Phycocyanin composite hydrogel with acid stability and preparation method thereof

Abstract

The invention discloses a phycocyanin composite hydrogel with acid stability and a preparation method thereof, belonging to the technical field of biomaterial preparation. After a phycocyanin aqueous solution, a gelatin solution and a sodium alginate solution are prepared, the solutions are sequentially mixed to obtain a formed gel, and then cross-linking is performed to obtain the phycocyanin composite hydrogel. Gelatin and sodium alginate are used as basic wall materials to protect the phycocyanin and thus improve its acid resistance activity. Adding a gum arabic solution to the initially formed and non-cross-linked gel can further improve the protective effect on the phycocyanin, so that the prepared gel product has good acid stability and mechanical properties. The product after acid treatment still has high antioxidant performance, thereby solving the acid instability problem of phycocyanin, improving the utilization rate of phycocyanin when acting on the human body, and effectively expanding the application scene and scope of phycocyanin.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterial preparation, and particularly relates to a phycocyanin composite hydrogel with acid stability and a preparation method thereof. Background Art

[0002] Phycocyanin is a bioactive compound extracted from algae such as spirulina, cyanobacteria, and Nostoc. It is rich in nutritional value and can provide the human body with the necessary proteins and vitamins. In addition to its efficient light energy capture ability, phycocyanin also has anti-tumor, anti-radiation, antioxidant, anti-inflammatory, neuroprotective, immune system enhancement, and hemolysis inhibition effects. On this basis, phycocyanin also has anti-cancer, blood cell regeneration, ovarian care, and elastin synthesis in the human body. At the beginning of the 21st century, phycocyanin was widely used as a high-grade natural pigment for food and cosmetics in Europe, America, Japan and other countries, and was made into biochemical drugs. Phycocyanin is also used to prepare rehabilitation drugs and nutritional foods for cancer patients, leukemia patients, etc., and has achieved outstanding combined therapeutic effects.

[0003] Although phycocyanin has excellent antioxidant properties and is believed to have good immune-promoting and anti-disease functions, it is unstable to heat, light, and acid. It is stable under weak acidity and neutral conditions (pH = 4.5-8), but will precipitate under acidic conditions (pH = 4.2), making it more prone to deterioration and difficult to efficiently exert its nutritional value. In recent years, there have been few studies on phycocyanin encapsulation systems. Generally speaking, macromolecular delivery systems suitable for protein encapsulation mainly include microcapsule encapsulation, protein polysaccharide delivery system encapsulation, liposome encapsulation, nanocarrier encapsulation, and gel encapsulation. If you want to better apply phycocyanin to the human body, constructing an encapsulation system will help prevent phycocyanin from being consumed excessively in gastric acid, allowing more phycocyanin to be smoothly absorbed by the intestines, thereby improving the utilization rate of phycocyanin.

[0004] Wang Mingchao et al. published an article entitled "Preparation and Research of an Antioxidant Phycocyanin / Collagen Composite Hydrogel", which pointed out that by mixing collagen with different concentrations of phycocyanin, a composite hydrogel can be prepared by utilizing the self-assembly characteristics of collagen. The dual-protein gel system can simultaneously possess the antioxidant and anti-inflammatory properties of phycocyanin and the cell nutrition of collagen; and a series of antioxidant performance tests confirmed the above conclusion. However, it should be noted that the performance of the obtained product after acid treatment was not tested. In other words, it is unclear whether the gel system can maintain the above performance or whether the antioxidant performance can be maintained without significant decrease under acidic conditions. When the gel system encapsulating phycocyanin acts on the human body, due to the presence of gastric acid in the human body, if the gel system does not have a certain acid stability, the product will be consumed excessively in the gastric acid environment, making it difficult to reach the end to play a role. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of poor acid stability of phycocyanin in the prior art, and to provide a phycocyanin composite hydrogel and a preparation method thereof. The sample formed by embedding phycocyanin in a suitable gel system can still maintain a high antioxidant activity after acid treatment, thereby improving the utilization rate of phycocyanin in the human body.

