown in Table 1, Examples 1 to 3 consistently yielded high polysaccharide and phycoerythrin yields while maintaining high purity and activity of the final product. The results of the three examples under different parameter combinations were similar, indicating that the method of the present invention has good robustness and reproducibility within the provided parameter range.

[0097] The low protein residue data of the polysaccharide products confirms the effectiveness of the hydrophobic flocculant combined with subsequent acid washing and alcohol precipitation purification steps. Comparative Example 1, using ordinary chitosan, showed a significant decrease in polysaccharide yield and a substantial increase in protein residue to 8.6%. This directly demonstrates that the synergistic effect of the quaternary ammonium salt groups and hydrophobic long chains in the self-made hydrophobic flocculant plays a crucial role in improving polysaccharide capture selectivity and reducing phycoerythrin entrainment. Comparative Example 3 omitted the purification step; although the polysaccharide yield was slightly higher, the protein residue was as high as 15.3%, and the product had a dark color and many impurities, failing to meet application requirements. This highlights the necessity of the purification step of washing with dilute hydrochloric acid to remove bound proteins and purifying by ethanol precipitation and solidification.

[0098] Regarding phycoerythrin, all three examples achieved purity ratios higher than 4.0 and fluorescence intensity retention rates exceeding 93%. In Comparative Example 2, replacing phenylboronic acid-functionalized polyethylene glycol with ordinary polyethylene glycol significantly reduced the phycoerythrin yield and purity ratio, and decreased fluorescence activity. This confirms that the molecular recognition capability provided by the reversible covalent bonding of phenylboronic acid groups is indispensable for achieving highly selective and active precipitation of phycoerythrin. Comparative Example 4 omitted tangential flow ultrafiltration purification; although the yield was acceptable, the purity ratio dropped to 3.0, and fluorescence activity was significantly reduced. This indicates that this step is crucial for removing impurities such as salts and free phenylboronic acid-functionalized polyethylene glycol, thereby improving purity and protecting activity.

[0099] Comparative Example 5, with a modified process sequence, showed significantly worse performance than Example 3 across all indicators. This demonstrates the rationality of the sequence of first flocculating polysaccharides and then precipitating phycoerythrin. Removing a large amount of polysaccharides first effectively reduces the complexity of the subsequent system and avoids interference from polysaccharides on the specific precipitation of phycoerythrin. Conversely, if phycoerythrin is precipitated first, the high concentration of polysaccharide colloids will severely interfere with the precipitation process, leading to a decrease in the separation efficiency of both products. This proves the synergistic advantages of the overall process design of this invention.

[0100] Table 1 Analysis of test results:

[0101] project Polysaccharide yield (%) Polysaccharide-protein residue (%) Phycoerythrin yield (%) Phycoerythrin purity (A565 / A280) Fluorescence intensity retention rate (%) Example 1 88.2 2.1 85.7 4.3 94.5 Example 2 86.5 2.4 84.9 4.1 93.8 Example 3 87.8 2.0 86.1 4.4 95.1 Comparative Example 1 72.3 8.6 78.5 3.1 88.2 Comparative Example 2 86.0 2.2 70.4 2.9 81.5 Comparative Example 3 90.1 15.3 85.0 4.2 93.0 Comparative Example 4 87.5 2.1 83.3 3.0 75.6 Comparative Example 5 79.6 7.8 76.2 3.3 85.4

[0102] Test results show that the present invention has constructed a sequential separation process by using a self-made hydrophobic flocculant and a composite precipitation inducer, which enables the simultaneous acquisition of high-purity polysaccharide products and highly active phycoerythrin concentrate from the water extract of degreased algal residue of Haematococcus pluvialis.

