Preparation method of algal cell wall material and method and application of purifying kelp polyphenols by algal cell wall

Abstract

The application discloses a kind of algal cell wall material preparation method and algal cell wall purifies kelp polyphenol method and application, algal cell wall purifies kelp polyphenol method is as follows: preparation algal cell wall material;Kelp polyphenol crude extract is dissolved in buffer to form mixed solution, adjust the pH value of mixed solution to 4.0~6.0, the total phenol concentration in mixed solution is 100~500mg / L;Algal cell wall material is mixed with mixed solution, keep oscillation, adsorb certain time;After the mixture is filtered, collect algal cell wall residue, algal cell wall residue is mixed with 50% concentration ethanol aqueous solution, keep oscillation, desorption certain time;After the mixture is filtered, collect eluent, evaporate and concentrate, freeze-drying, obtain kelp polyphenol.The application uses algal cell wall to adsorb and desorb kelp polyphenol, realizes kelp polyphenol separation and extraction, realizes high-value utilization of algal residue and kelp polyphenol, simple operation, high efficiency.

Description

Technical Field

[0001] This invention belongs to the field of phenolic substance separation and purification technology, specifically relating to a method for preparing algal cell wall materials and a method for purifying kelp polyphenols using algal cell walls, as well as their applications. Background Technology

[0002] Kelp is a large edible seaweed. Studies have shown that kelp contains a variety of effective components such as polyphenols, polysaccharides, and active iodine. Among them, kelp polyphenols, as an important class of polyphenol compounds in brown algae kelp, mainly exist in polymer form with phloroglucinol as the structural unit. Its unique structure endows kelp polyphenols with a variety of biological activities, including antioxidant, antitumor, and antibacterial effects. Therefore, kelp polyphenols are receiving increasing attention.

[0003] Currently, extraction methods for kelp polyphenols include solvent extraction, ultrasonic extraction, and microwave-assisted extraction. Among these, ultrasonic-assisted ethanol extraction is the most common. This method uses ultrasonic oscillation to loosen the kelp structure, releasing polyphenols and improving extraction efficiency. However, current extraction methods are primarily designed for raw kelp, resulting in low effective polyphenol concentrations, requiring the addition of numerous reagents, and producing extracts with low polyphenol purity. Therefore, the extraction process for kelp polyphenols needs further improvement. This invention establishes a kelp polyphenol adsorption technology based on algal cell walls, enabling high-value utilization of algal cell walls, reducing waste from seaweed extraction residues, and minimizing the use of chemical reagents. Simultaneously, it allows for the development of dietary fiber products based on seaweed polyphenols.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing algal cell wall materials and a method for purifying kelp polyphenols using algal cell walls, as well as their applications. The method in this invention can effectively purify kelp polyphenols with high purification efficiency.

[0006] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0007] A method for preparing algal cell wall material includes the following steps:

[0008] Step 1: Mix algae powder with 48-52% ethanol aqueous solution at a ratio of 1:(28-32)g / mL, boil, filter, and collect the residue;

[0009] Step 2: Mix the residue with a 58-63% ethanol aqueous solution at a ratio of 1:(28-32)g / mL, centrifuge, and collect the residue;

[0010] Step 3: Repeat steps 1 and 2 until no soluble sugars can be detected in the supernatant;

[0011] Step 4: Suspend the residue obtained in Step 3 in a 58-63% ethanol aqueous solution and centrifuge, and repeat this step several times.

[0012] Step 5: The residue obtained in step 4 is suspended in acetone solution at a ratio of 1:(4-6) g / mL, centrifuged and dried to obtain algal cell wall material.

[0013] In one or more embodiments of the present invention, the algae powder is selected from kelp powder, nori powder and wakame powder.

[0014] Another specific embodiment of the present invention provides the following technical solution:

[0015] A method for purifying kelp polyphenols from algal cell walls includes the following steps:

[0016] S1. Adsorption of kelp polyphenols: Dissolve crude kelp polyphenol extract in buffer solution to prepare a mixture, adjust the pH of the mixture to 4.0-6.0, and the total phenol concentration in the mixture to 100-500 mg / L; mix algal cell wall material with the mixture, keep shaking, and adsorb for a certain period of time.

[0017] S2. Desorption of kelp polyphenols: Filter the adsorbed mixture, collect the algal cell wall residue, mix the algal cell wall residue with an ethanol aqueous solution with a concentration of 40-70%, keep shaking, and desorb for a certain period of time.

[0018] S3. Collect kelp polyphenols: Filter the desorbed mixture, collect the eluent, evaporate and concentrate it, and freeze-dry it to obtain purified kelp polyphenols.