[0006] The present invention is achieved by: a method for preparing a phycocyanin composite hydrogel, comprising the following steps:

[0007] 1) dissolving phycocyanin powder in deionized water and mixing well to obtain a phycocyanin aqueous solution;

[0008] 2) Place gelatin and sodium alginate in deionized water respectively, stir and dissolve at 50°C to obtain gelatin solution and sodium alginate solution, and cool to room temperature;

[0009] 3) adding the phycocyanin aqueous solution obtained in step 1) to the sodium alginate solution obtained in step 2) and stirring evenly;

[0010] 4) adding the gelatin solution obtained in step 2) dropwise to the mixed system obtained in step 3), and stirring to form a gel;

[0011] 5) Adding calcium chloride solution to the gel system obtained in step 4) for cross-linking to obtain phycocyanin composite hydrogel.

[0012] Furthermore, after completing step 4) to obtain the formed gel and before performing step 5), that is, before cross-linking, the gum arabic solution is added dropwise to the formed gel to enhance the antioxidant property of the gel.

[0013] Furthermore, the gum arabic solution is obtained by adding gum arabic to deionized water, stirring and dissolving at 80° C., and then cooling to room temperature before use.

[0014] Furthermore, in the obtained phycocyanin composite hydrogel, the mass percentage concentration of sodium alginate is 0.05%-0.1%, the mass percentage concentration of gelatin is 1.9%-3.8%, and the mass percentage concentration of calcium chloride is 0.4%.

[0015] Furthermore, in the obtained phycocyanin composite hydrogel, the mass percentage concentration of gum arabic is 0.4%-0.8%.

[0016] Furthermore, in the obtained phycocyanin composite hydrogel, the mass percentage concentration of sodium alginate is 0.1%, the mass percentage concentration of gelatin is 3.8%, and the mass percentage concentration of gum arabic is 0.4%.

[0017] In the phycocyanin composite hydrogel prepared by the above method, phycocyanin is encapsulated in a gel system constructed with sodium alginate, gelatin and gum arabic as main raw materials. Gelatin and sodium alginate as basic wall materials protect phycocyanin. After gum arabic is added to the hydrogel system, it also protects phycocyanin, making phycocyanin acid-stable. The composite gel after acid treatment can still maintain high antioxidant properties after acid treatment.

[0018] Beneficial effects:

[0019] 1. The phycocyanin composite hydrogel prepared in the present application encapsulates phycocyanin in a gel system constructed by gelatin, gum arabic and sodium alginate, so that the gel system not only has the antioxidant and anti-inflammatory properties of phycocyanin, but also gives phycocyanin the ability to adapt to an acidic environment, so that the phycocyanin hydrogel after acid treatment can still maintain a high antioxidant activity;

[0020] 2. Since the phycocyanin composite hydrogel prepared in the present application has good acid stability, it can adapt to the gastric acid environment of the human body and will not be consumed excessively in the gastric acid environment. Therefore, it can be used in food and medical treatment to improve the effective utilization rate of phycocyanin in the human body;

[0021] 3. The phycocyanin composite hydrogel disclosed in the present application uses sodium alginate and gelatin as main raw materials to construct the gel wall material. The raw materials used have good biocompatibility and degradability, and can meet the requirements of oral materials;

[0022] 4. When preparing the gel system, the present application not only uses sodium alginate and gelatin, two common gel materials, but also adds a certain proportion of gum arabic thereto. Gum arabic not only has good biocompatibility and will not affect the application of the gel, but also the addition of a suitable proportion of gum arabic can enhance the protective effect on phycocyanin, thereby further effectively improving the antioxidant properties of the gel material. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a flow chart of the preparation of phycocyanin hydrogel;

