Phycocyanin with high stability and process extraction method thereof

Abstract

The invention discloses phycocyanin with high stability and a process extraction method thereof. The method comprises the following steps: 1) preparing a spirulina solution, adding a zinc salt solution into the spirulina solution, fully and uniformly mixing, freezing and thawing for 3-4 times, centrifuging, collecting supernate, and freeze-drying to obtain a phycocyanin-Zn < 2 + > compound crude product; 2) adding water into the phycocyanin-Zn < 2 + > compound crude product to prepare a phycocyanin crude extracting solution, slowly adding ammonium sulfate into the phycocyanin crude extracting solution, standing at 3-4 DEG C for 1.5-2.5 hours, centrifuging, dissolving in ultrapure water, dialyzing and freeze-drying to obtain a phycocyanin-Zn < 2 + > compound; and 3) dissolving the phycocyanin-Zn < 2 + > compound in ultrapure water to obtain a phycocyanin-Zn < 2 + > compound solution, mixing the ellagic acid solution and the phycocyanin-Zn < 2 + > compound solution in proportion, fully and uniformly mixing in a dark place, centrifuging, collecting supernate, carrying out rotary evaporation, and freeze-drying to obtain the phycocyanin-Zn < 2 + >-ellagic acid compound.

Description

Technical Field

[0001] This invention relates to the field of phycocyanin extraction. More specifically, it relates to a highly stable phycocyanin and its extraction process. Background Technology

[0002] Natural pigments are natural coloring components extracted from plants, animals, or minerals. They have gained widespread attention due to their low toxicity and good biocompatibility. Blue is one of the three primary colors and has a wide range of uses. Blue pigments are abundant in nature, but most blue pigments used in food processing are synthetic, and naturally occurring blue pigments that fully meet the needs of the food industry are rare.

[0003] Phycocyanin (PC) from Spirulina platensis, a water-soluble pigment protein with a linear tetrapyrrole chromophore (phycocyanin), shows great promise for applications in food coloring and drug delivery due to its unique blue fluorescence and antioxidant activity. However, its tetrapyrrole conjugated system is susceptible to temperature, light, and oxidative stress during extraction, processing, and storage, leading to chromophore degradation and functional loss, which severely restricts its industrial application. Traditional color protection methods focus on post-extraction processing, such as adding antioxidants or encapsulation techniques, but neglect the fact that the chromophore may already be irreversibly damaged by mechanical or chemical treatment during extraction, potentially destroying the chromophore itself. Therefore, developing a pretreatment method that can protect the PC structure from the source is crucial, and a molecular protection mechanism needs to be established in the initial extraction stage. Summary of the Invention

[0004] To address the above-mentioned shortcomings, the first objective of this invention is to provide a process for extracting phycocyanin from Spirulina while simultaneously providing pigment protection. This process utilizes freeze-thaw cycles, metal ion pre-stabilization techniques, and ellagic acid intervention to fuse phycocyanin with Zn... 2+ The ions coordinate with ellagic acid, ensuring that phycocyanin is effectively protected during both pre- and post-extraction processes, thus significantly improving the pigment preservation rate.

[0005] The second objective of this invention is to provide a phycocyanin with high stability obtained by the process described above.

[0006] To achieve the first objective mentioned above, the present invention adopts the following technical solution:

[0007] This invention discloses a process for extracting phycocyanin from Spirulina while simultaneously providing pigment protection, comprising the following steps:

[0008] 1) Prepare a spirulina solution by adding zinc salt solution, mixing thoroughly, freezing and thawing 3-4 times, centrifuging, collecting the supernatant, and freeze-drying to obtain phycocyanin-Zn. 2+ Crude compound;

[0009] 2) In phycocyanin-Zn 2+ A crude extract of phycocyanin was prepared by adding water to the crude complex. Ammonium sulfate was then slowly added to the crude phycocyanin extract, and the mixture was allowed to stand at 3-4°C for 1.5-2.5 hours. After centrifugation, the extract was dissolved in ultrapure water, dialyzed, and freeze-dried to obtain phycocyanin-Zn. 2+ complex;

