New method for preparing anthocyanin-phycocyanin peptide gel based on green health concept

Abstract

This invention discloses a novel method for preparing anthocyanin-phycocyanin peptide gel based on the concept of green health, belonging to the field of food processing and ingredient technology. The invention involves microwave-assisted enzymatic hydrolysis of phycocyanin to obtain phycocyanin peptides, followed by molecular weight fractionation of the phycocyanin peptides, and finally mixing them with anthocyanins and gellan gum to prepare the anthocyanin-phycocyanin peptide gel. This invention innovatively proposes a novel method for preparing anthocyanin-phycocyanin peptide gel. In the current trend of the food industry pursuing natural, healthy, and sustainable development, this method solves the problem of easy oxidation of anthocyanins during processing. It is a green and efficient preparation process, providing an innovative solution for the preparation of functional food ingredients. Furthermore, the overall process is green and safe, requires no large and complex equipment, and is simple to operate.

Description

Technical Field

[0001] This invention relates to a novel method for preparing anthocyanin-phycocyanin peptide gel based on the concept of green health, and belongs to the field of food processing and ingredient technology. Background Technology

[0002] Phycocyanin (C-PC) is a phycobiliprotein found in cyanobacteria, red algae, and some cryptophytes. Its protein backbone is covalently linked to the chromophore phycocyanobilin, an open-chain tetrapyrrole bile pigment, which imparts its characteristic absorption peak and blue fluorescence (Chen et al., 2022). Phycocyanin and its enzymatically hydrolyzed peptides exhibit good water solubility and biocompatibility. Related studies have shown that it demonstrates antioxidant, anti-inflammatory, and immunomodulatory activities both in vitro and in vivo (Li, 2022), indicating broad application prospects in functional foods and health ingredients.

[0003] Anthocyanins belong to the flavonoid family and are common water-soluble pigments found in nature. Numerous studies have shown that anthocyanins possess antioxidant functions such as scavenging free radicals, chelating metal ions, and donating hydrogen (electrons) (Enaru et al., 2021). In terms of cardiovascular protection, anthocyanins and their metabolites can promote the production of nitric oxide (NO) in vascular endothelium, improving arterial compliance and vasodilation (Xu et al., 2004). In terms of anti-inflammatory effects, anthocyanins can inhibit the NF-κB signaling pathway and reduce the levels of various pro-inflammatory mediators, thereby helping to alleviate inflammatory responses (Karlsen et al., 2007). Furthermore, anthocyanins can interact with rhodopsin, increasing rhodopsin stability and improving dark adaptation and low-light vision (Kalt et al., 2010).

[0004] However, anthocyanins are easily degraded or inactivated by factors such as pH, temperature, light, oxidants, and digestive enzymes in the gastrointestinal environment and during food processing (Pez Jaeschke et al., 2021; Zang et al., 2022), leading to a decrease in their antioxidant capacity and color stability. Currently, strategies for improving the stability of anthocyanins mainly include the following categories: Spray-dried microencapsulation, using maltodextrin, gum arabic, or gelatin as wall materials, is a simple process, but high temperatures (150-220°C) easily cause anthocyanin thermal degradation, and the powder is highly hygroscopic and has poor acid stability (Mohammadalinejhad & Kurek, 2021). Ionogel methods, using alginate or its combination with pectin, protein, etc., to form gel beads, can delay release to some extent, but the system is highly sensitive to pH and ionic strength, prone to initial burst release, and the gel is mainly polysaccharide, with limited molecular interaction with anthocyanins (Seke et al., 2022). Liposome systems can improve storage stability, but the encapsulation efficiency is low (≤50%), sensitive to oxidation and bile salts, prone to leakage or discoloration, and some preparation processes involve organic solvents, which does not meet the requirements of green food processing (Guldiken et al., 2017). Cyclodextrin inclusion complexes can improve photothermal stability and have mature processing techniques, but their loading capacity is limited and significantly affected by solvent composition and pH, making it difficult to achieve on-demand release under digestion conditions, and some processes still require organic solvents (Ali et al., 2024). In general, existing anthocyanin encapsulation and delivery systems suffer from problems such as high energy consumption at high temperatures, dependence on chemical cross-linking or organic solvents, difficulty in balancing mechanical strength and stability, and burst release due to pH sensitivity, limiting their application in food processing and the gastrointestinal environment. Therefore, there is an urgent need for a green and healthy delivery system that uses food-grade natural peptides as a backbone, constructs a three-dimensional network in aqueous phase and under mild conditions, requires no chemical cross-linking agents, and possesses both acid-resistant stability and controllable release performance. This invention addresses these problems by proposing a method for preparing anthocyanin-phycocyanin peptide gels based on a green and healthy concept, to achieve highly stable loading and targeted release of anthocyanins. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a method for preparing anthocyanin-phycocyanin peptide gel based on the concept of green health. This anthocyanin-phycocyanin peptide gel with antioxidant properties can solve the technical problem that the application of phycocyanin in terms of antioxidant activity is limited due to its large molecular weight and poor solubility. At the same time, it can protect anthocyanins and improve their application characteristics.