[0024] Figure 2 The figures are the results of the determination of hydroxyl radical scavenging rate, wherein (a) shows the results of hydroxyl radical scavenging rate of phycocyanin solutions of different concentrations before and after acid treatment; (b) shows the results of hydroxyl radical scavenging rate of phycocyanin hydrogels of different components before and after acid treatment;

[0025] Figure 31 is a graph showing the results of DPPH clearance, wherein (a) shows the DPPH clearance results of phycocyanin solutions of different concentrations before and after acid treatment, and (b) shows the DPPH clearance results of phycocyanin hydrogels of different components before and after acid treatment;

[0026] Figure 4 ABTS free radical scavenging rate measurement results are shown in the figure, wherein (a) shows the ABTS free radical scavenging rate results of phycocyanin solutions of different concentrations before and after acid treatment, and (b) shows the ABTS free radical scavenging rate results of phycocyanin hydrogels of different components before and after acid treatment;

[0027] Figure 5 This is a graph showing the results of measuring the swelling properties and moisturizing properties of phycocyanin hydrogels with different components;

[0028] Figure 6 This is a graph showing the results of measuring the encapsulation rate and release rate of phycocyanin hydrogels with different components. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0030] Experimental Materials:

[0031] Phycocyanin was purchased from Binmei Biotechnology Co., Ltd. (Zhejiang), gelatin was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai), sodium alginate was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai), gum arabic was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai), anhydrous calcium chloride was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai), and 95% ethanol was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai).

[0032] Embodiment 1

[0033] Preparation of raw materials:

[0034] 1) Preparation of phycocyanin aqueous solution: dissolving a corresponding amount of phycocyanin powder in deionized water and mixing well to prepare a phycocyanin aqueous solution;

[0035] 2) Preparation of gelatin aqueous solution: Add gelatin powder to deionized water, stir in a 50°C constant temperature water bath for 30 min until uniform, and let stand to room temperature;

[0036] 3) Preparation of sodium alginate aqueous solution: Sodium alginate was placed in deionized water, stirred in a 50°C constant temperature water bath for 30 min until uniform, and allowed to stand to room temperature;

[0037] 4) Preparation of gum arabic aqueous solution: Place gum arabic in deionized water, stir in a constant temperature water bath at 80°C for 30 min until uniform, and let stand to room temperature;

[0038] Construction of gel system (see Figure 1 ):

[0039] The phycocyanin aqueous solution is added dropwise to the sodium alginate aqueous solution, and the mixture is stirred continuously until uniform; the gelatin aqueous solution is added dropwise to the system, and the mixture is stirred continuously until uniform to form a hydrogel; the gum arabic aqueous solution is added dropwise to the system, and the mixture is stirred continuously until uniform to give the hydrogel antioxidant properties; a 2% calcium chloride solution is added dropwise to the system, and the mixture is cross-linked for 12 hours to obtain a phycocyanin composite hydrogel, which is then stored at 4°C.

[0040] Different gel systems can be obtained by changing the addition amount of each component. As shown in the following table, after the components are compounded into gel according to the above method, five groups of phycocyanin hydrogels with different component concentrations can be prepared (the data in the table represent the mass percentage concentration of each component in the prepared gel product).

[0041]

[0042] Related performance measurement

[0043] 1. Determination of the antioxidant properties of phycocyanin solution and phycocyanin hydrogel before and after acid treatment

[0044] Prepare a phycocyanin solution K of a certain concentration as a control (the concentrations of the phycocyanin solutions of groups 1 to 5 are 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively); take the same mass of gel from the five groups of phycocyanin hydrogels with different concentrations prepared in Example 1 as samples, and the samples are in duplicate (correspondingly recorded as A1, A2, B1, B2, C1, C2, D1, D2, E1, and E2), and place the phycocyanin hydrogel samples A1, B1, C1, D1, and E1 in an aqueous solution of pH = 2.5 for 3 minutes for acid treatment, and take them out after acid treatment, and samples A2, B2, C2, D2, and E2 are not subjected to acid treatment;

[0045] A portion of the phycocyanin solution with different concentrations was taken out as a sample, and the samples were in duplicate (correspondingly recorded as K1, K1', K2, K2', K3, K3', K4, K4', K5, K5'). Buffer was added to the K1, K2, K3, K4, and K5 samples to adjust the pH to 2.5, and the other five samples were not treated with acid;

[0046] The antioxidant properties of phycocyanin solution and phycocyanin hydrogel before and after acid treatment were measured.