[0019] In one or more embodiments of the present invention, in step S1, the algal cell wall material and the mixture are mixed at a ratio of (9-11):1 g / L.

[0020] In one or more embodiments of the present invention, in step S1, the buffer solution is citrate-disodium hydrogen phosphate buffer.

[0021] In one or more embodiments of the present invention, in step S1, the oscillation is performed at a speed of 130 to 160 rpm for 10 to 60 minutes.

[0022] In one or more embodiments of the present invention, in step S2, algal cell wall residues are mixed with an aqueous ethanol solution at a ratio of (18-22):1 g / L.

[0023] In one or more embodiments of the present invention, in step S2, the oscillation is performed at a speed of 130 to 160 rpm for 5 to 10 minutes.

[0024] In one or more embodiments of the present invention, in step S3, the evaporation and concentration temperature is 42-50°C.

[0025] Another specific embodiment of the present invention provides the following technical solution:

[0026] The algal cell wall material prepared by the above-mentioned method is used in food processing by adsorbing kelp polyphenols into the algal cell wall material.

[0027] Compared with existing technologies, this invention utilizes the cell walls of algae to adsorb and desorb kelp polyphenols, while optimizing the adsorption and desorption conditions. By limiting the solution pH, polyphenol concentration, and ethanol concentration of the eluent, kelp polyphenols with high purity can be obtained. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 These are scanning electron microscope (SEM) results of the kelp cell wall material in this invention at different magnifications.

[0030] Figure 2 These are scanning electron microscope (SEM) results of the laver cell wall material in this invention at different magnifications.

[0031] Figure 3 These are scanning electron microscope (SEM) results of the Undaria pinnatifida cell wall material at different magnifications in this invention.

[0032] Figure 4 The images show the infrared scan spectra of the kelp cell wall material before and after adsorption in this invention.

[0033] Figure 5 The images show the infrared scan spectra of the laver cell wall material before and after adsorption in this invention.

[0034] Figure 6 The images show the infrared scan spectra of the *Undaria pinnatifida* cell wall material before and after adsorption in this invention.

[0035] Figure 7 This is a comparison chart of the adsorption performance of different adsorbent materials for kelp polyphenols in Examples 1-6 of the present invention;

[0036] Figure 8This is a comparison diagram of the adsorption capacity of kelp cell wall materials for kelp polyphenols under different ionic intensities in Examples 7-16 of the present invention and Examples 1 and 4.

[0037] Figure 9 This is a comparison diagram of the adsorption capacity of the laver cell wall for kelp polyphenols under different ionic intensities in Examples 17-26 and Examples 2 and 5 of the present invention.

[0038] Figure 10 This is a comparison of the adsorption capacity of the Wakame seaweed cell wall material for kelp polyphenols under different ionic intensities in Examples 27-36 and Examples 3 and 6 of the present invention.

[0039] Figure 11 The effect of polyphenol solutions of different concentrations at an ionic strength of 0.1 mol / L on the adsorption capacity of kelp polyphenols is shown in Examples 1-6 and Examples 37-60 of this invention.

[0040] Figure 12 The effect of different concentrations of polyphenol solutions at an ionic strength of 0.5 mol / L on the adsorption capacity of kelp polyphenols is shown in Examples 1-6 and Examples 37-60 of this invention.

[0041] Figure 13 This is a comparison diagram of the sample states before and after adsorption in this invention. Detailed Implementation

[0042] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0043] In algae processing, the focus is mainly on the extraction and utilization of intracellular substances. However, the algal cell walls, after extraction, often fail to achieve high-value utilization, resulting in resource waste. The main components of algal cell walls include cellulose, pectin, hemicellulose, and lignin, which can serve as excellent adsorption carriers for polyphenols. This invention utilizes algal cell wall materials for the extraction and enrichment of kelp polyphenols, and also has significant potential value in the development of polyphenol-based functional foods.

[0044] The crude kelp polyphenol extract used in this invention is a commercially available product with a polyphenol purity of 19.68%. The relevant parameters in this invention are calculated as follows:

[0045] 1. The method for determining the total polyphenol content in the analyte is as follows:

[0046] Gallic acid standard solutions of varying concentrations were used to plot a standard curve based on absorbance measurements, i.e., Y = 0.00481X + 0.07384(R). 2 =0.99257), and then calculate the total polyphenol content based on the measured absorbance of the analyte and the standard curve.