[0010] 3) Phycocyanin-Zn 2+ The complex was dissolved in ultrapure water to obtain phycocyanin-Zn. 2+ The complex solution consists of ellagic acid solution and phycocyanin-Zn 2+ The complex solutions were mixed in proportion, thoroughly mixed in the dark, centrifuged, and the supernatant was collected, rotary evaporated, and freeze-dried to obtain phycocyanin-Zn. 2+ - Ellagic acid complex.

[0011] In the process of this invention, phycocyanin in Spirulina undergoes three main pigment protection processes, specifically: First, a freeze-thaw extraction method is used to extract phycocyanin. Traditional extraction processes (alkali dissolution and acid precipitation) easily damage the conjugated system of chromophores, leading to pigment loss and decreased bioactivity, while the freeze-thaw method can reduce the damage to phycocyanin during extraction; Second, the metal ion Zn is added before extraction. 2+ Metal ions Zn 2+ Phycocyanin is regulated by charge neutralization effect, forming a coordination bond with aspartic acid near the chromophore, Zn 2+ The secondary structure of phycocyanin is stabilized by coordination bonds, while β-sheets are increased. Freeze-thaw cycles avoid interference from chemical solvents. Metal ions provide dual protection to the chromophore conjugated system by inhibiting free radical chain reactions and enhancing the hydrogen bond network, thus preserving the native conformation of phycocyanin. To further improve color protection, ellagic acid is used to further treat phycocyanin after extraction. Ellagic acid's antioxidant properties scavenge free radicals, inhibiting oxidative stress damage to the chromophores and reducing pigment degradation. It can also bind to phycocyanin through hydrogen bonds or hydrophobic interactions, further stabilizing its tertiary structure, especially protecting the microenvironment surrounding the chromophores. Furthermore, it has UV absorption capabilities, acting as a "sunscreen" to reduce direct UV damage to phycocyanin and enhance photostability. With the above triple color protection, not only can phycocyanin be extracted from Spirulina efficiently, but the phycocyanin is also protected for pigment, which significantly enhances the intensity of the 620nm characteristic absorption peak and the fluorescence emission efficiency, and significantly improves the pigment retention rate under high temperature (70℃) and ultraviolet light irradiation (60h).

[0012] Furthermore, the concentration of the spirulina solution is 0.02-0.03 g / mL.

[0013] Furthermore, the zinc salt contained in the zinc salt solution is selected from zinc sulfate, and its concentration is 0.02-0.1 mol / L.

[0014] Furthermore, the volume ratio of spirulina solution to zinc salt solution is 5-9:1.

[0015] Furthermore, the concentration of the crude phycocyanin extract is 4-5 mg / mL.

[0016] Furthermore, the freeze-thaw temperature is -18 to -15°C, and the freeze-thaw time is 24-36 hours.

[0017] Furthermore, phycocyanin-Zn 2+ The concentration of the complex solution was 0.9-1.1 mg / mL.

[0018] Furthermore, the solvent used in the ellagic acid solution is ethanol, and the concentration of the ellagic acid solution is 0.4-0.5 mg / mL.

[0019] Furthermore, ellagic acid solution and phycocyanin-Zn 2+ The volume ratio of the complex solution is 1:5.

[0020] Furthermore, in steps 1) and 3), the mixing speed is 500-1000 r / min and the stirring time is 1-2 h.

[0021] Furthermore, in steps 1) and 3), the centrifugation speed is 10000-12000 r / min.

[0022] Furthermore, in steps 2) and 3), the freeze-drying temperature is -60℃ to -50℃, and the freeze-drying time is 48-72h.

[0023] Furthermore, the dialysis time is 36-72 hours, and the dialysis cutoff is 3500 Da.

