Preparation method of algae cell wall material, method for purifying kelp polyphenol through algae cell wall and application

Abstract

The invention discloses a preparation method of an algae cell wall material, a method for purifying kelp polyphenol through the algae cell wall and application. The method for purifying the kelp polyphenol through the algae cell wall comprises the steps that the algae cell wall material is prepared; the kelp polyphenol crude extract is dissolved in a buffer solution to prepare a mixed solution, the pH value of the mixed solution is adjusted to 4.0-6.0, and the total phenol concentration in the mixed solution is 100-500 mg / L; mixing an algae cell wall material with the mixed solution, keeping oscillation, and adsorbing for a certain time; filtering the adsorbed mixture, collecting algae cell wall residues, mixing the algae cell wall residues with an ethanol water solution with the concentration of 50%, keeping oscillation, and desorbing for a certain time; and filtering the desorbed mixture, collecting the eluent, evaporating and concentrating, and freeze-drying to obtain the kelp polyphenol. According to the method, the kelp polyphenol is adsorbed and desorbed through the algae cell walls, separation and extraction of the kelp polyphenol are achieved, high-value utilization of the algae residues and the kelp polyphenol is achieved, operation is easy, and efficiency is high.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of phenolic substances, and specifically relates to a method for preparing an algae cell wall material and a method and application of purifying kelp polyphenols from an algae cell wall. Background Art

[0002] Kelp is a large edible seaweed. Studies have shown that kelp contains a variety of effective ingredients such as polyphenols, polysaccharides, and active iodine. Among them, kelp polyphenols, as an important type of polyphenol compounds in brown algae kelp, mainly exist in the form of polymers with phloroglucinol as the structural unit. Its unique structure gives kelp polyphenols a variety of biological activities, including antioxidant, anti-tumor, antibacterial, etc. Therefore, kelp polyphenols are receiving more and more widespread attention.

[0003] At present, the extraction methods of kelp polyphenols include solvent extraction, ultrasonic extraction, microwave-assisted extraction, etc., among which ultrasonic-assisted ethanol extraction is the most common, which can loosen the kelp structure by ultrasonic oscillation, release polyphenols, and improve extraction efficiency, but the current extraction method is basically for kelp raw material processing, polyphenol effective concentration is low, more reagents need to be added, and the polyphenol purity in the obtained extract is low, so the extraction process for kelp polyphenols needs to be further improved. The present invention establishes a kelp polyphenol adsorption technology based on algae cell wall, which can realize the high-value utilization of algae cell wall, reduce the waste of seaweed extraction residues, reduce the use of chemical reagents, and develop dietary fiber products based on seaweed polyphenols at the same time.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing an algae cell wall material and a method and application of purifying kelp polyphenols from algae cell walls. The method of the present invention can effectively purify kelp polyphenols with high purification efficiency.

[0006] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0007] A method for preparing an algae cell wall material comprises the following steps:

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

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

[0010] Step 3, repeat steps 1 and 2 until no soluble sugar can be detected in the supernatant;

[0011] Step 4, suspending the residue obtained in step 3 in 58-63% ethanol aqueous solution and centrifuging, and repeating this step several times;

[0012] Step 5, suspending the residue obtained in step 4 in an acetone solution at a ratio of 1: (4-6) g / mL, centrifuging and drying to obtain algae cell wall material.

[0013] In one or more embodiments of the present invention, the algal flour is selected from kelp flour, laver flour and wakame flour.

[0014] Another specific embodiment of the present invention provides a technical solution as follows:

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

[0016] S1. Adsorption of kelp polyphenols: dissolving the crude extract of kelp polyphenols in a buffer solution to prepare a mixed solution, adjusting the pH value of the mixed solution to 4.0-6.0, and the total phenol concentration in the mixed solution to 100-500 mg / L; mixing the algae cell wall material with the mixed solution, maintaining oscillation, and adsorbing for a certain period of time;

[0017] S2, desorption of kelp polyphenols: filtering the adsorbed mixture, collecting algae cell wall residues, mixing the algae cell wall residues with a 40-70% ethanol aqueous solution, maintaining oscillation, and desorption for a certain period of time;

[0018] S3. Collecting kelp polyphenols: filtering the desorbed mixture, collecting the eluate, evaporating and concentrating it, and freeze-drying it to obtain purified kelp polyphenols.