[0047] 2. Adsorption capacity q A The calculation method is as follows:

[0048]

[0049] In the formula: C A,L,0 The initial concentration (mg / L) of total phenols in the polyphenol solution during adsorption is indicated; C A,L V represents the total phenol concentration (mg / L) in the polyphenol solution during the adsorption process; A The volume of the solution during the adsorption process is represented in mL; m represents the mass of the cell wall material during the experiment (g).

[0050] 3. Desorption capacity q D Desorption rate Q D The calculation method is as follows:

[0051]

[0052] In the formula: C D,L This indicates the total phenol concentration (mg / L) in the eluent during desorption; C D,L,0 V represents the initial concentration (mg / L) of total phenolic substances in the eluent during desorption; D q represents the solution volume (mL) during the desorption process; D q represents the mass (mg / g) of phenolic substances desorbed from the cell wall material after adsorption; A This indicates the mass (mg / g) of phenolic substances adsorbed by the cell wall material.

[0053] 4. The ionic strength of the solution during adsorption is adjusted by adding NaCl. The calculation method for ionic strength I is as follows:

[0054]

[0055] In the formula, Σ represents the summation function symbol; C i Z represents the molar concentration of ion "i". i It represents the charge number carried by the ion "i".

[0056] 5. The method for detecting soluble sugars in the supernatant is as follows:

[0057] Take 1 mL of the supernatant, add 2.5 mL of 6 mol / L HCl solution, then dilute with distilled water to approximately twice the original volume. Heat in a 70°C water bath, then add 2 mL of 5 mol / L NaOH solution and heat to 100°C in the water bath. After the sample in the test tube shows a color reaction, measure its absorbance at a wavelength of 540 nm. Calculate the soluble sugar content based on the glucose standard curve obtained. The glucose standard curve was plotted using conventional methods.

[0058] A specific embodiment of the present invention provides a method for preparing algal cell wall material, comprising:

[0059] Algal powder was mixed with a 48–52% ethanol aqueous solution at a ratio of 1:(28–32) g / mL. The mixture was then sealed and boiled in an electric thermostatic water bath for 28–30 min. The residue was filtered through eight layers of gauze and collected. The residue was then mixed with a 58–63% ethanol aqueous solution at a ratio of 1:(28–32) g / mL and centrifuged at 9000–10000 rpm for 28–30 min. The residue was collected again. This process was repeated until no soluble sugars were detected in the supernatant. The residue was then suspended in a 58–63% ethanol aqueous solution and centrifuged at 9000–10000 rpm for 28–30 min. This step was repeated several times to remove residual impurities. The residue was then suspended in an acetone solution at a ratio of 1:(4–6) g / mL to promote the drying process through solvent exchange. Finally, the centrifuged residue was dried at 50–60°C for 11–13 h to obtain algal cell wall material.

[0060] Specifically, the algal powder is selected from laver powder, kelp powder, and wakame powder. Algal cell walls have fibrous chambers mainly formed by cellulose microfibers, and cellulose can act as a carrier for polyphenols. Polyphenols can bind to the cellulose in the cell wall, thus demonstrating that the algal cell wall material can adsorb polyphenols. This invention selects laver powder, kelp powder, and wakame powder to prepare the cell wall material, which can effectively ensure the adsorption of kelp polyphenols by the cell wall material, thereby ensuring the purification effect of kelp polyphenols.

[0061] Another specific embodiment of the present invention provides a method for purifying kelp polyphenols from algal cell walls, including steps 1-3.

[0062] Step 1, adsorption of kelp polyphenols: Dissolve crude kelp polyphenol extract in buffer solution to prepare a mixture, adjust the pH of the mixture to 4.0-6.0, and the total phenol concentration in the mixture to 100-500 mg / L; mix the above algal cell wall material with the mixture, keep shaking, and adsorb for a certain period of time.

[0063] Specifically, in step 1, the buffer solution is a citrate-disodium hydrogen phosphate buffer with a pH of 3.8. The algal cell wall material is mixed with the mixture at a ratio of (9–11):1 g / L, and the pH of the mixture is adjusted to 4.0–6.0 using 1 mol / L HCl solution and 5 mol / L NaOH solution. The pH of the mixture can be adjusted to 4.0, 5.0, or 6.0. Then, it is shaken at 130–160 rpm for 10–60 min. Shaking at a certain speed can promote sufficient contact between the cell wall material and the polyphenols and help the cell wall material adsorb the polyphenols.

[0064] Step 2, desorbing kelp polyphenols: Filter the adsorbed mixture, collect the algal cell wall residue, mix the algal cell wall residue with an ethanol aqueous solution with a concentration of 40-70%, keep shaking, and desorb for a certain period of time.

