Method for improving the lipid-based dispersibility of phycocyanin and modified phycocyanin

Abstract

The present application relates to the technical field of protein modification, and in particular to a method for improving the dispersibility of phycocyanin in lipid base and modified phycocyanin. The present application first prepares a chitosan-phycocyanin complex by electrostatic complexation of phycocyanin and chitosan under weakly acidic conditions, then uses dodecanal as a hydrophobic modification reagent to graft a dodecyl hydrophobic chain onto the amino group outside the chitosan coating layer through a reductive amination reaction, and obtains the modified phycocyanin after dialysis purification and lyophilization protection. The shielding effect of the chitosan coating layer on the inner core phycocyanin makes the hydrophobic modification mainly occur on the amino group outside the chitosan, which can reduce the interference of the reaction reagent on the color performance of phycocyanin, and at the same time improve the dispersibility of the product in the lipid base.

Description

Technical Field

[0001] This invention relates to the field of protein modification technology, specifically to a method for improving the liposome dispersibility of phycocyanin and a modified phycocyanin. Background Technology

[0002] Phycocyanin is a water-soluble light-harvesting pigment protein, insoluble in ethanol and lipids. Phycocyanin consists of a protein moiety and a phycocyanin bile moiety, covalently linked by thioether bonds. The protein moiety contains two subunits: an α-subunit of 162 amino acid residues and a β-subunit of 172 amino acid residues. These subunits typically exist in vivo as trimers or hexamers, and their activity is generally affected by pH. Phycocyanin exhibits a characteristic absorption peak at 615-620 nm and a fluorescence emission peak at 635-647 nm.

[0003] Phycocyanin is essentially a pigment protein, and therefore its stability is often affected by factors such as temperature, pH, and light conditions. Phycocyanin is relatively stable below 40℃, while increasing the temperature accelerates pigment degradation. The isoelectric point of phycocyanin is around 4.5, but there are some differences in the isoelectric point of phycocyanin obtained from different strains. Phycocyanin solutions are relatively stable at pH 5-8, and adding a certain concentration of sucrose or citric acid to the solution can improve the stability of phycocyanin.

[0004] Chinese patent application CN121421192A discloses a stable Pickering emulsion of phycocyanin-rosmarinic acid conjugate and its application in surimi products. It utilizes laccase to oxidize the catechol structure of rosmarinic acid into a highly active o-quinone intermediate, which can undergo Michael addition / Schiff base reaction with nucleophilic groups on the protein surface (such as the amino group of lysine) to achieve stable covalent conjugation. Using the conjugate product as an emulsifier and corn oil as the oil phase, the emulsion is prepared through homogenization. This emulsion is added to surimi in a specific ratio, and the surimi product is obtained through chopping, stuffing, and two-stage heating. It can serve as a fat substitute, effectively filling and strengthening the three-dimensional network of the gel. Chinese patent CN119073564B discloses a method for preparing emulsion gels using a phycocyanin-starch composite emulsifier. The method involves adding phycocyanin and starch to distilled water, stirring at room temperature to hydrate, and obtaining a mixture. The mixture is then heated in a water bath and cooled to room temperature to obtain the phycocyanin-starch composite emulsifier. Finally, soybean oil is added, and the mixture is homogenized by high-speed shearing to obtain the emulsion gel. Phycocyanin and starch are combined through hydrogen bonds and hydrophobic interactions. This method combines phycocyanin with other substances to create an amphiphilic compound, improving the emulsifying properties of phycocyanin and enhancing its lipophilicity, but it does not truly achieve lipid dispersion of phycocyanin.