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

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

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

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

[0023] In one or more embodiments of the present invention, in step S2, the mixture is shaken 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 concentration temperature is 42-50°C.

[0025] Another specific embodiment of the present invention provides a technical solution as follows:

[0026] The algae cell wall material prepared by the algae cell wall material preparation method is used in food, and the algae cell wall material is used in food production after absorbing kelp polyphenols.

[0027] Compared with the prior art, the present invention utilizes algae cell walls to adsorb and desorb kelp polyphenols, optimizes the adsorption and desorption conditions, and can obtain kelp polyphenols with higher purity by limiting conditions such as solution pH value, polyphenol concentration and elution solution ethanol concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The scanning electron microscope results of the kelp cell wall material of the present invention at different magnifications;

[0030] Figure 2 The scanning electron microscopy results of the laver cell wall material in the present invention at different magnifications;

[0031] Figure 3 The scanning electron microscope results of the kelp cell wall material of the present invention at different magnifications;

[0032] Figure 4 This is an infrared scanning spectrum of the kelp cell wall material before and after adsorption in the present invention;

[0033] Figure 5 The infrared scanning spectrum of the laver cell wall material before and after adsorption in the present invention;

[0034] Figure 6 This is an infrared scanning spectrum diagram of the kelp cell wall material before and after adsorption;

[0035] Figure 7 A comparison chart of the adsorption performance of kelp polyphenols by different adsorption materials in Examples 1-6 of the present invention;

[0036] Figure 8It is a comparison chart of the adsorption capacity of kelp polyphenols by kelp cell wall materials under different ionic strengths in Examples 7-16 of the present invention and Examples 1 and 4;

[0037] Fig. 9 It is a comparison diagram of the adsorption capacity of laver cell wall to kelp polyphenols under different ionic strengths in Examples 17-26 of the present invention and Examples 2 and 5;

[0038] Fig.10 It is a comparison chart of the adsorption capacity of kelp polyphenols by the undaria pinnatifida cell wall material under different ionic strengths in Examples 27-36 of the present invention and Examples 3 and 6;

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

[0040] Fig.12 The effect of polyphenol solutions of different concentrations on the adsorption of kelp polyphenols at 0.5 mol / L ionic strength in Examples 1-6 and Examples 37-60 of the present invention;

[0041] Fig.13 This is a comparison diagram of the sample states before and after adsorption in the present invention. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0043] During the processing of algae, the main focus is on the extraction and utilization of algae intracellular substances, while the algae cell wall after extraction cannot be used in a high-value manner, resulting in a waste of resources. The main components of the algae cell wall include cellulose, pectin, hemicellulose and lignin, which can be used as a good adsorption carrier for the adsorption of polyphenols. The present invention utilizes algae cell wall materials to extract and enrich kelp polyphenols, and can also have good value in the development of polyphenol functional foods.

[0044] The kelp polyphenol crude extract used in the present invention is a conventional commercial product, and the polyphenol purity is 19.68%; the calculation method of the relevant parameters in the present invention is:

[0045] 1. The method for determining the total polyphenol content in the test object is:

[0046] Using a variety of gallic acid standard solutions of different concentrations, the gallic acid standard curve was obtained by measuring the absorbance, that is, Y = 0.00481X + 0.07384 (R 2 =0.99257), and then the total polyphenol content was calculated based on the measured absorbance of the analyte and the standard curve.