[0024] Furthermore, the amount of ammonium sulfate added is 30-40 wt% of the saturation amount of ammonium sulfate in the crude phycocyanin extract.

[0025] To achieve the second objective mentioned above, the present invention adopts the following technical solution:

[0026] This invention discloses a highly stable phycocyanin extracted using the process described above.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention discloses a process for extracting phycocyanin from Spirulina while simultaneously providing pigment protection. In this process, Zn... 2+ Forming coordination bonds with aspartic acid near the chromophore significantly increases the β-sheet content, thereby stabilizing the secondary structure of phycocyanin and inhibiting pigment degradation under high temperature (70℃) and ultraviolet light. Simultaneously, the freeze-thaw method reduces damage to phycocyanin during extraction. The synergistic effect of these two methods effectively enhances the intensity of characteristic absorption peaks and thermo-photostability. Furthermore, to further improve the color-protecting effect, ellagic acid is used to further treat phycocyanin after extraction. Through its antioxidant properties, ellagic acid can scavenge free radicals, inhibiting oxidative stress damage to the phycocyanin chromophore, thus reducing pigment degradation. It can also bind to phycocyanin through hydrogen bonds or hydrophobic interactions, further stabilizing its tertiary structure, especially protecting the microenvironment around the chromophore. It also has ultraviolet absorption capabilities, acting as a "sunscreen" to reduce direct damage to phycocyanin from ultraviolet light and enhance photostability. Attached Figure Description

[0029] Figure 1 The DSC curves are for the phycocyanin-metal ion binary complexes of Example 1 and Comparative Examples 1-5.

[0030] Figure 2 The images show the ultraviolet spectra of the supernatants obtained after crude extraction in Examples 1 and 1-5.

[0031] Figure 3a This is one of the fluorescence spectra of the supernatant obtained after crude extraction in Example 1 and Comparative Examples 1-5.

[0032] Figure 3b The second fluorescence spectrum is shown for the supernatant obtained after crude extraction in Example 1 and Comparative Examples 1-5.

[0033] Figure 4 The image shows the DSC curve of the ternary complex of phycocyanin-metal ion-polyphenol in Example 4.

[0034] Figure 5 This is a comparison chart of pigment retention rates for samples from Examples 1 and 4. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0037] Example 1

[0038] 1) Crude extraction: A 0.025 g / mL Spirulina solution was prepared using water as a solvent and mixed with a 0.06 mol / L zinc sulfate solution at a volume ratio of 9:1. The mixture was stirred at 1000 r / min for 1.5 h. The mixture was then frozen at -18 °C for 24 h and rapidly thawed in a 37 °C water bath. This freeze-thaw process was repeated 4 times. The mixture was then centrifuged at 10000 r / min for 10 min, and the supernatant was collected and freeze-dried to obtain phycocyanin-Zn. 2+ Crude compound.

[0039] 2) Purification: Take phycocyanin-Zn 2+ The crude compound was prepared into a crude phycocyanin extract with a concentration of 5 mg / mL. Ammonium sulfate solid was slowly added until the ammonium sulfate reached 40% of its saturation in the system. The mixture was stirred until completely dissolved, allowed to stand at 4°C for 2 hours, centrifuged at 10000 rpm for 15 minutes, and the precipitate was collected. The precipitate was dissolved in ultrapure water, dialyzed for 48 hours, and then freeze-dried to obtain phycocyanin-Zn. 2+ Binary complex, for later use, abbreviated as PC-Zn 2+ .

[0040] Example 2

[0041] The preparation method is the same as in Example 1, except that the zinc sulfate solution is changed to 0.02 mol / L.

[0042] Example 3

[0043] The preparation method is the same as in Example 1, except that the zinc sulfate solution is changed to 0.1 mol / L.

[0044] Comparative Example 1

[0045] The preparation method is the same as in Example 1, except that the zinc sulfate solution is replaced with a sodium sulfate solution, abbreviated as PC-Na. + .