[0047] (1) Determination of hydroxyl radical scavenging rate

[0048] 1.5 mL of deionized water was used as a blank control group. 1.5 mL of each of the 20 samples (including the phycocyanin solution samples and the phycocyanin hydrogel samples before and after acid treatment) was taken as a test sample (1.5 mL of the phycocyanin solution sample can be directly measured as a test sample, and the phycocyanin gel test sample is prepared by first weighing 1.25 g of the phycocyanin gel sample and then dissolving it in 1.5 mL of deionized water). The test sample was prepared in duplicate, and 1 mL, 2.5 mmol / L of salicylic acid, 1 mL, 5 mmol / L of ferrous sulfate, and 2 mL of deionized water were added in sequence. Then, 1 mL of 5 mol / L hydrogen peroxide solution was added to one of the test samples, and 1 mL of deionized water was added to the other as a substitute.

[0049] After reacting in a 37°C warm water bath for 30 min, the absorbance was measured at 510 nm. Each absorbance data was measured three times. The entire experiment was performed in a dark environment as much as possible.

[0050] The absorbance of the test sample (with H2O2) solution is A1; the absorbance of the test sample (without H2O2) solution is A2; and the absorbance of the blank control group is A3. The hydroxyl radical scavenging rate is calculated according to the following formula:

[0051] Hydroxyl free radical scavenging rate / % = A1-A2 / A3-A2×100%

[0052] (2) Determination of DPPH free radical scavenging rate

[0053] The test sample solution was prepared as above, in duplicate, each containing 2 mL of the test sample, deionized water as the blank control group, 2 mL of 0.1 mmol / L DPPH 95% ethanol solution was added to one test sample, 2 mL of anhydrous ethanol solution was added to the other test sample solution, and 2 mL of 0.1 mmol / L DPPH 95% ethanol solution was added to the blank control group. The mixture was thoroughly mixed, reacted at room temperature for 30 min, and the absorbance was measured at 517 nm. Each absorbance data was measured three times. The entire experiment was performed in a dark environment as much as possible.

[0054] Among them, the absorbance meter of the test sample group (with DPPH) is A1; the absorbance meter of the test sample group (without DPPH) is A2; the absorbance meter of the blank control group is A3. Calculate the DPPH free radical scavenging rate according to the formula:

[0055] DPPH free radical scavenging rate / % = A3-A1+A2 / A3×100%

[0056] (3) Determination of ABTS free radical scavenging rate

[0057] Prepare the test sample solution as above, mix 100mL of ABTS solution (7.4mmol / L) with 100mL of K2S2O4 solution (2.6mmol / L), and let it stand for 12h at 20℃ and in the dark. When using, dilute the mixed solution with anhydrous ethanol to an absorbance range of 0.7±0.02 and it can be used for subsequent tests; mix the diluted ABTS solution and the test sample solution added to the test tube in a volume ratio of 1:1, and immediately measure the absorbance of the mixed solution at 734nm, recorded as A1, and mix the diluted ABTS solution with 60% ethanol solution in the same ratio, and measure the absorbance at 734nm as A2. Each absorbance data is measured three times. The following formula calculates the ABTS free radical scavenging rate:

[0058] ABTS free radical scavenging rate / % = A2-A1 / A2×100%

[0059] Comparison of the antioxidant results of standard phycocyanin solution and phycocyanin hydrogel before and after acid treatment is shown in Figure 2 , 3 4. From the observation of the (b) panels of the three figures, it can be seen that the hydrogel clearance rates of groups A and C are higher than those of group B, indicating that gum arabic has a certain effect on improving the clearance rate, that is, after gum arabic is added to the hydrogel system, it also provides corresponding protection for phycocyanin.