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

[0048]

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

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

[0051]

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

[0053] 4. During the adsorption process, the ionic strength of the solution is adjusted by adding NaCl. The calculation method of the ionic strength I is:

[0054]

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

[0056] 5. The method for detecting soluble sugar in the supernatant is:

[0057] 1 mL of supernatant was taken, and 2.5 mL of 6 mol / L HCl solution was added first, and then distilled water was added to dilute it to about 2 times, and then heated in a 70°C water bath, and 2 mL of 5 mol / L NaOH solution was added again, and heated to 100°C in a water bath, and after the color of the sample in the test tube developed, its absorbance was measured at a wavelength of 540 nm, and its soluble sugar content was calculated according to the glucose standard curve obtained. The glucose standard curve was drawn using conventional methods.

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

[0059] The algae powder is mixed with a 48-52% ethanol aqueous solution at a ratio of 1: (28-32) g / mL, and then the mixture is sealed and boiled in an electric constant temperature water bath for 28-30 minutes; the residue is collected by filtering with eight layers of gauze; the residue is mixed with a 58-63% ethanol aqueous solution at a ratio of 1: (28-32) g / mL, centrifuged at 9000-10000 rpm for 28-30 minutes, and the residue is collected; the above process is repeated until no soluble sugar can be detected in the supernatant. Then, the residue is suspended in a 58-63% ethanol aqueous solution and centrifuged at 9000-10000 rpm for 28-30 minutes, and this step is repeated several times to remove residual impurities. The residue is then suspended in an acetone solution at a ratio of 1: (4-6) g / mL, and the drying process is promoted by solvent exchange. Finally, the residue collected by centrifugation is dried at 50-60°C for 11-13 hours to obtain algae cell wall material.

[0060] Specifically, the algae powder is selected from laver powder, kelp powder and wakame powder. The algae cell wall has a fiber chamber mainly formed by cellulose microfibrils, and cellulose can be used as a carrier of polyphenols. Polyphenols can be combined with cell wall cellulose, which is reflected in that the algae cell wall material can adsorb polyphenols. The present invention selects laver powder, kelp powder and wakame powder to prepare cell wall materials, which can effectively ensure the adsorption of kelp polyphenols by the cell wall materials, and further ensure the purification effect of kelp polyphenols.

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

[0062] Step 1, adsorbing kelp polyphenols: dissolving the crude kelp polyphenol extract in a buffer solution to prepare a mixed solution, adjusting the pH value of the mixed solution to 4.0-6.0, and the total phenol concentration in the mixed solution to 100-500 mg / L; mixing the above algae cell wall material with the mixed solution, maintaining oscillation, and adsorbing for a certain period of time.

[0063] Specifically, in step 1, the buffer is a citric acid-sodium hydrogen phosphate buffer with a pH value of 3.8, the algae cell wall material and the mixed solution are mixed at a ratio of (9-11): 1g / L, and the pH value of the mixed solution is adjusted to 4.0-6.0 using a 1mol / L HCl solution and a 5mol / L NaOH solution. The pH value of the mixed solution can be specifically adjusted to 4.0, 5.0 or 6.0. Then, the mixture is shaken at a speed of 130-160rpm for 10-60min. Oscillation at a certain speed can promote full contact between the cell wall material and the polyphenolic substance and help the cell wall material adsorb the polyphenolic substance.

[0064] Step 2, desorption of kelp polyphenols: filtering the adsorbed mixture, collecting algae cell wall residues, mixing the algae cell wall residues with 40-70% ethanol aqueous solution, maintaining oscillation, and desorption for a certain period of time.

[0065] Specifically, in step 2, the algae cell wall residues and the ethanol aqueous solution are mixed in a ratio of (18-22): 1 g / L, and oscillated at a speed of 130-160 rpm for 5-10 min. Oscillation at a certain speed can promote the detachment of polyphenols adsorbed on the cell wall from the cell wall, which helps to increase the yield of polyphenols. The concentration of the ethanol aqueous solution can be specifically selected to be 40%, 50%, 60% or 70%.

[0066] Step 3, collecting kelp polyphenols: filtering the desorbed mixture, collecting the eluate, evaporating and concentrating it, and freeze-drying it to obtain purified kelp polyphenols.

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

[0068] Another specific embodiment of the present invention provides the use of the algae cell wall material prepared by the above algae cell wall material preparation method in food, specifically, the algae cell wall material is used in food production after absorbing kelp polyphenols.