[0046] Comparative Example 2

[0047] The preparation method is the same as in Example 1, except that the zinc sulfate solution is replaced with a magnesium sulfate solution, abbreviated as PC-Mg. 2+ .

[0048] Comparative Example 3

[0049] The preparation method is the same as in Example 1, except that the zinc sulfate solution is replaced with a manganese sulfate solution, abbreviated as PC-Mn. 2+ .

[0050] Comparative Example 4

[0051] The preparation method is the same as in Example 1, except that the zinc sulfate solution is replaced with a copper sulfate solution, abbreviated as PC-Cu. 2+ .

[0052] Comparative Example 5

[0053] The preparation method is the same as in Example 1, except that the zinc sulfate solution is replaced with an ferric sulfate solution, abbreviated as PC-Fe. 3+ .

[0054] Test Example 1

[0055] 1. Phycocyanin yield

[0056] The results are shown in Table 1. It can be seen that the process method of the present invention can achieve a higher yield.

[0057] Table 1

[0058] sample Yield / % Example 1 15.48±0.14 Example 2 13.46±0.04 Example 3 14.17±0.13 Comparative Example 1 12.58±0.03 Comparative Example 2 11.85±0.11 Comparative Example 3 9.58±0.05 Comparative Example 4 7.39±0.07 Comparative Example 5 6.12±0.10

[0059] 2. Color difference analysis

[0060] The supernatant obtained after crude extraction in Examples 1-3 and Comparative Examples 1-5 was measured using a colorimeter. The sample was thoroughly shaken before measurement and calibrated using a white plate. The photometer was calibrated before use.

[0061] The results are shown in Table 2. It can be seen that the coordination of metal ions with phycocyanin has a significant impact on color; a positive b-value indicates yellow, and a negative b-value indicates blue. The b-value of PC (blank group) without added metal ions is -2. Among the formulations with added metal ions, those with added zinc ions (Examples 1-3) have the smallest b-values ​​and the bluest color. This phenomenon is mainly due to the specific action mechanism of zinc ions on the phycocyanin structure. Molecular docking simulation studies show that Zn... 2+ The binding site to phycocyanin monomers may be closer to the chromophore than that of other metal ions. The chromophore is the core region determining the blue light absorption of phycocyanin (absorption peak at 620 nm). Zn 2+ By binding to amino acid residues surrounding the chromophore, the conjugated structure or electron distribution of the chromophore may be optimized, thereby enhancing the absorption efficiency of blue light and making the blue color purer and more pronounced. The binding energy is higher than that of other metal ions, resulting in a more stable coordination structure. Zinc ions may induce increased hydrophobicity on the protein surface, reducing interference from water molecules around the chromophore and maintaining the stability of the color-emitting environment; enhanced hydrophobicity may also reduce protein molecule aggregation or light scattering effects in the aqueous phase, resulting in a purer blue color.

[0062] Table 2

[0063]

[0064]

[0065] 3. Stability

[0066] 1) Effect of temperature on stability: The supernatant obtained after crude extraction in Examples 1-3 and Comparative Examples 1-5 was placed in a water bath and heated at 70°C for 10 min. The absorbance before and after heating was measured, and the pigment retention rate of different samples was calculated.

[0067] 2) Effect of light on stability: The phycocyanin-metal ion binary complexes of Examples 1-3 and Comparative Examples 1-5 were placed in test tubes and irradiated under ultraviolet light with a wavelength of 620 nm for 60 h to test the pigment retention rate of the samples.

[0068] The results are shown in Table 3.

[0069] Table 3

[0070]

[0071] 3) DSC test: Differential scanning calorimetry was used for measurement. 1 mg of phycocyanin-metal ion binary complex was accurately weighed into a solid crucible and compressed into a tablet for measurement. The measurement conditions were set as follows: initial temperature 30℃, final temperature 100℃, and heating rate 10℃ / min.