[0065] Specifically, in step 2, the algal cell wall residue is mixed with an ethanol-water solution at a ratio of (18–22):1 g / L, and shaken at 130–160 rpm for 5–10 minutes. Shaking at this speed promotes the detachment of polyphenols adsorbed on the cell wall, thus increasing the yield of polyphenols. The concentration of the ethanol-water solution can be selected as 40%, 50%, 60%, or 70%.

[0066] Step 3, collecting kelp polyphenols: Filter the desorbed mixture, collect the eluent, evaporate and concentrate it, and freeze-dry it to obtain purified kelp polyphenols.

[0067] Specifically, in step 3, the ethanol is removed by rotary evaporation at 42-50°C. The concentrate is then frozen in a -20°C freezer and then dried in a freeze dryer for 48 hours.

[0068] Another specific embodiment of the present invention provides the application of algal cell wall material prepared by the above-described method in food, specifically, the algal cell wall material is used in food production after adsorbing kelp polyphenols.

[0069] Specifically, current algae processing mainly involves the extraction of active substances from algae. Large-scale production generates a large amount of byproducts such as seaweed residue, primarily composed of algal cell walls. These seaweed residues are a valuable resource that can be utilized through various pathways to maximize resource utilization and achieve sustainable environmental development. Kelp polyphenols possess a variety of biological activities, such as antibacterial, antiviral, antioxidant, and antitumor activities. This invention establishes a seaweed cell wall preparation method and a cell wall-based kelp polyphenol adsorption technology to enhance the nutritional value of algal cell walls. This allows for the development of cell wall polyphenol-based noodle products and multifunctional beverages, providing technical support for the green development and comprehensive utilization of polyphenol-based food ingredients.

[0070] The present invention will be further described in detail below with reference to specific embodiments.

[0071] Example of preparation of kelp cell wall material

[0072] Commercially available kelp was ground into powder. The kelp powder was mixed with a 50% ethanol aqueous solution at a ratio of 1:30 (g / mL). The mixture was sealed and boiled in an electric thermostatic water bath for 30 minutes. The solution was then filtered through eight layers of gauze, and the remaining residue was collected. The residue was mixed with a 60% ethanol aqueous solution at a ratio of 1:30 (g / mL), and the mixture was centrifuged at 10,000 rpm for 30 minutes. The residue was collected, and this process was repeated until no soluble sugars were detected in the supernatant. The residue was then suspended in a 60% ethanol aqueous solution and centrifuged at 10,000 rpm for 30 minutes. This process was repeated several times to thoroughly remove any remaining impurities. The residue was then centrifuged at 10,000 rpm for 30 minutes and collected. The residue was then dried at 55°C for 12 hours to obtain kelp cell wall material. Scanning electron microscopy results of the kelp cell wall material at different magnifications are shown below. Figure 1 As shown.

[0073] Example of preparation of laver cell wall material

[0074] Commercially available laver was ground into laver powder. The laver powder was mixed with a 50% ethanol aqueous solution at a ratio of 1:30 (g / mL). The mixture was sealed and boiled in an electric thermostatic water bath for 30 min. Then, the solution was filtered through eight layers of gauze, and the remaining residue was collected. The residue was mixed with a 60% ethanol aqueous solution at a ratio of 1:30 (g / mL), and the mixture was centrifuged at 10,000 rpm for 30 min. The residue was collected, and this process was repeated until no soluble sugars were detected in the supernatant. Then, the residue was suspended in a 60% ethanol aqueous solution and centrifuged at 10,000 rpm for 30 min. This process was repeated several times to thoroughly remove residual impurities. Then, the residue was centrifuged at 10,000 rpm for 30 min, collected, and dried at 55℃ for 12 h to obtain laver cell wall material. The scanning electron microscopy results of the laver cell wall material at different magnifications are shown below. Figure 2 As shown.

[0075] Preparation example of wakame cell wall material

[0076] Commercially available wakame seaweed was ground into powder. The wakame powder was mixed with a 50% ethanol aqueous solution at a ratio of 1:30 (g / mL). The mixture was sealed and boiled in a thermostatic water bath for 30 min. The solution was then filtered through eight layers of gauze, and the remaining residue was collected. The residue was mixed with a 60% ethanol aqueous solution at a ratio of 1:30 (g / mL), and the mixture was centrifuged at 10,000 rpm for 30 min. The residue was collected, and this process was repeated until no soluble sugars were detected in the supernatant. The residue was then suspended in a 60% ethanol aqueous solution and centrifuged at 10,000 rpm for 30 min. This process was repeated several times to thoroughly remove any remaining impurities. The residue was then centrifuged at 10,000 rpm for 30 min, collected, and dried at 55°C for 12 h to obtain wakame cell wall material. Scanning electron microscopy results of the wakame cell wall material at different magnifications are shown below. Figure 3 As shown.