[0069] Specifically, the current processing of algae is mainly the extraction of algae active substances. Large-scale preparation will produce a large amount of by-products such as seaweed residues, the main component of which is algae cell walls. These seaweed residues are a valuable resource that can be utilized in a variety of ways to achieve maximum resource utilization and sustainable development of the environment. Kelp polyphenols have a variety of biological activities, such as antibacterial and antiviral activity, antioxidant activity, antitumor activity, etc. The present invention improves the nutritional value of algae cell walls by establishing the preparation of seaweed cell walls and the kelp polyphenol adsorption technology based on cell walls, and can develop cell wall polyphenol noodles and multifunctional beverages, providing technical support for the green development and comprehensive utilization of polyphenol-based food raw materials.

[0070] The present invention is further described in detail below in conjunction with specific embodiments.

[0071] Preparation example of kelp cell wall material

[0072] Take conventional commercially available kelp and grind it into kelp powder. Mix the kelp powder with a 50% ethanol aqueous solution at a ratio of 1:30 (g / mL). Seal the mixture and boil it in an electric constant temperature water bath for 30 minutes. Then, filter the solution through eight layers of gauze, and collect the remaining residue. Mix the residue with a 60% ethanol aqueous solution at a ratio of 1:30 (g / mL), and then centrifuge the mixture at 10,000 rpm for 30 minutes, collect the residue, and repeat the process until no soluble sugar can be detected in the supernatant. Then, suspend the residue in a 60% ethanol aqueous solution and centrifuge it at 10,000 rpm for 30 minutes. Repeat the process several times to fully remove the remaining impurities. Then centrifuge at 10,000 rpm for 30 minutes and collect the residue, and dry it at 55°C for 12 hours to obtain the kelp cell wall material. The scanning electron microscopy results of the kelp cell wall material at different magnifications are shown as follows. Figure 1 shown.

[0073] Example of Preparation of Porphyra Cell Wall Material

[0074] Take conventional commercially available laver and grind it into laver powder. Mix the laver powder with a 50% ethanol aqueous solution at a ratio of 1:30 (g / mL). Seal the mixture and boil it in an electric constant temperature water bath for 30 minutes. Then, filter the solution through eight layers of gauze, and collect the remaining residue. Mix the residue with a 60% ethanol aqueous solution at a ratio of 1:30 (g / mL), centrifuge the mixture at 10,000 rpm for 30 minutes, collect the residue, and repeat the process until no soluble sugar can be detected in the supernatant. Then, suspend the residue in a 60% ethanol aqueous solution and centrifuge it at 10,000 rpm for 30 minutes. Repeat the process several times to fully remove the remaining impurities. Then centrifuge at 10,000 rpm for 30 minutes and collect the residue, and dry it at 55°C for 12 hours to obtain the laver cell wall material. The scanning electron microscopy results of the laver cell wall material at different magnifications are shown as follows. Figure 2 shown.

[0075] Preparation example of Undaria pinnatifida cell wall material

[0076] Take conventional commercially available kelp and grind it into kelp powder. Mix the kelp powder with a 50% ethanol aqueous solution at a ratio of 1:30 (g / mL). Seal the mixture and boil it in an electric constant temperature water bath for 30 minutes. Then, filter the solution through eight layers of gauze, and collect the remaining residue. Mix the residue with a 60% ethanol aqueous solution at a ratio of 1:30 (g / mL), and then centrifuge the mixture at 10,000 rpm for 30 minutes, collect the residue, and repeat the process until no soluble sugar can be detected in the supernatant. Then, suspend the residue in a 60% ethanol aqueous solution and centrifuge it at 10,000 rpm for 30 minutes. Repeat the process several times to fully remove the remaining impurities. Then centrifuge at 10,000 rpm for 30 minutes and collect the residue, and dry it at 55°C for 12 hours to obtain the kelp cell wall material. The scanning electron microscopy results of the kelp cell wall material at different magnifications are shown as follows. Figure 3 shown.