[0006] This invention provides a method for preparing anthocyanin-phycocyanin peptide gel, which mainly includes the following steps: S1: Microwave-assisted solubilization of phycocyanin A certain amount of commercially available phycocyanin was weighed and added to an appropriate amount of deionized water to prepare a phycocyanin aqueous solution. Subsequently, the solution was placed in a microwave field for treatment. Through the synergistic effect of the thermal and non-thermal effects of microwaves, the interactions within the phycocyanin molecules were altered, thereby achieving a solubilization effect and obtaining a phycocyanin solution with a looser structure and improved solubility.

[0007] S2: Enzymatic hydrolysis and product separation of phycocyanin The phycocyanin solution obtained in step S1 was transferred to a constant temperature device and incubated at a set temperature to allow the phycocyanin to reach a suitable state for enzymatic hydrolysis. Subsequently, an appropriate amount of a specific biological enzyme was added to the solution, and the enzymatic hydrolysis reaction was initiated. After terminating the enzyme activity using an appropriate enzyme inactivation method, the enzyme-inactivated solution was centrifuged.

[0008] S3: Molecular weight classification of phycocyanin peptides The phycocyanin peptide solution obtained in step S2 was fractionated using ultrafiltration technology to achieve different molecular weight ranges.

[0009] S4: Gel preparation based on phycocyanin peptides Accurately weigh appropriate amounts of anthocyanins and gellan gum, and add them to the phycocyanin peptide solution with a specific molecular weight range obtained in step S3. Transfer the mixed solution to a rotating device in an incubator for rotation incubation. Under suitable temperature and phycocyanin peptide concentration conditions, anthocyanins, gellan gum, and phycocyanin peptides form a gel with specific structure and properties.

[0010] In one embodiment of the present invention, in S1, the ratio of phycocyanin to water is 1:4~8, g / mL, the microwave field parameters are 100~600W, and the microwave treatment time is 5~30min.

[0011] In one embodiment of the present invention, in S2, the temperature for heating and incubation is 45~55°C.

[0012] In one embodiment of the present invention, in S2, the bioenzyme is one of papain, flavorzyme, bromelain, and neutral protease; the amount of protease added is 1-10 units / mg (based on substrate mass). Wherein, U (Unit) represents an enzyme activity unit, defined as: the amount of enzyme required to catalyze the conversion of 1 μmol of substrate into the corresponding product per minute under specified reaction conditions. To ensure consistent measurement standards, the definition and measurement conditions of enzyme activity units in the present invention are as follows: For papain, flavorzyme, bromelain, and neutral protease, using casein as a substrate, reacting at pH 7.0 and 37°C for 10 min, 1U is defined as above. Under the above definition, an enzyme activity addition amount of 1-10 U / mg means: adding an enzyme preparation with 1-10 enzyme activity units per milligram of phycocyanin substrate. The setting of the enzyme activity ratio aims to ensure that the protein is fully enzymatically hydrolyzed under mild conditions, avoiding excessive hydrolysis that could lead to peptide chain damage or loss of functional groups.

[0013] In one embodiment of the present invention, in S2, the enzymatic hydrolysis conditions are: temperature of 45~60℃ and time of 0.5~1.5h.

[0014] In one embodiment of the present invention, in S2, the enzyme inactivation treatment conditions are as follows: temperature is 95~100℃, and time is 10~20min.

[0015] In one embodiment of the present invention, in S2, the centrifugation conditions are as follows: temperature is 4~15℃, rotation speed is 8000~10000rpm, and time is 10~30min.

[0016] In one embodiment of the present invention, in S3, the phycocyanin peptides with different molecular weight ranges include phycocyanin peptides with molecular weights of <10kDa, 10kDa~30kDa, and >30kDa.

[0017] In one embodiment of the present invention, in S4, the amount of gellan gum added is 5-20% of the amount of phycocyanin protein, and the amount of anthocyanin added is 10-20% of the amount of phycocyanin protein.

[0018] In one embodiment of the present invention, in step S4, the rotation speed is 100~200 rpm, the temperature is 15~45℃, and the time is 0.5~2 h, until the anthocyanins are completely dissolved and form a gel.

[0019] The present invention provides anthocyanin-phycocyanin peptide gel prepared by the method described above.

[0020] This invention provides the application of the anthocyanin-phycocyanin peptide gel described above in the biomaterials, food, and cosmetics industries.

[0021] [Beneficial Effects] (1) Phycocyanin peptide gel can provide a relatively stable environment for anthocyanins, reducing their loss during processing and storage. The combination of the two can improve their stability and utilization in the food system. Specifically, the gel network structure formed by the cross-linking of phycocyanin peptides provides a physical barrier for anthocyanins, reducing the direct contact between anthocyanins and external factors such as oxygen, moisture, and light, and reducing the oxidation and hydrolysis of anthocyanins by these factors, thereby improving the stability of anthocyanins.

[0022] (2) The prepared phycocyanin-phycocyanin peptide has a certain swelling property. It can absorb a certain amount of water and swell in aqueous solution. The pore structure of the gel is relatively uniform and the pore size is moderate. The diffusion of anthocyanin molecules in the pores of the gel is restricted, which increases the diffusion distance and can prolong its storage time.