[0077] Example 1

[0078] Crude kelp polyphenol extract was dissolved in a citrate-disodium hydrogen phosphate buffer solution at pH 3.8, resulting in a total phenol concentration of 400 mg / L. The pH of the mixture was adjusted to 4.0. Kelp cell wall material was added to the crude kelp polyphenol extract solution at a ratio of 10:1 g / L. The Erlenmeyer flask containing the mixture was placed in a constant-temperature shaking incubator at 25°C and 150 rpm for 60 min for adsorption. An ionic strength of 0.1 mol / L was used during the adsorption process, and the ionic strength was adjusted using NaCl.

[0079] After the polyphenol adsorption was complete, the resulting mixture was collected, filtered, and the separated algal cell wall residue was collected. The cell wall residue was mixed with a 50% ethanol aqueous solution at a ratio of 20:1 g / L and placed in an Erlenmeyer flask. The mixture was then placed in a constant temperature shaking incubator at 25°C and 150 rpm for desorption for 10 min.

[0080] After desorption, the resulting cell wall-elution mixture was filtered, the eluent was collected, and concentrated by rotary evaporation at 45°C to remove ethanol. The concentrate was then frozen at -20°C and subsequently dried in a freeze dryer for 48 hours to obtain purified kelp polyphenols.

[0081] Example 2

[0082] Crude kelp polyphenol extract was dissolved in a citrate-disodium hydrogen phosphate buffer solution at pH 3.8, resulting in a total phenol concentration of 400 mg / L. The pH of the mixture was adjusted to 4.0. Porphyra cell wall material was added to the crude kelp polyphenol extract solution at a ratio of 10:1 g / L. The Erlenmeyer flask containing the mixture was placed in a constant-temperature shaking incubator at 25°C and 150 rpm for 60 min for adsorption. An ionic strength of 0.1 mol / L was used during adsorption, and the ionic strength was adjusted using NaCl.

[0083] After the polyphenol adsorption was completed, the adsorption mixture was collected, filtered, and the separated algal cell wall residue was collected. The cell wall residue was mixed with a 50% ethanol aqueous solution at a ratio of 20:1 g / L and placed in an Erlenmeyer flask. The mixture was then placed in a constant temperature shaking incubator at 25°C and 150 rpm for desorption for 10 min.

[0084] After desorption, the resulting cell wall-elution mixture was filtered, the eluent was collected, and concentrated by rotary evaporation at 45°C to remove ethanol. The concentrate was then frozen at -20°C and subsequently dried in a freeze dryer for 48 hours to obtain purified kelp polyphenols.

[0085] Example 3

[0086] Crude kelp polyphenol extract was dissolved in a citrate-disodium hydrogen phosphate buffer solution at pH 3.8, resulting in a total phenol concentration of 400 mg / L. The pH of the mixture was adjusted to 4.0. Wakame seaweed cell wall material was added to the crude kelp polyphenol extract solution at a ratio of 10:1 g / L. The Erlenmeyer flask containing the mixture was placed in a constant-temperature shaking incubator at 25°C and 150 rpm for 60 min for adsorption. An ionic strength of 0.1 mol / L was used during adsorption, and the ionic strength was adjusted using NaCl.

[0087] After the polyphenol adsorption was complete, the resulting mixture was collected and filtered through eight layers of gauze to collect and separate algal cell wall residues. The cell wall residues were mixed with a 50% ethanol aqueous solution at a ratio of 20:1 g / L and placed in an Erlenmeyer flask. The mixture was then placed in a constant temperature shaking incubator at 25°C and 150 rpm for desorption for 10 min.

[0088] After desorption, the resulting cell wall-elution mixture was filtered, the eluent was collected, and concentrated by rotary evaporation at 45°C to remove ethanol. The concentrate was then frozen at -20°C and subsequently dried in a freeze dryer for 48 hours to obtain purified kelp polyphenols.

[0089] Examples 4-6

[0090] The difference between Examples 4-6 and Examples 1-3 is that the ionic strength is 0.5 mol / L. The adsorption capacity and desorption rate of algal cell walls under different ionic strengths are shown in Table 1 below.

[0091] Table 1. Adsorption capacity and desorption rate of cell walls of three algae under different ionic strengths.

[0092]

[0093] Examples 7-16

[0094] The difference between Examples 7-16 and Examples 1 and 4 is that different ionic strengths and adsorption times were used, as shown in Table 2:

[0095]

[0096]

[0097] Examples 17-26

[0098] The difference between Examples 17-26 and Examples 2 and 5 lies in the use of different ionic strengths and adsorption times, as detailed in Table 2.