[0077] Example 1

[0078] The crude extract of kelp polyphenols was dissolved in a citric acid-sodium hydrogen phosphate buffer solution with a pH of 3.8, the total phenol concentration in the mixed solution was 400 mg / L, and the pH value of the mixed solution was adjusted to 4.0. The kelp cell wall material was added to the crude extract of kelp polyphenols at a ratio of 10:1 g / L, and the conical flask containing the mixture was placed in a constant temperature shaking shaker at 25°C and 150 rpm for adsorption for 60 minutes. During the adsorption process, an ionic strength of 0.1 mol / L was used, and the ionic strength was adjusted by NaCl.

[0079] After the polyphenol adsorption is completed, the adsorption mixture is collected and filtered to collect the separated algae cell wall residues. The cell wall residues are mixed with a 50% ethanol aqueous solution at a ratio of 20:1 g / L and placed in a conical flask, and also placed in a constant temperature shaking shaker at 25°C and 150 rpm for desorption for 10 minutes.

[0080] After desorption, the obtained cell wall-eluate mixture was filtered, and the eluate was collected and concentrated by rotary evaporation at 45°C to remove ethanol. After that, the concentrate was first frozen in a -20°C refrigerator, and then placed in a freeze dryer for 48 hours to obtain purified kelp polyphenols.

[0081] Example 2

[0082] The crude extract of kelp polyphenols was dissolved in a citric acid-sodium hydrogen phosphate buffer solution with a pH of 3.8, the total phenol concentration in the mixed solution was 400 mg / L, and the pH value of the mixed solution was adjusted to 4.0. The laver cell wall material was added to the crude extract of kelp polyphenols at a ratio of 10:1 g / L, and the conical flask containing the mixture was placed in a constant temperature shaking shaker at 25°C and 150 rpm for adsorption for 60 minutes. During the adsorption process, an ionic strength of 0.1 mol / L was used, and the ionic strength was adjusted by NaCl.

[0083] After the polyphenol adsorption is completed, the adsorption mixture is collected and filtered to collect the separated algae cell wall residues. The cell wall residues are mixed with a 50% ethanol aqueous solution at a ratio of 20:1 g / L and placed in a conical flask, and also placed in a constant temperature shaking shaker at 25°C and 150 rpm for desorption for 10 minutes.

[0084] After desorption, the obtained cell wall-eluate mixture was filtered, and the eluate was collected and concentrated by rotary evaporation at 45°C to remove ethanol. After that, the concentrate was first frozen in a -20°C refrigerator, and then placed in a freeze dryer for 48 hours to obtain purified kelp polyphenols.

[0085] Example 3

[0086] The crude extract of kelp polyphenols was dissolved in a citric acid-sodium hydrogen phosphate buffer solution with a pH of 3.8, the total phenol concentration in the mixture was 400 mg / L, and the pH value of the mixture was adjusted to 4.0. The cell wall material of Undaria pinnatifida was added to the crude extract of kelp polyphenols at a ratio of 10:1 g / L, and the conical flask containing the mixture was placed in a constant temperature shaking shaker at 25°C and 150 rpm for adsorption for 60 minutes. During the adsorption process, an ionic strength of 0.1 mol / L was used, and the ionic strength was adjusted by NaCl.

[0087] After the polyphenol adsorption is completed, the adsorption mixture is collected and filtered through eight layers of gauze to collect and separate the algae cell wall residues. The cell wall residues are mixed with a 50% ethanol aqueous solution at a ratio of 20:1 g / L and placed in a conical flask, and also placed in a constant temperature shaking shaker at 25°C and 150rpm for desorption for 10 minutes.

[0088] After desorption, the obtained cell wall-eluate mixture was filtered, and the eluate was collected and concentrated by rotary evaporation at 45°C to remove ethanol. After that, the concentrate was first frozen in a -20°C refrigerator, and then placed in a freeze dryer for 48 hours to obtain purified kelp polyphenols.