[0023] (3) The raw materials used to prepare the anthocyanin-phycocyanin peptide gel are natural and safe.

[0024] (4) From a functional perspective, the prepared anthocyanin-phycocyanin peptide gel has multiple beneficial effects. First, anthocyanins, as a natural and powerful antioxidant, can effectively scavenge free radicals in the human body, reduce oxidative stress damage to cells, and help prevent various chronic diseases. The gel formed by its combination with phycocyanin peptides can be slowly released in the gastrointestinal environment, prolonging the action time of anthocyanins and improving their bioavailability. Second, phycocyanin peptides themselves are rich in a variety of amino acids and have good nutritional properties, which can provide the human body with necessary nutrients and enhance the body's immunity. In addition, the special structure of the gel gives it good water retention and stability, and it can be used as a high-quality thickener and stabilizer in food processing to improve the texture and taste of food, and has broad application prospects in food innovation and development. Attached Figure Description

[0025] Figure 1 Images of the anthocyanin-phycocyanin peptide gels in Examples 1-4, and cross-sectional scanning electron microscope (SEM) images at 500 μm, 200 μm, and 100 μm. Figure 2 SDS gel electrophoresis images of the phycocyanin peptides prepared in Examples 1-4; Figure 3 Images showing the appearance of anthocyanin-phycocyanin peptide gels in comparative examples 1-5; Figure 4 This is a flowchart illustrating the preparation process of the anthocyanin-phycocyanin peptide gel of the present invention. Detailed Implementation

[0026] Materials used in the examples: 1. Phycocyanin is sourced from Binmei Biotechnology Co., Ltd., catalog number E30, with a purity of 3.

[0027] 2. Papain is derived from Maclean's, CAS number 9001-73-4, with an enzyme activity of 200 U / mg. Enzyme activity is defined as the amount of enzyme required, expressed as u / g, to produce trichloroacetic acid-soluble substances at a wavelength of 275 nm at a rate equivalent to the absorbance of 1 microgram of tyrosine, using ultraviolet spectrophotometry under the following conditions (37±0.2℃; pH 7.0). 3. Bromelain is derived from Maclean's, CAS number 9001-00-7, with an enzyme activity of 300 U / mg. Enzyme activity is defined as: the amount of enzyme required to release trichloroacetic acid-soluble matter from casein per minute at a wavelength of 275 nm with an absorbance equivalent to that of 1 microgram of tyrosine, using ultraviolet spectrophotometry under the following conditions (37±0.2℃; pH 7.0). This amount is expressed as u / g.

[0028] 4. Flavorzyme is derived from Maclean's, CAS number 9001-92-7, with an enzyme activity of 100 U / mg. Enzyme activity is defined as the amount of enzyme required, expressed as u / g, when the absorbance of the trichloroacetic acid-soluble substance released per minute from hydrolyzed casein is equivalent to that of 1 microgram of tyrosine at a wavelength of 275 nm, measured by ultraviolet spectrophotometry under the following conditions (37±0.2℃; pH 7.0).

[0029] 5. Neutral protease is derived from Maclean's, CAS number 9068-59-1, with an enzyme activity of 200 U / mg. Enzyme activity is defined as the amount of enzyme required, expressed as u / g, to produce trichloroacetic acid-soluble matter at a wavelength of 275 nm at a measured temperature of 37±0.2℃ and pH 7.0, when the absorbance is equivalent to that of 1 microgram of tyrosine.

[0030] Test method: (1) Scanning electron microscopy test After the samples were frozen and fractured in liquid nitrogen, surface and cross-sectional samples were taken and sputtered with gold. The microstructure of the gel at 500 μm, 200 μm, and 100 μm was then analyzed by scanning electron microscopy (SEM, S-4800, Hitachi Co., Tokyo, Japan) at an accelerating voltage of 5 kV.

[0031] (2) Polyacrylamide gel electrophoresis (SDS-PAGE) analysis SDS gel electrophoresis, or sodium dodecyl sulfate polyacrylamide gel electrophoresis, is mainly used for the separation and analysis of proteins, and includes the following steps: 1. Prepare reagents and materials: Prepare acrylamide, methylenebisacrylamide, SDS, Tris-HCl buffer, ammonium persulfate, TEMED (tetramethylethylenediamine), protein samples, loading buffer, markers, etc. 2. Gel preparation: Mix acrylamide and methylenebisacrylamide in the specified proportions, add SDS, Tris-HCl buffer, etc., and bring to a final volume with deionized water. Add ammonium persulfate and TEMED to initiate the polymerization reaction, first preparing the separating gel, and then preparing the stacking gel after it solidifies. 3. Processing protein samples: Mix the protein sample with loading buffer, heat at 100°C or in a boiling water bath for 3-5 minutes, immerse the gel in staining solution (such as Coomassie Brilliant Blue staining solution) for a period of time, and then destain with destaining solution until the protein bands are clearly visible, thus denaturing the protein. 4. Sample loading: Add the denatured protein sample and marker to the sample loading wells of the gel; 5. Electrophoresis: Turn on the power and start electrophoresis at a lower voltage (e.g., 80V). After the sample enters the separating gel, increase the voltage (e.g., 120V) and continue electrophoresis until the bromophenol blue indicator migrates to the vicinity of the bottom of the gel. 6. Staining and destaining: SDS gel electrophoresis can separate proteins based on their molecular weight. The operation is relatively simple and it is widely used in the field of protein research.