[0103] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue from Haematococcus pluvialis, characterized in that, The method includes the following steps: S1. Pretreatment: The defatted algal residue of *Hylocereus pluvialis* is subjected to water extraction treatment, and the pH value of the resulting water extract of defatted algal residue of *Hylocereus pluvialis* is adjusted to 5.0-5.8; S2. Deep flocculation and primary separation of polysaccharides: A hydrophobic flocculant is added to the aqueous extract of degreased algal residue obtained in S1. After flocculation reaction at 20-30℃, solid-liquid separation is performed to obtain a flocculated wet cake containing polysaccharides and a first supernatant. The flocculated wet cake containing polysaccharides is then refined to obtain a solid polysaccharide product. S3. Specific precipitation of phycoerythrin: A composite precipitation inducer containing phenylboronic acid-functionalized polyethylene glycol and ammonium sulfate is added to the first supernatant obtained in S2. The mixture is allowed to stand at 2-8°C to induce precipitation, followed by solid-liquid separation to obtain phycoerythrin precipitate. The phycoerythrin precipitate is then purified to obtain phycoerythrin concentrate. In step S2, the step of refining the polysaccharide-containing flocculent wet cake is as follows: the polysaccharide-containing flocculent wet cake obtained by solid-liquid separation is stirred and washed with a dilute hydrochloric acid solution with a pH of 2.5-3.5, the solid is collected after centrifugation, and the solid is dispersed in a 60%-75% ethanol aqueous solution, stirred, centrifuged, and dried to obtain a solid polysaccharide product. In step S2, the hydrophobic flocculant is prepared by the following steps: chitosan is dissolved in an acetic acid solution with a mass fraction of 1%-2%, and glycidyltrimethylammonium chloride and dodecane oxide are added successively under nitrogen protection and at 60-70°C for 8-12 hours. The pH value is maintained at 8.5-9.5 throughout the reaction. After the reaction is completed, the product is precipitated, washed and dried to obtain the hydrophobic flocculant. In step S3, the purification of the phycoerythrin precipitate is as follows: the obtained phycoerythrin precipitate is dissolved in a buffer solution with a pH of 7.0-7.5 and containing 0.1-0.5M sorbitol; the resulting solution is subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30-50 kDa; desalting and concentration are performed to obtain a concentrated phycoerythrin solution.

2. The method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of *Haemaphysalis* according to claim 1, characterized in that, In step S1, the water extraction process includes the following steps: the astaxanthin-extracted defatted algal residue of *Hydnocarpus erythropoietin* is added to a phosphate buffer solution with a pH of 6.5-7.0 at a material-to-liquid ratio of 1:20 to 1:30 (g / mL), and the mixture is stirred and extracted at 60-70℃ for 1.5-2.5 hours. The supernatant is then centrifuged and collected as the water extract of the defatted algal residue of *Hydnocarpus erythropoietin*.

3. The method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of Haematococcus pluvialis according to claim 1, characterized in that, The amount of dilute hydrochloric acid solution used is 3-5 times the volume of the polysaccharide-containing flocculent wet cake, and the washing time is 10-20 minutes; the amount of ethanol aqueous solution used is 8-12 times the volume of the solid mass after washing.

4. The method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of Haematococcus pluvialis according to claim 1, characterized in that, The molar ratio of glycidyltrimethylammonium chloride to the amino group in chitosan is 1.0-1.2:1, and the amount of dodecyl oxide added is 0.2 to 0.3 times the molar amount of the amino group in chitosan.

5. The method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of Haematococcus pluvialis according to claim 1, characterized in that, In step S3, the phenylboronic acid-functionalized polyethylene glycol is prepared by the following steps: 3-carboxyphenylboronic acid pinacol ester is dissolved in an anhydrous solvent and reacted with thionyl chloride at 0-30°C for 2-5 hours in the presence of a catalytic amount of N,N-dimethylformamide; the resulting acyl chloride intermediate solution is added to an anhydrous dichloromethane solution of monomethoxy polyethylene glycol in the presence of an ice bath and triethylamine, and the reaction is continued at 25-35°C for 6-12 hours; after the reaction is completed, the product is precipitated, washed, and dried to obtain phenylboronic acid-functionalized polyethylene glycol.

6. The method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of Haematococcus pluvialis according to claim 5, characterized in that, The molar ratio of the hydroxyl terminus of the 3-carboxyphenylboronic acid pinacol ester to monomethoxy polyethylene glycol is 1.10-1.25:

1.

7. The method for sequentially separating polysaccharides and phycoerythrin from the aqueous extract of defatted algal residue of Haematococcus pluvialis according to claim 1, characterized in that, In step S3, the composite precipitation inducer is added as follows: first, phenylboronic acid functionalized polyethylene glycol is added to the first supernatant to make its final mass concentration reach 5%-8%, and then ammonium sulfate is added under stirring to make the saturation of ammonium sulfate in the system reach 52%-60%.