[0072] See results Figure 1 The addition of metal ions generally improves the thermal stability of PC; the DSC curve of PC shows a distinct melting peak at 71.3℃. After the addition of metal ions, the melting point temperature order is PC-Zn. 2+ (Example 1) > PC-Na + (Comparative Example 1) > PC-Cu 2+ (Comparative Example 4) > PC-Mg 2+ (Comparative Example 2) > PC-Mn 2+ (Comparative Example 3) > PC-Fe 3+ (Comparative Example 5). PC-Zn 2+ A distinct melting peak appears at 74.7℃, which is the highest temperature among the peaks, indicating that Zn... 2+ The addition of Zn improves PC stability. This is because Zn 2+ Zn is a transition metal ion with high charge density that can form coordinate bonds with certain amino acids in proteins. This interaction significantly stabilizes the local protein structure. Furthermore, Zn...2+ By combining inducible colocal conformational changes in proteins, the β-sheet content is increased, making their structure more compact.

[0073] 4. Particle size and zeta potential analysis

[0074] The particle size of the supernatant obtained after crude extraction in Examples 1-3 and Comparative Examples 1-5 was analyzed using a dynamic light scattering device. The potential and particle size of the PC solution were measured in automatic mode at an equilibrium temperature of 25°C.

[0075] The results are shown in Table 4. The particle size of PC in the blank group was approximately 163 nm. The addition of metal ions increased the particle size. + (Comparative Example 1) has a particle size of approximately 192 nm, PC-Mg 2+ (Comparative Example 2) has a particle size of approximately 202 nm, PC-Mn 2+ (Comparative Example 3) has a particle size of approximately 221 nm, PC-Zn 2+ (Example 1) has a particle size of 214 nm, PC-Zn 2+ (Example 2) has a particle size of 195 nm, PC-Zn 2+ (Example 3) has a particle size of 215 nm, PC-Cu 2+ (Comparative Example 4) has a particle size of 231 nm, PC-Fe 3+ The particle size of (Comparative Example 5) was 242 nm. This increase is attributed to the positive charge of the metal ions, which can neutralize some of the negatively charged groups on the PC molecules. In solution, the positively charged metal ions interact with the negatively charged carboxylic acid groups on the PC molecules, promoting PC aggregation and increasing the PC particle size. Among all metal ions, PC-Zn... 2+ The PDI is the lowest, ranging from 0.28 to 0.32, indicating that it is more uniformly dispersed and more stable.

[0076] Table 4

[0077] sample Particle size / nm PDI Blank group 163.83±1.71 0.32 Example 1 214.74±2.71 0.32 Example 2 195.29±3.44 0.28 Example 3 215.62±2.75 0.30 Comparative Example 1 192.96±3.61 0.33 Comparative Example 2 202.45±5.17 0.45 Comparative Example 3 221.36±5.89 0.35 Comparative Example 4 231.69±5.50 0.40 Comparative Example 5 242.81±4.17 0.41

[0078] 5. Ultraviolet and fluorescence spectroscopy analysis

[0079] The ultraviolet spectra of the supernatants obtained after crude extraction in Example 1 and Comparative Examples 1-5 were analyzed in the range of 200-700 nm using an ultraviolet spectrophotometer at a temperature of 25°C.

[0080] The fluorescence spectra of the supernatants obtained after crude extraction in Examples 1-3 and Comparative Examples 1-5 were analyzed using a fluorescence spectrophotometer (F-7100). The excitation wavelengths were 330 nm and 645 nm, respectively, and the emission wavelengths were 280-380 nm and 600-700 nm, respectively. The slit width was set to 10 nm.