[0005] Therefore, improving the dispersibility of phycocyanin lipids is an urgent need in the biopharmaceutical industry. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for improving the lipid dispersibility of phycocyanin and a modified phycocyanin. First, chitosan and phycocyanin are electrostatically combined to form a chitosan-phycocyanin complex. Then, hydrophobic alkyl chains are modified onto the chitosan-phycocyanin complex through Schiff base bonds formed by aldehyde and amino groups. Subsequently, most of the Schiff bases are reduced to stable CN bonds, thereby improving the lipid dispersibility of phycocyanin without damaging its color development properties.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for improving the dispersibility of phycocyanin lipid groups, comprising the following steps:

[0009] S1, dispersing phycocyanin in pure water to obtain a phycocyanin solution; dispersing chitosan in an aqueous acetic acid solution to obtain a chitosan solution;

[0010] S2, add phycocyanin solution dropwise to the stirred chitosan solution, and react after the addition is complete to obtain chitosan-phycocyanin complex;

[0011] S3, adjust the pH of the chitosan-phycocyanin complex with sodium hydroxide solution, add dodecyl aldehyde anhydrous ethanol solution dropwise, stir after the addition is complete, add the first part of sodium cyanoborohydride aqueous solution, mix, add the second part of sodium cyanoborohydride aqueous solution, stir to reduce, and obtain the reduced solution;

[0012] S4, the reducing solution is dialyzed to obtain the dialysate, trehalose is added to the dialysate, and the mixture is stirred to obtain the pre-frozen solution. The pre-frozen solution is pre-frozen, freeze-dried, and dried to obtain the modified phycocyanin.

[0013] Further, in S1, the mass-to-volume ratio of phycocyanin to pure water is (0.5~1) g: 100 mL.

[0014] Furthermore, in S1, the number-average molecular weight of the chitosan is (100~200) kDa, and the degree of deacetylation of the chitosan is ≥85%.

[0015] Furthermore, in S1, the pH of the acetic acid aqueous solution is 3.8 to 4.2.

[0016] Further, in S1, the mass-to-volume ratio of chitosan to aqueous acetic acid solution is (0.5~1) g: 100 mL.

[0017] Phycocyanin is a natural protein pigment that is highly soluble in water and has a negative charge on its molecular surface; chitosan is a cationic polysaccharide whose amino groups are protonated in a weakly acidic environment and carry a large number of positive charges.

[0018] Furthermore, in step S2, the stirring speed is 200~400 rpm.

[0019] Furthermore, in step S2, the dropping rate is 0.5~1 mL / min.

[0020] When a negatively charged phycocyanin solution is dropped into a large amount of excess positively charged chitosan solution, each drop of phycocyanin solution enters an environment with a high concentration of chitosan. The local chitosan concentration is much higher than the phycocyanin concentration. Driven by electrostatic force, chitosan tends to coat the surface of phycocyanin, completing the coating of phycocyanin before the phycocyanin molecules self-aggregate, forming a chitosan-phycocyanin complex. The chitosan molecular chains are wrapped around the phycocyanin, which on the one hand stabilizes the native conformation of phycocyanin through steric hindrance, preventing phycocyanin aggregation, and on the other hand introduces a large number of active amino functional groups on the surface of phycocyanin, providing reaction sites for subsequent functional modification.

[0021] The coating of chitosan on the surface of phycocyanin is a stepwise self-assembly process. When the phycocyanin solution is dropped into the chitosan solution, the chitosan and phycocyanin at the liquid-liquid interface come into contact. A protonated amino group on the chitosan chain forms an ion pair with a carboxyl group on the surface of phycocyanin, anchoring the chitosan chain around the phycocyanin. Subsequently, adjacent amino groups on the chitosan chain continue to form ion pairs with adjacent carboxyl groups on the surface of phycocyanin, forming continuous linker segments on the surface of phycocyanin. Finally, a coating structure composed of several linker segments is formed on the surface of phycocyanin.

[0022] Furthermore, in step S2, the volume ratio of the chitosan solution to the phycocyanin solution is (3~5):1.

[0023] Further, in step S2, when adding the phycocyanin solution, the pH of the system is maintained at 4.8-5.2 using sodium hydroxide solution or acetic acid solution; the concentration of the sodium hydroxide solution is 0.01-0.05 mol / L; and the volume concentration of the acetic acid solution is 0.01-0.05%.

[0024] By controlling the pH of the system during the dropwise addition, it can be ensured that the amino groups of chitosan are fully protonated and uniformly complexed with phycocyanin.

[0025] Furthermore, in step S2, the reaction speed is 200-400 rpm, and the reaction time is 30-60 min.