[0089] Embodiment 4-6

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

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

[0092]

[0093] Embodiment 7-16

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

[0095]

[0096]

[0097] Examples 17-26

[0098] The difference between Examples 17-26 and Examples 2 and 5 is that different ionic strengths and adsorption times are used, as shown 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 are used, as shown in Table 2:

[0103] Example Algae cell wall materials used Ionic strength (mol / L) Adsorption time (min) Example 3 Undaria pinnatifida cell wall material 0.1 60 Embodiment 27 Undaria pinnatifida cell wall material 0.1 10 Embodiment 28 Undaria pinnatifida cell wall material 0.1 20 Embodiment 29 Undaria pinnatifida cell wall material 0.1 30 Embodiment 30 Undaria pinnatifida cell wall material 0.1 40 Embodiment 31 Undaria pinnatifida cell wall material 0.1 50 Example 6 Undaria pinnatifida cell wall material 0.5 60 Embodiment 32 Undaria pinnatifida cell wall material 0.5 10 Embodiment 33 Undaria pinnatifida cell wall material 0.5 20 Embodiment 34 Undaria pinnatifida cell wall material 0.5 30 Embodiment 35 Undaria pinnatifida cell wall material 0.5 40 Embodiment 36 Undaria pinnatifida cell wall material 0.5 50

[0104] Embodiment 37

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

[0106] Embodiment 38

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

[0108] Embodiment 39

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

[0110] Embodiment 40

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

[0112] Embodiment 41

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

[0114] Embodiment 42

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

[0116] Embodiment 43

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

[0118] Embodiment 44

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

[0120] Embodiment 45

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

[0122] Embodiment 46

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

[0124] Embodiment 47

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

[0126] Embodiment 48

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

[0128] Embodiment 49

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

[0130] Embodiment 50

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

[0132] Embodiment 51

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

[0134] Embodiment 52

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

[0136] Embodiment 53

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

[0138] Embodiment 54

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

[0140] Embodiment 55

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

[0142] Embodiment 56

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

[0144] Embodiment 57

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

[0146] Embodiment 58

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

[0148] Embodiment 59

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

[0150] Embodiment 60

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

[0152] When the polyphenol adsorption is carried out at an ionic strength of 0.1 mol / L in the present invention, the average particle size of the kelp cell wall material is 1305 nm, the average particle size of the laver cell wall material is 1475 nm, and the average particle size of the kelp cell wall material is 1748 nm; when the polyphenol adsorption is carried out at an ionic strength of 0.5 mol / L, the average particle size of the kelp cell wall material is 1861 nm, the average particle size of the laver cell wall material is 4373 nm, and the average particle size of the kelp cell wall material is 2782 nm.

[0153] Reference Figure 1 , Figure 2 and Figure 3 It can be seen that the intracellular substances of the Laminaria cell wall material, Porphyra cell wall material and Undaria pinnatifida cell wall material prepared by the present invention have been completely dissolved out of the cell body, and only a small amount of intracellular solutes such as polysaccharides and proteins remain on the cell wall surface.

[0154] The cell wall of kelp presents a relatively obvious porous structure, the electron microscope structure of the cell wall of laver presents an irregular reticular structure, and the cell wall of wakame presents a dual structure of porous and reticular. The cell wall of algae has a fiber chamber mainly formed by cellulose microfibrils, and cellulose can be used as a carrier of polyphenols and can adsorb polyphenols. The reticular and porous structures of the cell wall of kelp, laver, and wakame selected by the present invention help to improve the combination of cellulose and polyphenols, thereby ensuring the adsorption capacity of polyphenols.