[0060] The clearance rates of hydrogels in groups D and E were lower than those in group A, which verified that gelatin and sodium alginate as basic wall materials would play a certain protective role on phycocyanin. When preparing gel products, the dosage of gelatin and sodium alginate needs to reach a certain level.

[0061] It can be seen from the three accompanying drawings that the decrease in the clearance rate of the hydrogel products before and after acid treatment is generally smaller than that of the standard protein group, and in the three antioxidant activity test experiments of hydroxyl free radicals, DPPH free radicals, and ABTS free radicals, the comprehensive change in the clearance rate of group A hydrogel before and after acid treatment is the smallest, that is, when gelatin accounts for 3.8%, sodium alginate accounts for 0.1%, and gum arabic accounts for 0.4%, the composite hydrogel can play a better protective role on phycocyanin, while group C with a higher proportion of gum arabic only has a slight increase in the hydroxyl free radical clearance rate, and a slight decrease in the DPPH free radical and ABTS free radical clearance rates.

[0062] 2. Evaluation of swelling rate and moisture retention rate of phycocyanin hydrogel

[0063] (1) freeze-drying the prepared composite hydrogel, taking out the freeze-dried powder and weighing it, which is recorded as W0;

[0064] (2) Place the pellets in a buffer solution to swell according to the group, take them out after 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, and 3 h, dry the surface water, and weigh them;

[0065] (3) Record the mass of each group of hydrogels at each time period, recorded as W t , calculate the swelling ratio (SR) of each group of hydrogels, and calculate the swelling ratio according to the formula:

[0066] SR / %=W t -W0 / W0×100%

[0067] (1) Take out the prepared composite hydrogel, carefully dry the surface water, and weigh it, which is recorded as W0;

[0068] (2) Place the hydrogels in a culture dish according to the group, away from light and wind, and maintain room temperature at 25°C. Take them out and weigh them at 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 12 h, and 24 h, and record the mass of each group of hydrogels at each time period, which is recorded as W. t , calculate the gel moisturizing rate according to the moisturizing rate calculation formula:

[0069] Moisture retention rate / % = W0-W t / W0×100%

[0070] The swelling performance of phycocyanin hydrogels with different components is as follows Figure 5 As shown in the middle panel a, it can be seen from the figure that gum arabic and gelatin have a greater influence on the swelling properties, but increasing the mass proportion of gum arabic too much will reduce the swelling properties of the freeze-dried powder.

[0071] The moisturizing properties of phycocyanin hydrogels with different components are shown in Figure 5(b). It can be seen from the figure that gum arabic and gelatin can enhance the moisturizing properties of the hydrogel to a certain extent and reduce the surface water loss. With the increase in the amount of gum arabic and gelatin, the moisturizing rate is improved to a certain extent, and sodium alginate has little effect on the moisturizing rate.

[0072] On the whole, the gel has better moisturizing and swelling properties when the mass proportion of gelatin is 3.8%, the mass proportion of sodium alginate is 0.1%, and the mass proportion of gum arabic is 0.4%. If the proportion of the components is too high, it will cause changes in the embedding structure, thereby causing a decrease in related performance.