[0026] Further, in step S3, the concentration of the sodium hydroxide solution is 0.01~0.05 mol / L.

[0027] Furthermore, in step S3, the pH of the chitosan-phycocyanin complex after adjustment is 6.0~6.5.

[0028] The pH of the system was adjusted to 6.0-6.5 using a low-concentration sodium hydroxide solution, which preserved the reactivity of some amino groups on chitosan while avoiding the destruction of the phycocyanin structure by a strongly alkaline environment.

[0029] Furthermore, in step S3, the dropping rate is 0.1~0.5 mL / min.

[0030] Furthermore, in step S3, the mass concentration of the anhydrous ethanol solution of dodecanal is 3-5%.

[0031] Further, in step S3, the volume ratio of the dodecyl anhydrous ethanol solution and the chitosan-phycocyanin complex is (4.55~4.7):100.

[0032] Dodecyl is an organic aldehyde compound, a colorless to pale yellow oily liquid at room temperature, with a strong fatty and citrus aroma. Its molecule consists of a hydrophobic dodecyl chain and a reactive aldehyde group. When dodecyl is added to a chitosan-phycocyanin complex, the aldehyde group of dodecyl reacts with the primary amine groups on the surface of the chitosan-phycocyanin complex to form an unstable Schiff base, thus initially binding the hydrophobic dodecyl chain to the surface of the chitosan-phycocyanin complex.

[0033] Furthermore, in step S3, the stirring speed is 150~220 rpm, and the stirring time is 30~60 min.

[0034] Furthermore, in step S3, the concentration of the first sodium cyanoborohydride aqueous solution is 50~100 g / L.

[0035] Furthermore, in step S3, the mixing speed is 150~220 rpm, and the mixing time is 30~60 min.

[0036] Furthermore, in step S3, the concentration of the second sodium cyanoborohydride aqueous solution is 50~100 g / L.

[0037] Furthermore, in step S3, the stirring and reduction speed is 150~220 rpm, and the stirring and reduction time is 90~180 min.

[0038] Further, in step S3, the mass ratio of the first part of sodium cyanoborohydride aqueous solution to the second part of sodium cyanoborohydride aqueous solution is 3:(1~3).

[0039] Further, in S3, the ratio of the total mass of the first sodium cyanoborohydride aqueous solution and the second sodium cyanoborohydride aqueous solution to the mass of the dodecyl aldehyde anhydrous ethanol solution is (33~38):175.

[0040] Sodium cyanoborohydride is a mild reducing agent widely used for the reduction of aldehydes and ketones, especially for the reductive amination of ketones. Sodium cyanoborohydride is added in two portions to the mixture forming Schiff bases to reduce the generated Schiff base bonds to more stable carbon-nitrogen single bonds. The first portion of sodium cyanoborohydride is added when a large amount of Schiff base is generated, selectively reducing the already formed Schiff base to a stable secondary amine under weakly acidic conditions. The remaining aldehyde continues to react with the amino group to generate a Schiff base, ensuring sufficient hydrophobic alkyl chains are grafted onto the surface of the chitosan-phycocyanin complex. The second portion of sodium cyanoborohydride is used to reduce the remaining and newly generated Schiff base in the system, ensuring that the hydrophobic alkyl chains are grafted onto the surface of the chitosan-phycocyanin complex, constructing a hydrophobic region on the surface of the complex.

[0041] Further, in step S4, the specific operation of dialysis is as follows: the reducing solution is transferred into a dialysis bag with a molecular weight cutoff of 1000~5000 Da, and dialyzed at 0~5℃ with a pH of 6.2~6.8 and a concentration of 8~15 mM phosphate buffer. The solution is changed every 6 hours, and dialysis is continued until the conductivity of the dialysate is stable and the conductivity is <50 μS / cm.

[0042] By removing unreacted small molecules such as dodecaldehyde and sodium cyanoborohydride from the system through dialysis, a relatively pure product is obtained.

[0043] Furthermore, the dialysis bag is preferably a dialysis bag with a molecular weight cutoff of 3500 Da.