[0155] Fourier transform infrared spectrometer was used to characterize and analyze the kelp cell wall material, laver cell wall material and wakame cell wall material before adsorption and the cell wall material after adsorption in Examples 1-6. The chemical composition of the samples was analyzed according to the characteristic absorption peaks of the functional organic groups of the samples. The specific infrared scanning spectra are shown in FIG. Figure 4-6 As shown, Figure 4-6 The horizontal axis is the wave number, and the vertical axis is the transmittance. The main characteristic absorption of alcohols and phenols is the stretching vibration absorption of OH and CO; the stretching vibration of free radical OH is between 3650 and 3600 cm -1 The absorption peak is sharp in the range of 3500-3200cm-1. -1 The absorption peak is broad in the range; the CO stretching vibration absorption peak is 1300~1000cm -1 OH out-of-plane bending absorption peak ranges from 769 to 659 cm -1 Within the range. Figure 4-6 It can be seen that the three cell wall materials are between 1300 and 1000 cm -1 The stretching vibration of CO functional group appeared in the range, indicating that the three cell wall materials contained polyphenols, which explained that the polyphenols were selectively adsorbed on the cell wall surface during the entire adsorption process, proving that the three cell wall materials all had the ability to adsorb polyphenols.

[0156] Reference Figure 7In Examples 1-6, different cell walls were used to adsorb kelp polyphenols, and the adsorption capacities of kelp polyphenols were 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 the desorption rates were 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 higher purity.

[0157] Reference Figure 8-10 At the same time, combined with Table 1, it can be seen that the adsorption capacity of the three algae cell walls at 0.5 mol / L ionic strength is stronger than that at 0.1 mol / L ionic strength. When the adsorption time is 10 min, the adsorption capacity is basically in a balanced state.

[0158] Referring to Table 1, among the three algae cell wall materials, the adsorption capacity of the laver cell wall material is the highest, which is 22.87 mg / g at an ionic strength of 0.5 mol / L; the adsorption capacity of the kelp cell wall at an ionic strength of 0.5 mol / L is 17.39 mg / g; and the adsorption capacity of the kelp cell wall at an ionic strength of 0.5 mol / L is relatively low, with an adsorption amount of 13.56 mg / g.

[0159] In addition, regarding the desorption capacity of the three algae cell wall materials, it can be seen from Table 1 that the desorption capacity of kelp is the best, and its desorption rate reaches 89.50% and 89.70% at 0.1mol / L and 0.5mol / L ionic strengths, respectively. The desorption capacity of laver cell wall and kelp cell wall is moderate, and its desorption capacity is 71.74%, 71.28%, 74.84% and 82.74% at 0.1mol / L and 0.5mol / L ionic strengths, respectively.

[0160] Reference Figure 11-12 , the total concentration of polyphenols in Examples 1-6 and Examples 37-60 continued to increase. When the polyphenol concentration was 100 mg / L, the polyphenol adsorption capacity was low, only 4.34 mg / g. Then, the polyphenol adsorption capacity gradually increased with the continuous increase in the concentration of the polyphenol solution. When the polyphenol concentration reached 500 mg / L, the polyphenol adsorption capacity was the highest, at 24.90 mg / g. Analysis shows that under low concentration conditions, the cell wall has reached the adsorption saturation state and the adsorption capacity is too low; under high concentration conditions, the contact amount of the cell wall per unit surface area with the polyphenol will increase, and the adsorption amount will increase; at the same time, the impurities competing with the polyphenol for adsorption sites are also increasing, causing the cell wall adsorption capacity to gradually decrease.

[0161] In addition, the present invention controls the pH value of the kelp polyphenol crude extract solution to 4.0-6.0. As the pH value increases, the polyphenol adsorption capacity and adsorption amount of the algae cell wall gradually decrease. Analysis shows that this may be due to the fact that when the pH value of the sample solution is in the range of 4.0-6.0, it is in an acidic condition and can well maintain its molecular state, which is conducive to the adsorption of the kelp cell wall, but under alkaline conditions (7.0≤pH≤8.0), it cannot maintain its molecular state, which is not conducive to the adsorption of the kelp cell wall. Therefore, as the pH value increases, the cell wall's ability to adsorb polyphenols gradually decreases. Therefore, the present invention controls the pH value of the kelp polyphenol crude extract solution to 4.0-6.0 as the preferred pH value.