[0081] Figure 2 The images show the UV absorption spectra of phycocyanin (PC) and its binary complexes with metal ions. PC exhibits a characteristic absorption peak at 620 nm, due to the covalent bonding of its linear tetrapyrrole chromophore, phycocyanin, to the PC subunit via an ether bond. Proteins containing aromatic amino acids such as tryptophan and tyrosine show a characteristic absorption peak at 280 nm. Compared to PC, PC-Na... + (Comparative Example 1), PC-Mg 2+ (Comparative Example 2) and PC-Zn 2+ (Example 1) The absorption intensity of the composite at 620 nm increased, in which PC-Zn 2+ The highest absorption peak is found in PC-Mn. 2+ (Comparative Example 3), PC-Fe 3+ (Comparative Example 5) and PC-Cu 2+ (Comparative Example 4) The absorption peaks of the complex were all lower than those of PC. PC-Zn 2+ The enhanced absorption at 620 nm indicates that Zn 2+ It possesses strong coordination ability. Zn 2+ Coordination with Zn can enhance the conjugation effect of the chromophore or induce charge transfer, increasing the probability of electron transitions from the ground state to the excited state. This results in a stronger absorption peak. 2+ It may act as a structural stabilizer, binding to specific sites on PC to maintain the stability of its secondary structure. A stable conformation ensures a more ordered arrangement of chromophores, reduces non-radiative energy loss, and improves light absorption efficiency. Zn 2+ The binding can alter the hydrophobicity of amino acid residues surrounding the chromophore, reduce interference from solvent polarity, and further enhance absorbance. In contrast, Mn 2+ Cu 2+ and Fe 3+ Cu has weak coordination ability and poor ionic radius compatibility, and cannot effectively stabilize the microenvironment around the chromophore. 2+ and Fe 3+ Its strong oxidizing properties may cause oxidative stress or protein aggregation, damaging the chromophore structure and resulting in an absorption peak lower than that of natural PC.

[0082] See fluorescence spectrum Figure 3a and Figure 3b PC has two main sources of fluorescence. The first is intrinsic fluorescence, primarily derived from tyrosine residues, which are key components of the α and β subunits of PC and are excited at 330 nm. The second source is attributed to the tetrapyrrole chromophore, which emits characteristic fluorescence when excited at 645 nm. Figure 3a The fluorescence spectra of PC with different added metal ions at an excitation wavelength of 330 nm are shown. No peak shift was observed after adding different metal ions. PC-Zn 2+and PC-Na + The fluorescence intensity of the complex is higher than that of natural PC, while PC-Fe 3+ PC-Cu 2+ PC-Mn 2+ and PC-Mg 2+ The low fluorescence intensity of the complex indicates fluorescence quenching. Figure 3b The fluorescence spectra of PC with different metal ions at an excitation wavelength of 645 nm are shown, and their variation trends are similar to those of... Figure 3a Consistent. These results indicate that Zn 2+ and Na + It can improve the chromophore efficiency of PC. Na + The negative charge on the PC surface is neutralized through weak electrostatic interactions, reducing intermolecular collisions and dynamic quenching. Zn 2+ It forms stable coordination bonds with amino acids surrounding the PC chromophore, strengthening the hydrogen bond network on the β-sheet and improving fluorescence efficiency. Conversely, Fe... 3+ and Cu 2+ As a strong oxidizing agent, it oxidizes PC and disrupts its conjugated system. These changes alter the π-electron cloud distribution of the chromophore, reducing the energy required for electronic transitions. The conditions previously favorable for fluorescence production are disrupted, increasing non-radiative transitions and decreasing fluorescence intensity. Consequently, the concentration of phycocyanin in solution is significantly reduced, resulting in a marked decrease in the blue color of the solution.

[0083] To further improve the color-protecting effect, polyphenols were added to the system, and the specific research is as follows.