[0044] Furthermore, the preferred temperature for dialysis is 4°C.

[0045] Furthermore, in step S4, the mass ratio of trehalose to dialysis product is (4~6):1.

[0046] Trehalose is a non-reducing disaccharide widely found in lower plants, algae, bacteria, fungi, yeast, insects, and other invertebrates. It is both a storage sugar and an important product of stress metabolism. Adding trehalose to the dialysis product before lyophilization serves two purposes: firstly, it acts as a lyophilization protectant, replacing water molecules during dehydration and forming hydrogen bonds with phycocyanin to maintain its native conformation and prevent structural collapse, thus avoiding product color changes; secondly, when the modified phycocyanin is dispersed in lipid groups, trehalose creates a micro-aqueous environment within it, maintaining the electrostatic attraction between chitosan and phycocyanin. Simultaneously, through hydrophobic interactions, it removes the alkyl chain of dodecyl aldehyde from the micro-aqueous environment and extends into the lipid system, forming a lipid-based dispersion of the modified phycocyanin.

[0047] Furthermore, in step S4, the stirring speed is 200~400 rpm, and the stirring time is 5~12 min.

[0048] Furthermore, in S4, the pre-freezing is low-temperature pre-freezing or liquid nitrogen pre-freezing.

[0049] Furthermore, the temperature for low-temperature pre-freezing is -40 to -80°C, and the pre-freezing time is 2 to 12 hours.

[0050] Furthermore, the liquid nitrogen pre-freezing time is 30~60 min, and the volume ratio of liquid nitrogen to pre-freezing liquid is (5~10):1.

[0051] Furthermore, in step S4, the freeze-drying temperature is -20 to -40°C, the freeze-drying pressure is 0.1 to 0.2 mbar, and the endpoint of freeze-drying is a moisture content of <40%.

[0052] Traditional freeze-drying aims for complete dehydration, but this invention limits the endpoint of freeze-drying to a moisture content of <40%. At this point, the free ice formed by the freezing of free water in the system has been basically sublimated, and the remaining water is mainly bound water that forms hydrogen bonds with proteins and trehalose.

[0053] Furthermore, in step S4, the drying temperature is 0~10℃, the drying pressure is 0.05~0.15mbar, and the drying endpoint is when the moisture content of the material is 5~10%.

[0054] The freeze-dried material is dried at a low temperature of 0~10℃ until the moisture content of the material is 5~10%. This moisture content can maintain the hydrogen bond network of bound water around phycocyanin and maintain the extended structure of phycocyanin chromophores, thereby ensuring that the final product does not undergo serious color shift. Trehalose is in a rigid glassy state in a 5~10% moisture environment, and the molecular motion is frozen, which can fix the overall structure of modified phycocyanin.

[0055] In a second aspect, the present invention provides a modified phycocyanin prepared by the method described in the first aspect.

[0056] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0057] This invention provides a method for improving the liposome dispersibility of phycocyanin. Phycocyanin prepared by this method exhibits improved dispersibility in liposome systems and can be used to prepare liposome-based pharmaceutical formulations. This invention utilizes a stepwise process of chitosan electrostatic composite and dodecyl aldehyde surface hydrophobic modification to construct a chitosan-rich layer with hydrophobic alkyl chains on the exterior of phycocyanin, thereby improving the dispersibility of phycocyanin in liposome systems. The positively charged chitosan and the negatively charged phycocyanin are electrostatically attracted, and the chitosan is enriched on the phycocyanin surface, reducing the inward diffusion of reactants into the protein surface. Simultaneously, the surface of the chitosan-phycocyanin complex contains a large amount of chitosan. Due to the numerous amino groups on its molecular chain, chitosan becomes a preferential target for aldehyde reagents. Therefore, hydrophobic modification mainly occurs on the amino groups of chitosan, which is beneficial for maintaining the structural stability of the phycocyanin chromophores and reducing the negative impact of hydrophobic modification on the protein's color development. Attached Figure Description

[0058] Figure 1 This is a physical image of the modified phycocyanin prepared in Example 1 of the present invention.