[0162] In addition, the present invention controls the concentration of the ethanol aqueous solution used for desorbing kelp polyphenols to 40-70%. As the concentration of the ethanol aqueous solution increases, the polyphenol desorption capacity of the cell wall increases continuously, and when the concentration of the ethanol aqueous solution reaches a certain value, the polyphenol desorption capacity of the cell wall reaches the best, and when the concentration of the ethanol aqueous solution is increased again, the polyphenol desorption capacity of the cell wall shows a downward trend. Analysis shows that this may be due to the fact that the ethanol solvent with a lower concentration cannot effectively destroy the hydrogen bond formed between the polyphenol and the algae cell wall, thereby causing its desorption capacity to decrease. The ethanol solvent with a higher concentration can destroy the hydrogen bond formed between the polyphenol and the algae cell wall, but gradually reaches an adsorption saturation state with the change of time, and the ethanol solvent cannot be miscible with the polyphenol substance in the algae cell wall, thereby causing its desorption rate to decrease. Therefore, 40-70% is selected as the preferred elution concentration of 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0164] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing algae cell wall material, characterized in that: The steps include: Step 1, mixing algae powder with 48-52% ethanol aqueous solution at a ratio of 1: (28-32) g / mL, boiling, filtering, and collecting the residue; Step 2, mixing the residue with 58-63% ethanol aqueous solution at a ratio of 1:(28-32) g / mL, centrifuging, and collecting the residue; Step 3, repeat steps 1 and 2 until no soluble sugar can be detected in the supernatant; Step 4, suspending the residue obtained in step 3 in 58-63% ethanol aqueous solution and centrifuging, and repeating this step several times; Step 5, suspending the residue obtained in step 4 in an acetone solution at a ratio of 1: (4-6) g / mL, centrifuging and drying to obtain algae cell wall material.

2. The method for preparing algae cell wall material according to claim 1, characterized in that: The algae powder is selected from kelp powder, laver powder and wakame powder.

3. A method for purifying kelp polyphenols from algae cell walls, characterized in that: The steps include: S1. Adsorption of kelp polyphenols: dissolving the crude extract of kelp polyphenols in a buffer solution to prepare a mixed solution, adjusting the pH value of the mixed solution to 4.0-6.0, and the total phenol concentration in the mixed solution to 100-500 mg / L; mixing the algae cell wall material with the mixed solution, maintaining oscillation, and adsorbing for a certain period of time; S2, desorption of kelp polyphenols: filtering the adsorbed mixture, collecting algae cell wall residues, mixing the algae cell wall residues with a 40-70% ethanol aqueous solution, maintaining oscillation, and desorption for a certain period of time; S3. Collecting kelp polyphenols: filtering the desorbed mixture, collecting the eluate, evaporating and concentrating it, and freeze-drying it to obtain purified kelp polyphenols.

4. The method for purifying kelp polyphenols from algae cell walls according to claim 3, characterized in that: In step S1, the algae cell wall material and the mixed solution are mixed at a ratio of (9-11): 1 g / L.

5. The method for purifying kelp polyphenols from algae cell walls according to claim 3, characterized in that: In step S1, the buffer is citric acid-disodium hydrogen phosphate buffer.

6. The method for purifying kelp polyphenols from algae cell walls according to claim 3, characterized in that: In step S1, the mixture is shaken at a speed of 130 to 160 rpm for 10 to 60 min.

7. The method for purifying kelp polyphenols from algae cell walls according to claim 3, characterized in that: In step S2, the algae cell wall residues are mixed with an ethanol aqueous solution at a ratio of (18-22): 1 g / L.

8. The method for purifying kelp polyphenols from algae cell walls according to claim 3, characterized in that: In step S2, the mixture is shaken at a speed of 130 to 160 rpm for 5 to 10 minutes.

9. The method for purifying kelp polyphenols from algae cell walls according to claim 3, characterized in that: In step S3, the evaporation concentration temperature is 42-50°C.

10. Application of the algae cell wall material prepared by the method for preparing the algae cell wall material according to any one of claims 1 to 2 in food, characterized in that: The algae cell wall material is used in food production after absorbing kelp polyphenols.

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