[0073] 3. Evaluation of encapsulation rate and self-release rate of phycocyanin hydrogel

[0074] (1) Evaluation of packaging rate

[0075] 1) preparing the prepared hydrogel into a solution to fully release the phycocyanin;

[0076] 2) The hydrogel that fully released phycocyanin was centrifuged at a speed of 4000 rad / s for 10 min, and the absorbance of the solution was measured, wherein the amount of phycocyanin encapsulated in the hydrogel (mg) was calculated as W1; the amount of phycocyanin put into the hydrogel (mg) was calculated as W0, and the active phycocyanin concentration was calculated and the encapsulation rate was calculated by comparing with the standard protein absorbance concentration curve. The encapsulation rate calculation formula is as follows:

[0077] Packing rate / % = W1 / W0×100%

[0078] (2) Evaluation of self-release rate

[0079] 1) preparing a solution of the prepared hydrogel, and measuring the absorbance of the solution after the phycocyanin is fully released;

[0080] 2) Calculate the natural release rate of phycocyanin from the composite hydrogel. The amount of phycocyanin naturally released from the hydrogel in the solution (mg) is calculated as W1; the amount of phycocyanin put into the hydrogel (mg) is calculated as W0, and the release rate is calculated according to the formula:

[0081] Self-release rate / %=W1 / W0×100%

[0082] The encapsulation efficiency of phycocyanin hydrogels with different components is as follows Figure 6 As shown in the middle panel a, the phycocyanin composite hydrogels prepared using the three materials all have a high encapsulation rate, among which the encapsulation effect is best when the mass proportion of gelatin is 3.8%, the mass proportion of sodium alginate is 0.1%, and the mass proportion of gum arabic is 0.4%.

[0083] The self-release rate of phycocyanin hydrogels with different components is shown in Figure 6(b). It can be seen from the figure that when gelatin accounts for 3.8%, sodium alginate accounts for 0.1%, and gum arabic accounts for 0.4%, the natural release rate of phycocyanin in the buffer system is low, and it has higher medicinal activity and structural stability.

[0084] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a phycocyanin composite hydrogel, characterized in that: The steps include: 1) dissolving phycocyanin powder in deionized water and mixing well to obtain a phycocyanin aqueous solution; 2) Place gelatin and sodium alginate in deionized water respectively, stir and dissolve at 50°C to obtain gelatin solution and sodium alginate solution, and cool to room temperature; 3) adding the phycocyanin aqueous solution obtained in step 1) to the sodium alginate solution obtained in step 2) and stirring evenly; 4) adding the gelatin solution obtained in step 2) dropwise to the mixed system obtained in step 3), and stirring to form a gel; 5) adding the gum arabic solution dropwise to the formed gel to enhance the antioxidant property of the gel; 6) adding a calcium chloride solution to the gel system obtained in step 5) for cross-linking to obtain a phycocyanin composite hydrogel; In the obtained phycocyanin composite hydrogel, the mass percentage concentration of sodium alginate is 0.05%-0.1%, the mass percentage concentration of gelatin is 1.9%-3.8%, the mass percentage concentration of calcium chloride is 0.4%, and the mass percentage concentration of gum arabic is 0.4%-0.8%.

2. The method for preparing a phycocyanin composite hydrogel according to claim 1, characterized in that: The gum arabic solution is prepared by adding gum arabic to deionized water, stirring and dissolving the solution at 80° C., and then cooling the solution to room temperature before use.

3. The method for preparing a phycocyanin composite hydrogel according to claim 1, characterized in that: In the obtained phycocyanin composite hydrogel, the mass percentage concentration of sodium alginate is 0.1%, the mass percentage concentration of gelatin is 3.8%, and the mass percentage concentration of gum arabic is 0.4%.

4. A phycocyanin composite hydrogel, characterized in that: The phycocyanin composite hydrogel is prepared according to the preparation method of any one of claims 1 to 3.

5. The phycocyanin composite hydrogel according to claim 4, characterized in that: Phycocyanin is encapsulated in a gel system constructed with sodium alginate, gelatin and gum arabic as the main raw materials. Gelatin and sodium alginate as basic wall materials protect phycocyanin. After gum arabic is added to the hydrogel system, it also protects phycocyanin, making phycocyanin acid-stable. The composite gel after acid treatment can still maintain a high antioxidant property after acid treatment.

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