[0059] Figure 2 The image shows the UV-Vis absorption spectrum of the modified phycocyanin prepared in Example 1 of this invention dispersed in lipid groups. Detailed Implementation

[0060] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present invention and the specific features in the embodiments are detailed descriptions of the technical solution of the present invention, rather than limitations thereof.

[0061] Example 1

[0062] This embodiment provides a method for improving the dispersibility of phycocyanin lipid groups, including the following steps:

[0063] S1. Phycocyanin was dispersed in pure water at a mass-to-volume ratio of 0.8g:100mL to obtain a phycocyanin solution; chitosan with a number-average molecular weight of 150kDa and a degree of deacetylation ≥85% was dispersed in an acetic acid aqueous solution at pH=4, with a mass-to-volume ratio of chitosan to acetic acid aqueous solution of 0.8g:100mL to obtain a chitosan solution.

[0064] S2, the chitosan solution was stirred at 300 rpm, and phycocyanin solution was added dropwise to the chitosan solution at a rate of 0.7 mL / min. The volume ratio of chitosan solution to phycocyanin solution was 4:1. During the dropwise addition, the pH of the system was maintained at 5.0 with 0.03 mol / L sodium hydroxide solution. After the dropwise addition was completed, the mixture was stirred at 300 rpm for 45 min to obtain the chitosan-phycocyanin complex.

[0065] S3. The pH of the chitosan-phycocyanin complex was adjusted to 6.2 using a 0.03 mol / L sodium hydroxide solution. A 4.5% (w / w) anhydrous ethanol solution of dodecaldehyde was added dropwise at a rate of 0.3 mL / min, with a volume ratio of 4.66:100 between the anhydrous ethanol solution of dodecaldehyde and the chitosan-phycocyanin complex. After the addition was complete, the mixture was stirred at 200 rpm for 45 min. Then, a first portion of 80 g / L sodium cyanoborohydride aqueous solution was added, and the mixture was stirred at 200 rpm for 45 min. After that, a second portion of 80 g / L sodium cyanoborohydride aqueous solution was added, and the mixture was stirred at 200 rpm for 135 min to reduce the solution, thus obtaining the reduced solution. The mass ratio of the first portion of sodium cyanoborohydride aqueous solution to the second portion of sodium cyanoborohydride aqueous solution was 3:2, and the mass ratio of the total mass of the first portion of sodium cyanoborohydride aqueous solution to the second portion of sodium cyanoborohydride aqueous solution to the mass of the anhydrous ethanol solution of dodecaldehyde was 36:175.

[0066] S4. The reducing solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed at 4°C with 10 mM phosphate buffer (pH 6.5). The buffer was changed every 6 hours until the conductivity of the dialysate stabilized and was <50 μS / cm. The dialysate was collected from the dialysis bag. Trehalose was added to the dialysate at a mass ratio of 5:1, and the mixture was stirred at 300 rpm for 10 min to obtain a pre-freeze. The pre-freeze was then placed in liquid nitrogen for 45 min at a volume ratio of 7:1 (liquid nitrogen to pre-freeze). Subsequently, it was freeze-dried at 0.15 mbar until the moisture content was <40% at -30°C, and then dried at 0.07 mbar until the moisture content was 7% at 5°C to obtain the product. Figure 1 The modified phycocyanin shown, Figure 2 The image shows the UV-Vis absorption spectrum of the modified phycocyanin prepared in this embodiment dispersed in lipid groups. As can be seen from the image, the modified phycocyanin has a characteristic absorption peak at 610~625nm.

[0067] Example 2

[0068] This embodiment provides a method for improving the dispersibility of phycocyanin lipid groups, including the following steps:

[0069] S1. Phycocyanin is dispersed in pure water at a mass-to-volume ratio of 1g:100mL to obtain a phycocyanin solution; chitosan with a number-average molecular weight of 100kDa and a degree of deacetylation ≥85% is dispersed in an acetic acid aqueous solution at pH=4.2, with a mass-to-volume ratio of chitosan to acetic acid aqueous solution of 1g:100mL to obtain a chitosan solution.