[0099]

[0100]

[0101] Examples 27-26

[0102] The difference between Examples 27-26 and Examples 3 and 6 is that different ionic strengths and adsorption times were used, as shown in Table 2:

[0103] Example 3 wakame cell wall material 0.1 60 Example 27 wakame cell wall material 0.1 10 Example 28 wakame cell wall material 0.1 20 Example 29 wakame cell wall material 0.1 30 Example 30 wakame cell wall material 0.1 40 Example 31 wakame cell wall material 0.1 50 Example 6 wakame cell wall material 0.5 60 Example 32 wakame cell wall material 0.5 10 Example 33 wakame cell wall material 0.5 20 Example 34 wakame cell wall material 0.5 30 Example 35 wakame cell wall material 0.5 40 Example 36 wakame cell wall material 0.5 50

[0104] Example 37

[0105] The only difference between this embodiment and Example 1 is that the total phenol concentration is 100 mg / L.

[0106] Example 38

[0107] The only difference between this embodiment and Example 1 is that the total phenol concentration is 200 mg / L.

[0108] Example 39

[0109] The only difference between this embodiment and Example 1 is that the total phenol concentration is 300 mg / L.

[0110] Example 40

[0111] The only difference between this embodiment and Example 1 is that the total phenol concentration is 500 mg / L.

[0112] Example 41

[0113] The only difference between this embodiment and Example 2 is that the total phenol concentration is 100 mg / L.

[0114] Example 42

[0115] The only difference between this embodiment and Example 2 is that the total phenol concentration is 200 mg / L.

[0116] Example 43

[0117] The only difference between this embodiment and Example 2 is that the total phenol concentration is 300 mg / L.

[0118] Example 44

[0119] The only difference between this embodiment and Example 2 is that the total phenol concentration is 500 mg / L.

[0120] Example 45

[0121] The only difference between this embodiment and Example 3 is that the total phenol concentration is 100 mg / L.

[0122] Example 46

[0123] The only difference between this embodiment and Example 3 is that the total phenol concentration is 200 mg / L.

[0124] Example 47

[0125] The only difference between this embodiment and Example 3 is that the total phenol concentration is 300 mg / L.

[0126] Example 48

[0127] The only difference between this embodiment and Example 3 is that the total phenol concentration is 500 mg / L.

[0128] Example 49

[0129] The only difference between this embodiment and Example 4 is that the total phenol concentration is 100 mg / L.

[0130] Example 50

[0131] The only difference between this embodiment and Example 4 is that the total phenol concentration is 200 mg / L.

[0132] Example 51

[0133] The only difference between this embodiment and Example 4 is that the total phenol concentration is 300 mg / L.

[0134] Example 52

[0135] The only difference between this embodiment and Example 4 is that the total phenol concentration is 500 mg / L.

[0136] Example 53

[0137] The only difference between this embodiment and Example 5 is that the total phenol concentration is 100 mg / L.

[0138] Example 54

[0139] The only difference between this embodiment and Example 5 is that the total phenol concentration is 200 mg / L.

[0140] Example 55

[0141] The only difference between this embodiment and Example 5 is that the total phenol concentration is 300 mg / L.

[0142] Example 56

[0143] The only difference between this embodiment and Example 5 is that the total phenol concentration is 500 mg / L.

[0144] Example 57

[0145] The only difference between this embodiment and Example 6 is that the total phenol concentration is 100 mg / L.

[0146] Example 58

[0147] The only difference between this embodiment and Example 6 is that the total phenol concentration is 200 mg / L.

[0148] Example 59

[0149] The only difference between this embodiment and Example 6 is that the total phenol concentration is 300 mg / L.

[0150] Example 60

[0151] The only difference between this embodiment and Example 6 is that the total phenol concentration is 500 mg / L.

[0152] In this invention, when polyphenol adsorption is performed with an ionic strength of 0.1 mol / L, the average particle size of the kelp cell wall material used is 1305 nm, the average particle size of the nori cell wall material is 1475 nm, and the average particle size of the wakame cell wall material is 1748 nm; when polyphenol adsorption is performed with an ionic strength of 0.5 mol / L, the average particle size of the kelp cell wall material used is 1861 nm, the average particle size of the nori cell wall material is 4373 nm, and the average particle size of the wakame cell wall material is 2782 nm.

[0153] Reference Figure 1 , Figure 2 and Figure 3 It is known that the intracellular substances of the kelp cell wall material, laver cell wall material and wakame cell wall material prepared by the present invention have been completely dissolved in the cell body, and only a small amount of intracellular solutes such as polysaccharides and proteins remain on the cell wall surface.