[0032] (3) Full texture testing Texture profile analysis was performed using a texture analyzer. Fresh hydrogel was placed on a fixed base plate (P / 5 probe, 5 mm diameter) under the probe until deformation reached 60%. Speeds were set to 1.0, 0.5, and 1 mm / s before, during, and after the test, respectively. The trigger force was 5 g, and the time between loadings was 5 s. Bead texture (e.g., hardness, elasticity, cohesion, elasticity, gelatinousness, and chewiness) was calculated using Texture Expert software version 1.22.

[0033] (4) Determination of anthocyanin encapsulation efficiency (EE) The encapsulation efficiency of anthocyanins in the gel was determined using the pH differential method. The specific steps are as follows: 1. Extraction of free anthocyanins: Collect the supernatant and eluent from the gel surface during the preparation of anthocyanin-phycocyanin peptide gel.

[0034] 2. Extraction of total anthocyanins from the gel: Accurately weigh a certain mass of the prepared anthocyanin-phycocyanin peptide gel and place it in a methanol solution containing 0.1% hydrochloric acid. Under light-protected, ice-bath conditions, perform ultrasonic disruption (300W ultrasonic power, 20 min) to break down the three-dimensional network structure of the gel and completely release the embedded anthocyanins. Then, centrifuge at 8000 rpm for 10 min at 4℃ and collect the supernatant. Repeat the extraction 2-3 times until the gel is colorless. Combine the supernatants and bring the volume to a final volume.

[0035] 3. Content Determination: Take the above extracts and dilute them with 0.025 mol / L potassium chloride buffer (pH 1.0) or 0.4 mol / L sodium acetate buffer (pH 4.5). After standing at room temperature for 30 min in the dark, measure the absorbance at 520 nm and 700 nm using a UV-Vis spectrophotometer. The formula for calculating the absorbance difference A of anthocyanin content is as follows:

[0036] The formula for calculating anthocyanin concentration C (as cyanidin-3-glucoside, mg / L) is as follows:

[0037] Where: MW is the relative molecular mass of cyanidin-3-glucoside (449.2 g / mol); DF is the dilution factor; The molar extinction coefficient (26900 L·mol) -1 ·cm -1 L is the optical path length of the cuvette (1 cm).

[0038] 4. Encapsulation ratio calculation:

[0039] (5) Determination of retention rate under ultraviolet light irradiation This test aims to simulate a high-intensity light environment and evaluate the gel's ability to protect against anthocyanins.

[0040] Equal amounts of free anthocyanin powder and anthocyanin-phycocyanin peptide gels (containing equal amounts of anthocyanins) prepared in each example / comparative example were accurately weighed and spread evenly in uncovered transparent petri dishes. The petri dishes were placed in a UV irradiation chamber with pre-set parameters, using a 254 nm wavelength UV lamp as the light source (fixed light intensity), and continuously irradiated at room temperature (25℃). Samples were taken before irradiation (0 h) and 168 h after irradiation.

[0041] The residual anthocyanin concentration in the gel at the sampling point was determined according to the gel total anthocyanin extraction and pH differential method described in “(4)” above.

[0042] The calculation formula is as follows:

[0043] (6) 60℃ high temperature half-life (t) 1 / 2 Determination of ) This test was used to evaluate the thermal stability of anthocyanin-phycocyanin peptide gel under common thermal stresses during food processing and storage.

[0044] Accurately weigh several portions of the anthocyanin-phycocyanin peptide gel prepared in each example / comparative example, and seal them separately in light-proof glass ampoules or screw-cap centrifuge tubes. Place the sealed tubes in a constant temperature water bath or incubator set at 60°C and conduct accelerated thermal degradation tests under light-proof conditions. Remove the samples at preset time intervals (0, 4, 8, 12, 24, 48 h) and quickly immerse them in ice water to cool and terminate the thermal degradation reaction.

[0045] The residual anthocyanin concentration was extracted and determined according to the method described in "(4)". The data were fitted using a first-order reaction kinetic model:

[0046] Among them, C t Let Ct be the anthocyanin concentration at time t, C0 be the initial anthocyanin concentration, and k be the first-order degradation rate constant (ht). -1 ), where t is the heating time (h). Based on the fitted rate constant k, the time required for half of the anthocyanin to degrade is calculated, i.e., the half-life (t). 1 / 2 ):

[0047] Example 1 This embodiment provides a method for preparing anthocyanin-phycocyanin peptide gel, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin Weigh 20g of phycocyanin and add it to 100mL of deionized water to prepare an aqueous solution of phycocyanin. Then, place the solution in a microwave field at 200 W for 15 min to induce changes in the intramolecular interactions of phycocyanin, thereby achieving a solubilization effect and obtaining a phycocyanin solution with a looser structure and improved solubility.