[0070] S2, the chitosan solution was stirred at 200 rpm, and phycocyanin solution was added dropwise to the chitosan solution at a rate of 0.5 mL / min. The volume ratio of chitosan solution to phycocyanin solution was 5:1. During the dropwise addition, the pH of the system was maintained at 5.2 with 0.01 mol / L sodium hydroxide solution. After the dropwise addition was completed, the reaction was stirred at 200 rpm for 60 min to obtain the chitosan-phycocyanin complex.

[0071] S3. The pH of the chitosan-phycocyanin complex was adjusted to 6.5 using a 0.01 mol / L sodium hydroxide solution. A 5% (w / w) anhydrous ethanol solution of dodecaldehyde was added dropwise at a rate of 0.1 mL / min, with a volume ratio of 4.7:100 between the anhydrous ethanol solution of dodecaldehyde and the chitosan-phycocyanin complex. After the addition was complete, the mixture was stirred at 150 rpm for 60 min. Then, a first portion of sodium cyanoborohydride aqueous solution with a concentration of 100 g / L was added, and the mixture was stirred at 150 rpm for 60 min. After that, a second portion of sodium cyanoborohydride aqueous solution with a concentration of 100 g / L was added, and the mixture was stirred at 150 rpm for 180 min to reduce the solution, thus obtaining the reduced solution. The mass ratio of the first portion of sodium cyanoborohydride aqueous solution to the second portion of sodium cyanoborohydride aqueous solution was 3:1, and the ratio of the total mass of the first portion of sodium cyanoborohydride aqueous solution to the mass of the anhydrous ethanol solution of dodecaldehyde was 33:175.

[0072] S4. The reducing solution was transferred into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed at 0°C with 8 mM phosphate buffer at pH 6.8. The buffer was changed every 6 hours until the conductivity of the dialysate was stable and <50 μS / cm. The dialysate was collected from the dialysis bag. Trehalose was added to the dialysate at a mass ratio of 4:1. The mixture was stirred at 200 rpm for 12 minutes to obtain a pre-freeze solution. The pre-freeze solution was placed at -80°C for 3 hours and then freeze-dried at 0.2 mbar until the moisture content was <40% at -40°C. Finally, it was dried at 0.15 mbar until the moisture content was 10% at 10°C to obtain modified phycocyanin.

[0073] Example 3

[0074] This embodiment provides a method for improving the dispersibility of phycocyanin lipid groups, including the following steps:

[0075] S1. Phycocyanin was dispersed in pure water at a mass-to-volume ratio of 0.5g:100mL to obtain a phycocyanin solution; chitosan with a number-average molecular weight of 200kDa and a degree of deacetylation ≥85% was dispersed in an acetic acid aqueous solution at pH=3.8, with a mass-to-volume ratio of chitosan to acetic acid aqueous solution of 0.5g:100mL to obtain a chitosan solution.

[0076] S2, the chitosan solution was stirred at 400 rpm, and phycocyanin solution was added dropwise to the chitosan solution at a rate of 1 mL / min. The volume ratio of chitosan solution to phycocyanin solution was 3:1. During the dropwise addition, the pH of the system was maintained at 4.8 with 0.05 mol / L sodium hydroxide solution. After the dropwise addition was completed, the mixture was stirred at 400 rpm for 30 min to obtain the chitosan-phycocyanin complex.

[0077] S3. The pH of the chitosan-phycocyanin complex was adjusted to 6.0 using a 0.05 mol / L sodium hydroxide solution. A 3% (w / w) anhydrous ethanol solution of dodecaldehyde was added dropwise at a rate of 0.5 mL / min, with a volume ratio of 4.55:100 between the anhydrous ethanol solution of dodecaldehyde and the chitosan-phycocyanin complex. After the addition was complete, the mixture was stirred at 220 rpm for 30 min. Then, a first portion of sodium cyanoborohydride aqueous solution with a concentration of 50 g / L was added, and the mixture was stirred at 220 rpm for 30 min. After that, a second portion of sodium cyanoborohydride aqueous solution with a concentration of 50 g / L was added, and the mixture was stirred at 220 rpm for 90 min to reduce the solution, thus obtaining the reduced solution. The mass ratio of the first portion of sodium cyanoborohydride aqueous solution to the second portion of sodium cyanoborohydride aqueous solution was 3:3, and the ratio of the total mass of the first portion of sodium cyanoborohydride aqueous solution to the mass of the anhydrous ethanol solution of dodecaldehyde was 38:175.