[0154] Kelp cell walls exhibit a relatively obvious porous structure, while the electron microscopy structure of nori cell walls shows an irregular network structure, and wakame cell walls exhibit a dual structure of both porous and network. Algal cell walls possess fibrous chambers primarily formed by cellulose microfibers. Cellulose can act as a carrier for polyphenols, enabling the adsorption of these substances. The network and porous structures of the kelp, nori, and wakame cell walls selected in this invention help enhance the binding of cellulose with polyphenols, thereby ensuring the adsorption capacity for polyphenols.

[0155] Fourier transform infrared spectroscopy was used to characterize and analyze the kelp cell wall materials before adsorption, the laver cell wall materials, and the wakame cell wall materials after adsorption in Examples 1-6. The chemical composition of the samples was analyzed based on the characteristic absorption peaks of the functional organic groups. The specific infrared scan spectra are shown below. Figure 4-6 As shown, Figure 4-6 The horizontal axis represents wavenumber, and the vertical axis represents transmittance. The main characteristic absorption of alcohols and phenols is the stretching vibration absorption of OH and CO; the stretching vibration of the free radical OH occurs in the range of 3650–3600 cm⁻¹. -1 The absorption peaks are sharp within this range; the intermolecular hydrogen bond stretching vibrations (OH) occur between 3500 and 3200 cm⁻¹. -1 The absorption peak is broad within the range; the CO stretching vibration absorption peak is 1300–1000 cm⁻¹. -1 The out-of-plane bending absorption peak of OH is in the range of 769–659 cm⁻¹. -1 Within the scope. By Figure 4-6 It can be seen that the three cell wall materials are in the range of 1300–1000 cm. -1 The presence of stretching vibrations of CO functional groups within the range indicates that the three cell wall materials contain polyphenols. This demonstrates that polyphenols are selectively adsorbed onto the cell wall surface during the entire adsorption process, proving that all three cell wall materials have the ability to adsorb polyphenols.

[0156] Reference Figure 7Examples 1-6 employed different cell walls to adsorb kelp polyphenols, ultimately yielding adsorption capacities of 13.08 mg / g, 21.91 mg / g, 17.17 mg / g, 13.56 mg / g, 22.87 mg / g, and 17.39 mg / g, respectively; and desorption rates of 89.50%, 71.74%, 74.84%, 89.70%, 71.28%, and 82.74%, respectively. This indicates that the method of the present invention has excellent adsorption and desorption effects on kelp polyphenols, and can obtain kelp polyphenols with high purity.

[0157] Reference Figure 8-10 Furthermore, as shown in Table 1, the adsorption capacity of the cell walls of the three algae is stronger at an ionic strength of 0.5 mol / L than at an ionic strength of 0.1 mol / L. When the adsorption time is 10 min, the adsorption capacity is basically in equilibrium.

[0158] Referring to Table 1, among the three algal cell wall materials, the adsorption capacity of the nori cell wall material was the highest, at 22.87 mg / g at an ionic strength of 0.5 mol / L; the adsorption capacity of the wakame cell wall material was 17.39 mg / g at an ionic strength of 0.5 mol / L; while the adsorption capacity of the kelp cell wall material was relatively low at an ionic strength of 0.5 mol / L, with an adsorption capacity of 13.56 mg / g.

[0159] Furthermore, regarding the desorption capacity of the three algal cell wall materials, Table 1 shows that kelp exhibits the best desorption capacity, with desorption rates reaching 89.50% and 89.70% at ionic strengths of 0.1 mol / L and 0.5 mol / L, respectively. The cell walls of nori and wakame show moderate desorption capacity, with desorption rates of 71.74%, 71.28%, 74.84%, and 82.74% at ionic strengths of 0.1 mol / L and 0.5 mol / L, respectively.

[0160] Reference Figure 11-12 In Examples 1-6 and Examples 37-60, the total polyphenol concentration continuously increased. When the polyphenol concentration was 100 mg / L, its polyphenol adsorption capacity was low, only 4.34 mg / g. Subsequently, its polyphenol adsorption capacity gradually increased with the continuous increase of polyphenol solution concentration, reaching its highest value of 24.90 mg / g when the polyphenol concentration reached 500 mg / L. Analysis suggests that under low concentration conditions, the cell wall had already reached adsorption saturation, resulting in excessively low adsorption capacity; while under high concentration conditions, the contact area between the cell wall and polyphenol per unit surface area increased, leading to increased adsorption. Simultaneously, the number of impurities competing with polyphenol for adsorption sites also increased, causing a gradual decrease in the cell wall adsorption capacity.