[0048] S2: Enzymatic hydrolysis and product separation of phycocyanin The phycocyanin solution obtained in step S1 was transferred to a constant temperature device and incubated at 55 °C for 20 min to allow the phycocyanin to reach a suitable state for enzymatic hydrolysis. Subsequently, papain at 2% (w / w) of phycocyanin content was added to the solution, and the enzymatic hydrolysis reaction was carried out at 55 °C for 1 h. After terminating enzyme activity by treatment at 95 °C for 10 min, the enzyme-inactivated solution was centrifuged at (10000 rpm, 10 min, 4 °C) to obtain a phycocyanin peptide solution.

[0049] S3: Molecular weight classification of phycocyanin peptides The phycocyanin peptide solution obtained in step S2 was fractionated using an ultrafiltration tube to separate phycocyanin peptides with molecular weights of <10kDa, 10kDa~30kDa, and >30kDa.

[0050] S4: Gel preparation based on phycocyanin peptides Accurately weigh 4 g of anthocyanins and 1.5 g of gellan gum, and add them to the phycocyanin peptide solution with a molecular weight range of 10 kDa to 30 kDa obtained in step S3. Transfer the mixture to a rotating device in an incubator and incubate at 30°C and 200 rpm for 60 min. Anthocyanins, gellan gum, and phycocyanin peptides will form a gel with specific structure and properties.

[0051] Example 2 This embodiment provides a method for preparing anthocyanin-phycocyanin peptide gel, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The steps are the same as S1 in Example 1.

[0052] S2: Enzymatic hydrolysis and product separation of phycocyanin The phycocyanin solution obtained in step S1 was transferred to a constant temperature device and incubated at 50 °C for 20 min to allow the phycocyanin to reach a suitable state for enzymatic hydrolysis. Subsequently, 1.5% (w / w) of bromelain was added to the solution, and the enzymatic hydrolysis reaction was carried out at 50 °C for 1 h. After terminating enzyme activity by treatment at 95 °C for 10 min, the enzyme-inactivated solution was centrifuged at 10,000 rpm for 10 min at 4 °C to obtain the phycocyanin peptide solution.

[0053] S3: Molecular weight classification of phycocyanin peptides The steps are the same as S3 in Example 1.

[0054] S4: Gel preparation based on phycocyanin peptides Accurately weigh 4 g of anthocyanins and 1.5 g of gellan gum, and add them to the phycocyanin peptide solution with a molecular weight >30 kDa obtained in step S3. Transfer the mixed solution to a rotating device in an incubator and incubate at 30°C and 200 rpm for 60 min. Anthocyanins, gellan gum, and phycocyanin peptides will form a gel with specific structure and properties.

[0055] Example 3 This embodiment provides a method for preparing anthocyanin-phycocyanin peptide gel, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The steps are the same as S1 in Example 1.

[0056] S2: Enzymatic hydrolysis and product separation of phycocyanin The phycocyanin solution obtained in step S1 was transferred to a constant temperature device and incubated at 53 °C for 20 min to allow the phycocyanin to reach a suitable state for enzymatic hydrolysis. Subsequently, 1.5% (w / w) of flavor protease was added to the solution, and the enzymatic hydrolysis reaction was carried out at 53 °C for 1.5 h. After terminating enzyme activity by treatment at 95 °C for 10 min, the enzyme-inactivated solution was centrifuged at 10000 rpm for 10 min at 4 °C to obtain a phycocyanin peptide solution.

[0057] S3: Molecular weight classification of phycocyanin peptides The steps are the same as S3 in Example 1.

[0058] S4: Gel preparation based on phycocyanin peptides Accurately weigh 2 g of anthocyanin and 1 g of gellan gum, and add them to the phycocyanin peptide solution with a molecular weight >30 kDa obtained in step S3. Transfer the mixed solution to a rotating device in an incubator and incubate at 30 °C and 200 rpm for 60 min. A gel with specific structure and properties will form between the anthocyanin, gellan gum, and phycocyanin peptide.

[0059] Example 4 This embodiment provides a method for preparing anthocyanin-phycocyanin peptide gel, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The steps are the same as S1 in Example 1.

[0060] S2: Enzymatic hydrolysis and product separation of phycocyanin The phycocyanin solution obtained in step S1 was transferred to a constant temperature device and incubated at 45 °C for 20 min to allow the phycocyanin to reach a suitable state for enzymatic hydrolysis. Subsequently, 1.5% (w / w) of neutral protease was added to the solution, and the enzymatic hydrolysis reaction was carried out at 45 °C for 0.5 h. After terminating enzyme activity by treatment at 95 °C for 10 min, the enzyme-inactivated solution was centrifuged at 10,000 rpm for 10 min at 4 °C to obtain the phycocyanin peptide solution.

[0061] S3: Molecular weight classification of phycocyanin peptides The steps are the same as S3 in Example 1.

[0062] S4: Gel preparation based on phycocyanin peptides Accurately weigh 3 g of anthocyanin and 1 g of gellan gum, and add them to the phycocyanin peptide solution with a molecular weight of <10 kDa obtained in step S3. Transfer the mixed solution to a rotating device in an incubator and incubate at 40 °C and 150 rpm for 90 min. Anthocyanin, gellan gum, and phycocyanin peptides will form a gel with specific structure and properties.