[0078] S4. The reducing solution was transferred into a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed at 5°C with 15 mM phosphate buffer at pH 6.2. The buffer was changed every 6 hours until the conductivity of the dialysate was stable and <50 μS / cm. The dialysate was collected from the dialysis bag. Trehalose was added to the dialysate at a mass ratio of 6:1 and stirred at 400 rpm for 5 min to obtain a pre-freeze solution. The pre-freeze solution was placed in liquid nitrogen for 60 min at a volume ratio of 10:1 to liquid nitrogen. Then, it was freeze-dried at 0.1 mbar until the moisture content was <40% at -20°C. Finally, it was dried at 0.05 mbar until the moisture content was 5% at 0°C to obtain modified phycocyanin.

[0079] Comparative Example 1

[0080] A method for improving the dispersibility of phycocyanin lipid groups, which differs from Example 1 in that, in S2, the phycocyanin solution and chitosan solution are directly mixed and stirred at 300 rpm for 45 min, while other operating steps and process parameters are exactly the same as in Example 1.

[0081] Comparative Example 2

[0082] A method for improving the dispersibility of phycocyanin lipid groups, which differs from Example 1 in that S2 is omitted, and the pH of the phycocyanin solution is adjusted to 6.2 with 0.01 mol of sodium hydroxide. Other operating steps and process parameters are exactly the same as in Example 1.

[0083] Comparative Example 3

[0084] A method for improving the dispersibility of phycocyanin lipid groups, which differs from Example 1 in that, without step-by-step processing, the phycocyanin solution, chitosan solution and dodecyl aldehyde anhydrous ethanol solution are directly mixed and stirred at 300 rpm for 45 min. Other operating steps and process parameters are exactly the same as in Example 1.

[0085] Comparative Example 4

[0086] A method for improving the dispersibility of phycocyanin lipid groups, which differs from Example 1 in that chitosan is not used in the preparation process, and dodecyl aldehyde anhydrous ethanol solution is directly added dropwise to phycocyanin solution with pH=6.2. Other operating steps and process parameters are exactly the same as in Example 1.

[0087] The modified phycocyanin prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to performance tests, with blank examples used for comparison. The specific process is as follows:

[0088] The sample to be tested was dispersed in medium-chain triglycerides at a mass concentration of 1%, and stirred at 400 rpm for 30 min. The homogeneity of the system was observed, and the absorbance of the lipid phase was measured at 0 h, 6 h, and 24 h after stirring at a wavelength of 620 nm. The layering and precipitation of the system were observed. Unmodified phycocyanin was used as a blank control group.

[0089] Table 1. Performance test results of the modified phycocyanin prepared in the examples and comparative examples.

[0090]

[0091] As shown in Table 1, the modified phycocyanin prepared by the method of the present invention has good lipid dispersibility. The lipid dispersibility of phycocyanin can be achieved by stirring and can be maintained for 24 hours.

[0092] The initial absorbance of Comparative Example 1 was lower than that of Example 1, and decreased significantly after 24 hours. This may be because the phycocyanin solution and chitosan solution in Comparative Example 1 were directly mixed, resulting in uneven chitosan coating and affecting the suspension stability of the final product. The initial absorbance of Comparative Example 2 was much lower than that of Example 1, and the color was blue-green. The absorbance decreased significantly after 24 hours, and there was almost no effective dispersion in the lipid group. This may be because Comparative Example 2 lacked a chitosan coating layer, and the phycocyanin reacted directly with dodecyl aldehyde, leading to protein denaturation and chromophore destruction. The initial absorbance of Comparative Example 3 was lower than that of Example 1 and Comparative Example 1, but higher than that of Comparative Example 2. However, Comparative Example 3 had poor 24-hour stability. After 24 hours, the absorbance of the lipid phase decreased significantly, and a large amount of precipitation occurred. This may be because Comparative Example 3 directly mixed all raw materials, which could not form the ordered hierarchical structure of the present invention. Therefore, the lipid group dispersion uniformity was not as good as that of Example 1. The initial absorbance value of Comparative Example 4 was lower than that of Example 1 but higher than that of the blank control. This may be because some dodecylaldehyde came into contact with the amino groups on the surface of phycocyanin and reacted with them to form alkyl-grafted phycocyanin. The long alkyl chain provided a certain degree of lipid dispersibility for phycocyanin. After 6 hours, the absorbance value of Comparative Example 4 decreased significantly. This may be because the amino group density on the surface of phycocyanin was not high, resulting in insufficient grafting of dodecylaldehyde and inability to provide sufficient lipophilicity.