[0161] Furthermore, this invention controls the pH value of the crude kelp polyphenol extract solution to 4.0–6.0. ​​As the pH value increases, the polyphenol adsorption capacity and amount adsorbed by the algal cell wall gradually decrease. Analysis suggests this may be because the sample solution is in an acidic condition (pH 4.0–6.0), which effectively maintains the molecular state and is conducive to kelp cell wall adsorption. However, under alkaline conditions (pH 7.0 ≤ 8.0), the molecular state cannot be maintained, hindering kelp cell wall adsorption. Therefore, as the pH value increases, the cell wall's ability to adsorb polyphenols gradually decreases. Thus, this invention preferably controls the pH value of the crude kelp polyphenol extract solution to 4.0–6.0.

[0162] Furthermore, this invention controls the concentration of the ethanol-water solution used for desorbing kelp polyphenols to be 40-70%. As the concentration of the ethanol-water solution increases, the desorption capacity of the cell wall for polyphenols also increases. Moreover, when the concentration of the ethanol-water solution reaches a certain value, the desorption capacity of the cell wall for polyphenols reaches its optimum. Further increases in the concentration of the ethanol-water solution lead to a decrease in the desorption capacity of the cell wall for polyphenols. Analysis suggests that this may be because lower concentrations of ethanol solvent cannot effectively break the hydrogen bonds formed between polyphenols and algal cell walls, thus reducing its desorption capacity. Higher concentrations of ethanol solvent can break the hydrogen bonds formed between polyphenols and algal cell walls, but over time, adsorption saturation is reached, and the ethanol solvent becomes immiscible with the polyphenols within the algal cell walls, resulting in a decrease in the desorption rate. Therefore, 40-70% is selected as the preferred elution concentration for the ethanol eluent.

[0163] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0164] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for purifying kelp polyphenols from algal cell walls, characterized in that, The steps include the following: S1. Adsorption of kelp polyphenols: Dissolve crude kelp polyphenol extract in buffer solution to prepare a mixture, adjust the pH of the mixture to 4.0-6.0, and the total phenol concentration in the mixture to 100-500 mg / L; mix algal cell wall material with the mixture, keep shaking, and adsorb for a certain period of time. S2. Desorption of kelp polyphenols: Filter the adsorbed mixture, collect the algal cell wall residue, mix the algal cell wall residue with an ethanol aqueous solution with a concentration of 40-70%, keep shaking, and desorb for a certain period of time. S3. Collect kelp polyphenols: Filter the desorbed mixture, collect the eluent, evaporate and concentrate it, and freeze-dry it to obtain purified kelp polyphenols; The method for preparing algal cell wall materials includes the following steps: Step 1: Mix the algae powder with a 48-52% ethanol aqueous solution at a ratio of 1:(28-32) g / mL, boil, filter, and collect the residue; the algae powder is selected from kelp powder, nori powder and wakame powder; Step 2: Mix the residue with a 58-63% ethanol aqueous solution at a ratio of 1:(28-32) g / mL, centrifuge, and collect the residue; Step 3: Repeat steps 1 and 2 until no soluble sugars can be detected in the supernatant; Step 4: Suspend the residue obtained in Step 3 in a 58-63% ethanol aqueous solution and centrifuge, and repeat this step several times. Step 5: The residue obtained in step 4 is suspended in acetone solution at a ratio of 1:(4-6) g / mL, centrifuged and dried to obtain algal cell wall material.

2. The method for purifying kelp polyphenols from algal cell walls according to claim 1, characterized in that, In step S1, the algal cell wall material and the mixture are mixed at a ratio of (9-11):1 g / L.

3. The method for purifying kelp polyphenols from algal cell walls according to claim 1, characterized in that, In step S1, the buffer solution is citrate-disodium hydrogen phosphate buffer.

4. The method for purifying kelp polyphenols from algal cell walls according to claim 1, characterized in that, In step S1, oscillate at a speed of 130-160 rpm for 10-60 minutes.

5. The method for purifying kelp polyphenols from algal cell walls according to claim 1, characterized in that, In step S2, algal cell wall residues are mixed with an aqueous ethanol solution at a ratio of (18–22):1 g / L.

6. The method for purifying kelp polyphenols from algal cell walls according to claim 1, characterized in that, In step S2, oscillate at a speed of 130-160 rpm for 5-10 minutes.

7. The method for purifying kelp polyphenols from algal cell walls according to claim 1, characterized in that, In step S3, the evaporation and concentration temperature is 42–50°C.

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