[0084] Example 4

[0085] Preparation of the finished product: Ellagic acid was dissolved in anhydrous ethanol to obtain an ellagic acid solution with a concentration of 0.4 mg / mL. The phycocyanin-Zn obtained in Example 1 was then added... 2+ The complex was dissolved in ultrapure water and stirred magnetically in the dark to obtain phycocyanin-Zn with a concentration of 1 mg / mL. 2+ The complex solution consists of ellagic acid solution and phycocyanin-Zn 2+ The complex solutions were mixed at a volume ratio of 1:5 and stirred magnetically (1000 rpm) for 2 hours in the dark to ensure thorough mixing. The mixture was then centrifuged at 10000 rpm, and the supernatant was collected. Ethanol was removed by rotary evaporation at 35°C, followed by freeze-drying to obtain phycocyanin-Zn. 2+ - Ellagic acid ternary complex, abbreviated as PC-Zn 2 + -Ea.

[0086] Example 5

[0087] The preparation method is the same as in Example 4, except that the ellagic acid solution is changed to 0.1 mg / mL.

[0088] Example 6

[0089] The preparation method is the same as in Example 4, except that the ellagic acid solution is changed to 1 mg / mL.

[0090] Comparative Example 6

[0091] The preparation method is the same as in Example 4, except that the ellagic acid solution is replaced with a quercetin solution.

[0092] Comparative Example 7

[0093] The preparation method is the same as in Example 4, except that the ellagic acid solution is replaced with ferulic acid solution.

[0094] Test Example 2

[0095] 1. Color difference analysis

[0096] The testing procedure was the same as in Test Example 1, and the results are shown in Table 5. The experiment showed that the b-value decreased significantly after the addition of ellagic acid, which may be due to the combined effect of the saturation effect of polyphenol-protein interactions and steric hindrance. Phycocyanin has polyphenol-binding sites on its surface, and ellagic acid may bind to these sites through hydrogen bonds and hydrophobic interactions to form a protective complex, slowing down pigment degradation. When the concentration of ellagic acid reaches a certain level, the effective binding sites on the protein surface are completely occupied; further increasing the concentration of ellagic acid at this point cannot increase the binding amount, and the protective effect no longer strengthens. Furthermore, ellagic acid is a polyphenolic hydroxyl compound, and its conjugated double bonds and multiple phenolic hydroxyl groups give it extremely strong free radical scavenging ability. This strong antioxidant property can more effectively inhibit the oxidative degradation of phycocyanin, thereby maintaining pigment stability. The oxygen free radical absorption capacity of ellagic acid is generally higher than that of quercetin and ferulic acid. Ellagic acid can bind to the chromophore (phycocyanin) of phycocyanin through π-π stacking or hydrogen bonds to form a more stable complex. This binding can reduce the degradation of the chromophore exposed to light / heat, thereby maintaining the color difference b-value. Ellagic acid's planar rigid structure and polyphenolic hydroxyl groups make it easier to bind to the hydrophobic regions of proteins, while the flexible structure or steric hindrance of ferulic acid and quercetin may reduce binding efficiency.

[0097] Table 5

[0098] sample b value Blank group -2.00±0.01 Example 4 -4.13±0.01 Example 5 -4.05-±0.13 Example 6 -3.96±0.04 Comparative Example 6 -3.91±0.03 Comparative Example 7 -3.15±0.18

[0099] 2. Stability

[0100] 1) DSC test: The phycocyanin-Zn obtained in Example 4 was subjected to DSC test. 2+ The ellagic acid ternary complex was tested using the same procedure as in Test Example 1, and the results are shown below. Figure 4 .

[0101] 2) Effect of temperature on stability: The test procedure was the same as in Test Example 1, and the results are shown in Table 6. Ellagic acid can form stable complexes with protein molecules, thereby improving the stability of phycocyanin under acidic or high-temperature conditions. This interaction may include hydrogen bonding and hydrophobic interactions, which can effectively reduce protein degradation and denaturation. Although ferulic acid and quercetin also have some stabilizing effect, their intermolecular forces may be weaker or less specific than those of ellagic acid. Ferulic acid mainly relies on the binding of a single phenolic hydroxyl group and carboxyl group, resulting in a relatively weak force. Although quercetin can form hydrogen bonds, the flexibility of the flavonoid skeleton makes it prone to breakage at high temperatures, leading to the exposure of binding sites. The rigid structure of ellagic acid makes it less prone to deformation at high temperatures, maintaining the integrity of the binding interface. Ferulic acid (small molecule) and quercetin (flexible molecule) may easily undergo rotation or vibration at high temperatures, leading to the detachment of binding sites.