[0063] Figure 1 Images of the anthocyanin-phycocyanin peptide gels prepared in Examples 1-4, along with their cross-sectional microstructures under different magnifications (500 μm, 200 μm, and 100 μm), are presented. The SEM images show that the gels prepared in these examples all exhibit a continuous and uniform three-dimensional porous honeycomb network structure at the microscopic level. Example 4 exhibits the densest pore distribution with small and uniform pore sizes and thick, continuous pore walls. This highly dense microporous structure confirms the strong multiple non-covalent cross-linking between the low molecular weight peptides (<10 kDa) and gellan gum and anthocyanins, directly explaining the highest hardness, highest encapsulation efficiency, and best photothermal stability observed in this group during macroscopic testing. Examples 1 and 2 exhibit a more complete but slightly larger porous structure, with the high molecular weight peptides contributing better flexibility to the pore walls. In contrast, the comparative examples (see description of comparative examples) that were not enzymatically hydrolyzed or had an imbalanced formulation often exhibited structural collapse or a rough and disordered surface, making microscopic observation impossible and resulting in poor ability to effectively lock in internal active substances. The SEM results strongly confirm the success of the method of this invention in constructing a uniform and stable physical barrier.

[0064] Figure 2 These are SDS-PAGE gel electrophoresis images of the phycocyanin peptides prepared in Examples 1-4. Figure 2 It is evident that undigested native phycocyanin typically exhibits characteristic α and β subunit concentration bands around 17–20 kDa. However, in the lanes of this invention, after treatment with "microwave-assisted unfolding + combined enzymatic hydrolysis," the large protein bands completely disappear, replaced by continuous, diffuse polypeptide bands in the low molecular weight region. This fully demonstrates the thoroughness and efficiency of the enzymatic hydrolysis reaction in step S2 of this invention. Furthermore, comparing the ultrafiltration fraction lanes of different embodiments clearly shows that after ultrafiltration fractionation in step S3, the products are successfully and precisely retained within the target molecular weight range (e.g., <10 kDa fractions do not tail to the high molecular weight region, and >30 kDa fractions show no obvious bands in the low molecular weight region). SDS-PAGE results verify the accuracy and reliability of the fractionation process of this invention, providing a solid material basis and theoretical foundation for subsequent "on-demand customization of gel texture and embedding performance" by selecting polypeptides of different molecular weights.

[0065] Table 1. Complete texture table of anthocyanin-phycocyanin peptide gels prepared in Examples 1, 2, 3, and 4

[0066] As shown in Table 1, the textural properties (TPA) test data revealed that different enzymatic hydrolysis processes, peptide molecular weight distributions, and raw material ratios significantly affected the microstructure and macroscopic texture of the anthocyanin-phycocyanin peptide gel. Specific analysis follows: 1. Hardness, adhesiveness, and chewability analysis The hardness of the gel directly reflects the density and resistance to deformation of the three-dimensional network of the system. Test results showed that Example 4 had the highest hardness (183.296±2.936 g), adhesiveness (76.523±2.811%), and chewiness (33.474±4.522%) among the four groups. This indicates that although the amount of gellan gum used in Example 4 was relatively low (1 g), the use of low molecular weight phycocyanin peptides (<10 kDa) and sufficient anthocyanin addition (3 g) allowed the small peptides, with their smaller steric hindrance, to more fully expose their active groups, forming dense and strong non-covalent crosslinks (such as hydrogen bonds and hydrophobic interactions) with anthocyanins and polysaccharide molecules, thus constructing an extremely dense and rigid gel framework. Conversely, Example 3 had the lowest hardness (157.243±1.248 g) and chewiness (26.811±0.283%) among all groups. The reason is that this group not only used a lower amount of gellan gum (1g), but also reduced the amount of anthocyanin to 2g, and used high molecular weight peptides (>30KDa). High molecular weight peptides have significant steric hindrance, and coupled with insufficient concentration of anthocyanins acting as cross-linking agents, the resulting polysaccharide-protein-polyphenol gel network is relatively loose, and the structural support is reduced.

[0067] 2. Elasticity and Cohesion Analysis Elasticity reflects the ability of a gel to recover its original shape after being deformed under pressure. Comparing examples with consistent gellan gum and anthocyanin dosages (1.5 g gellan gum, 4 g anthocyanins), the elasticity of Example 2 (high molecular weight peptide >30 kDa) (0.142 ± 0.002%) was slightly higher than that of Example 1 (medium molecular weight peptide 10–30 kDa, 0.13 ± 0.008%). This indicates that long-chain, high molecular weight peptides provide better molecular chain flexibility and buffer space within the gel, giving it superior deformation recovery and flexibility; while the medium molecular weight peptides of Example 1 provide a moderate balance between rigidity and elasticity. Furthermore, the cohesiveness of each example remained stable between 0.417 ± 0.029% and 0.436 ± 0.044%, and the adhesiveness remained between 0.399 ± 0.004% and 0.421 ± 0.012%, with minimal fluctuations between groups. This confirms that the anthocyanin-phycocyanin peptide gel system described in this invention has excellent compatibility and gelation stability. Even under different component parameter adjustments, its internal binding force (cohesion) still maintains good uniformity.