[0093] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the dispersibility of phycocyanin lipid groups, characterized in that, Includes the following steps: S1, dispersing phycocyanin in pure water to obtain a phycocyanin solution; dispersing chitosan in an aqueous acetic acid solution to obtain a chitosan solution; S2, add phycocyanin solution dropwise to the stirred chitosan solution, and react after the addition is complete to obtain chitosan-phycocyanin complex; S3, adjust the pH of the chitosan-phycocyanin complex with sodium hydroxide solution, add dodecyl aldehyde anhydrous ethanol solution dropwise, stir after the addition is complete, add the first part of sodium cyanoborohydride aqueous solution, mix, add the second part of sodium cyanoborohydride aqueous solution, stir to reduce, and obtain the reduced solution; S4, the reducing solution is dialyzed to obtain the dialysate, trehalose is added to the dialysate, and the mixture is stirred to obtain the pre-frozen solution. The pre-frozen solution is pre-frozen, freeze-dried, and dried to obtain the modified phycocyanin.

2. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In S1, the mass-to-volume ratio of phycocyanin to pure water is (0.5~1) g: 100 mL; the mass-to-volume ratio of chitosan to acetic acid aqueous solution is (0.5~1) g: 100 mL.

3. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S2, the dropping rate is 0.5~1 mL / min; the volume ratio of the chitosan solution to the phycocyanin solution is (3~5):

1.

4. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S3, the pH of the chitosan-phycocyanin complex is 6.0~6.

5.

5. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S3, the dropping rate is 0.1~0.5 mL / min; the mass concentration of the dodecyl anhydrous ethanol solution is 3~5%; and the volume ratio of the dodecyl anhydrous ethanol solution to the chitosan-phycocyanin complex is (4.55~4.7):

100.

6. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S3, the concentration of the first sodium cyanoborohydride aqueous solution is 50-100 g / L; the concentration of the second sodium cyanoborohydride aqueous solution is 50-100 g / L; and the mass ratio of the first sodium cyanoborohydride aqueous solution to the second sodium cyanoborohydride aqueous solution is 3:(1-3).

7. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S3, the ratio of the total mass of the first sodium cyanoborohydride aqueous solution and the second sodium cyanoborohydride aqueous solution to the mass of the dodecyl aldehyde anhydrous ethanol solution is (33~38):

175.

8. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S4, the specific operation of dialysis is as follows: the reducing solution is transferred into a dialysis bag, and dialysis is performed at 0~5℃ using phosphate buffer solution with pH=6.2~6.8 and a concentration of 8~15mM. The solution is changed every 6 hours, and dialysis is performed until the conductivity of the dialysate is stable and the conductivity is <50μS / cm.

9. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S4, the mass ratio of trehalose to dialysis product is (4~6):1; the pre-freezing is low-temperature pre-freezing or liquid nitrogen pre-freezing, and the low-temperature pre-freezing temperature is -40~-80℃.

10. The method for improving the dispersibility of phycocyanin lipid groups according to claim 1, characterized in that, In step S4, the drying temperature is 0~10℃, the drying pressure is 0.05~0.15mbar, and the drying endpoint is when the moisture content of the material is 5~10%.

11. A modified phycocyanin, characterized in that, It is prepared by the method described in any one of claims 1-10.

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