[0102] Table 6

[0103]

[0104] In addition, the phycocyanin-Zn obtained in Example 4 will also be used. 2+ - Ellagic acid ternary complex and cyanin-Zn obtained in Example 1 2+ The pigment retention rates of the binary complexes were compared, and the results are shown in [reference needed]. Figure 5 It can be observed that in Example 4, phycocyanin-Zn 2+ - Ellagic acid ternary complex and blue protein-Zn obtained in Example 1 2+ The pigment retention rates of the binary complexes were not significantly different when heated at 60°C for 10 minutes, but after heating at 70°C for 10 minutes, the phycocyanin-Zn obtained in Example 4 showed a significant difference. 2+ The pigment retention rate of the ellagic acid ternary complex is higher than that of the cyanin-Zn obtained in Example 1. 2+ Binary complex.

[0105] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A process for extracting phycocyanin from Spirulina while simultaneously providing pigment protection, characterized in that, Includes the following steps: 1) Prepare a spirulina solution by adding zinc salt solution, mixing thoroughly, freezing and thawing 3-4 times, centrifuging, collecting the supernatant, and freeze-drying to obtain phycocyanin-Zn. 2+ Crude compound; 2) In phycocyanin-Zn 2+ A crude extract of phycocyanin was prepared by adding water to the crude complex. Ammonium sulfate was then slowly added to the crude phycocyanin extract, and the mixture was allowed to stand at 3-4°C for 1.5-2.5 hours. After centrifugation, the extract was dissolved in ultrapure water, dialyzed, and freeze-dried to obtain phycocyanin-Zn. 2+ complex; 3) Phycocyanin-Zn 2+ The complex was dissolved in ultrapure water to obtain phycocyanin-Zn. 2+ The complex solution consists of ellagic acid solution and phycocyanin-Zn 2+ The complex solutions were mixed in proportion, thoroughly mixed in the dark, centrifuged, and the supernatant was collected, rotary evaporated, and freeze-dried to obtain phycocyanin-Zn. 2+ - Ellagic acid complex.

2. The process method according to claim 1, characterized in that, The concentration of the spirulina solution is 0.02-0.03 g / mL; and / or The zinc salt contained in the zinc salt solution is selected from zinc sulfate, and its concentration is 0.02-0.1 mol / L.

3. The process method according to claim 2, characterized in that, The volume ratio of spirulina solution to zinc salt solution is 5-9:

1.

4. The process method according to claim 1, characterized in that, The concentration of the crude phycocyanin extract is 4-5 mg / mL.

5. The process method according to claim 1, characterized in that, Phycocyanin-Zn 2+ The concentration of the complex solution is 0.9-1.1 mg / mL; and / or The solvent used in the ellagic acid solution is ethanol, and the concentration of the ellagic acid solution is 0.4-0.5 mg / mL.

6. The process method according to claim 5, characterized in that, Ellagic acid solution and phycocyanin-Zn 2+ The volume ratio of the complex solution is 1:

5.

7. The process method according to claim 1, characterized in that, In steps 1) and 3), the mixing speed is 500-1000 r / min and the stirring time is 1-2 h.

8. The process method according to claim 1, characterized in that, In steps 1) and 3), the centrifugation speed is 10000-12000 r / min.

9. The process method according to claim 1, characterized in that, The amount of ammonium sulfate added should be 30-40 wt% of the saturation amount of ammonium sulfate in the crude phycocyanin extract.

10. A phycocyanin with high stability, characterized in that, It is obtained by the process method described in any one of claims 1-9.

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