[0068] Table 2. Encapsulation efficiency and stability of anthocyanin-phycocyanin peptide gels prepared in Examples 1, 2, 3, and 4.

[0069] Of all the examples, Example 4 exhibited the best performance (encapsulation efficiency 92.65 ± 1.18%, UV retention 72.33 ± 1.48%, high-temperature half-life 51.42 h). This is consistent with the highest hardness shown in its texture analysis. Due to the use of low molecular weight phycocyanin peptides (<10 kDa), their minimal steric hindrance allowed for full exposure of the active groups (amino acid residues), forming an extremely dense non-covalent cross-linked network with gellan gum and anthocyanins. This high-density "physical shield" significantly hindered water migration, oxygen permeation, and internal conduction of photothermal energy. Simultaneously, abundant hydrogen bonds stabilized the anthocyanin structure, giving the system the strongest resistance to photothermal degradation. Examples 1 and 2 also showed high encapsulation efficiency and stability, confirming that peptides of different molecular weights can form a good protective barrier when properly proportioned. Example 3, due to its large peptide molecular weight and low gellan gum concentration, had a relatively loose network, resulting in the lowest performance among the examples.

[0070] This invention effectively achieves customized gel texture through targeted enzymatic hydrolysis and fractionation of phycocyanin and dynamic control of formulation ratios. For a high-strength, chew-resistant gel system, a peptide of <10kDa combined with a high concentration of anthocyanins can be used (as in Example 4); for a gel with better elasticity and a softer texture, a high molecular weight peptide of >30kDa can be used (as in Example 2). This preparation method greatly expands the application potential of anthocyanin-phycocyanin peptide gels in different health food dosage forms (such as gummies, jellies, or swallowing aids).

[0071] Comparative Example 1 Phycocyanin cannot form a stable gel structure without enzymatic hydrolysis. The specific technical solution is as follows, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The same steps as S1 in Example 1 were followed to obtain a phycocyanin solution.

[0072] S2: Gel preparation based on phycocyanin solution.

[0073] Accurately weigh 2 g of anthocyanin and 1 g of gellan gum, and add them to the phycocyanin solution obtained in step S1. Transfer the mixture to a rotating device in an incubator and incubate at 40 °C and 150 rpm for 90 min.

[0074] Because phycocyanin has not undergone enzymatic hydrolysis, it retains its large molecular weight and high-order protein structure, resulting in significant steric hindrance. This intact or semi-incomplete macromolecular state means that its internal active functional groups (such as hydrophobic amino acid residues and hydrogen bond sites) are deeply embedded within the molecule and cannot be fully exposed. Therefore, unhydrolyzed phycocyanin struggles to engage in effective non-covalent interactions (such as electrostatic interactions, hydrogen bonds, and hydrophobic interactions) with small anthocyanins and polysaccharides (gellan gum). It cannot function as an effective "cross-linking node" to participate in the construction of a uniform three-dimensional gel network, leading to the components forming only loose suspensions or precipitates within the system, ultimately failing to form a stable gel structure.

[0075] Comparative Example 2 If the amount of gellan gum added is too small, a stable gel structure cannot be formed. The specific technical solution is as follows, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The same steps as S1 in Example 1 were followed to obtain a phycocyanin solution.

[0076] S2: Enzymatic hydrolysis and product separation of phycocyanin The same steps as S2 in Example 1 were followed to obtain a phycocyanin peptide solution.

[0077] S3: Molecular weight classification of phycocyanin peptides The same steps as S3 in Example 1 were followed to obtain phycocyanin peptides with different molecular weight distributions.

[0078] S4: Gel preparation based on phycocyanin peptides Accurately weigh 2 g of anthocyanins and 0.1 g of gellan gum, and add them to the phycocyanin peptide solution with a molecular weight <10 kDa obtained in step S3. Transfer the mixture to a rotating device in an incubator and incubate at 40 °C and 150 rpm for 90 min.

[0079] Gellan gum plays a crucial role in constructing the polysaccharide backbone and providing basic three-dimensional network support in gel systems. However, the amount of gellan gum added in this comparative example was too small (only 0.1 g, lower than the 5-20% ratio requirement of phycocyanin peptides specified in this invention), resulting in a polysaccharide concentration below the critical concentration required to form a continuous gel network. Under such extremely low polysaccharide concentrations, even though phycocyanin peptides and anthocyanins exhibit good binding ability, there are insufficient long polysaccharide chains to link, cross-link, and lock in moisture. Therefore, the system macroscopically only presents as a free liquid or loose flocculent dispersion, unable to solidify and form a solid shape, thus losing its physical encapsulation and barrier protection function for anthocyanins.

[0080] Comparative Example 3 Excessive amounts of gellan gum have been added. The specific technical solution is as follows, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The same steps as S1 in Example 1 were followed to obtain a phycocyanin solution.

[0081] S2: Enzymatic hydrolysis and product separation of phycocyanin The same steps as S2 in Example 1 were followed to obtain a phycocyanin peptide solution.

[0082] S3: Molecular weight classification of phycocyanin peptides The same steps as S3 in Example 1 were followed to obtain phycocyanin peptides with different molecular weight distributions.

[0083] S4: Gel preparation based on phycocyanin peptides Accurately weigh 2 g of anthocyanins and 5 g of gellan gum, and add them to the phycocyanin peptide solution with a molecular weight range of 10 kDa to 30 kDa obtained in step S3. Transfer the mixture to a rotating device in an incubator and incubate at 30°C and 200 rpm for 60 min.

[0084] Because the amount of gellan gum added was too large (far exceeding the 5-20% range of phycocyanin content specified in this invention), the viscosity of the system increased rapidly in a very short time, causing anthocyanins and phycocyanin peptides to fail to disperse uniformly in the solvent. The gel network became rigid, extremely brittle, and its pores shrank drastically due to excessive cross-linking of polysaccharides. This not only caused a large amount of anthocyanin to aggregate and precipitate, but also destroyed the original uniform swelling and controlled release ability of the gel system, indicating that only within the specified range of gellan gum addition can an anthocyanin-phycocyanin peptide gel with uniform texture and high encapsulation efficiency be obtained.

[0085] Comparative Example 4 Without microwave assistance, the specific technical solution is as follows, including the following steps: S1: Commercially available phycocyanin dissolution Weigh 20 g of phycocyanin and add it to 100 mL of deionized water to prepare an aqueous solution of phycocyanin. Do not microwave; simply stir and mix at room temperature for 15 min to obtain the phycocyanin solution.

[0086] S2: Enzymatic hydrolysis and product separation of phycocyanin The phycocyanin solution obtained in step S1 was transferred to a constant temperature device and incubated at 55 °C for 20 min. Subsequently, papain at 2% (w / w) of the phycocyanin content was added to the solution, and the enzymatic hydrolysis reaction was carried out at 55 °C for 1 h. After terminating the enzyme activity by treatment at 95 °C for 10 min, the enzyme-inactivated solution was centrifuged at 10,000 rpm for 10 min at 4 °C to obtain the phycocyanin peptide solution.

[0087] S3: Molecular weight classification of phycocyanin peptides The same steps as S3 in Example 1 were followed to obtain phycocyanin peptides with different molecular weight distributions.

[0088] S4: Gel preparation based on phycocyanin peptides The steps are the same as S4 in Example 1.

[0089] Because microwave-assisted processing was omitted, phycocyanin molecules failed to undergo effective unfolding through the thermal and non-thermal effects of microwaves, resulting in insufficient exposure of their internal hydrophobic groups and enzyme cleavage sites. This led to a significant reduction in enzymatic hydrolysis efficiency in S2, resulting in severely insufficient peptide yield and exposure of active groups. In the S4 cross-linking stage, the lack of sufficient active small peptides to engage in non-covalent interactions (such as hydrogen bonds and hydrophobic interactions) with gellan gum and anthocyanins resulted in a loose gel structure with extremely high water content that could not maintain a fixed shape, even exhibiting a semi-fluid state, greatly weakening the physical barrier protection for anthocyanins.

[0090] Comparative Example 5 Phycocyanin peptides are added directly to the phycocyanin peptide solution obtained in S2 without fractionation. The specific technical solution is as follows, including the following steps: S1: Microwave-assisted solubilization of commercially available phycocyanin The same steps as S1 in Example 2 were followed to obtain a phycocyanin solution.

[0091] S2: Enzymatic hydrolysis and product separation of phycocyanin The same steps as S2 in Example 2 were performed to obtain a mixed phycocyanin peptide solution containing all molecular weight fragments.

[0092] S3: Gel preparation based on mixed phycocyanin peptides Accurately weigh 2 g of anthocyanins and 1 g of gellan gum, and add them directly to the unfractionated mixed phycocyanin peptide solution obtained in step S2. Transfer the mixture to a rotating device in an incubator and incubate at 30 °C and 200 rpm for 60 min.

[0093] Because no ultrafiltration fractionation was performed, the mixed peptide solution contained high-molecular-weight peptides, low-molecular-weight peptides, and even incompletely hydrolyzed large protein residues. These molecules with different chain lengths and steric hindrances competed fiercely and bound randomly with the gellan gel, resulting in an extremely uneven distribution of cross-linking sites in the gel's three-dimensional network. Macroscopically, this manifested as a rough gel surface, highly unstable texture (locally overly hard, locally hydrolyzed), and a chaotic microscopic pore structure. This heterogeneous structure not only prevented targeted and controlled release of anthocyanins but also easily triggered an initial "burst release" phenomenon, making it difficult to meet the requirements for stable anthocyanin delivery.

[0094] Table 3. Encapsulation efficiency and stability of anthocyanin-phycocyanin peptide gels prepared in Comparative Examples 1, 2, 3, 4, and 5.

[0095] The performance indicators of Comparative Example 1 (undigested) decreased significantly (encapsulation efficiency was only 42.36 ± 2.15%). Due to the unfolding of the large protein molecules, the huge steric hindrance hindered effective cross-linking, and a large amount of anthocyanins were free outside the gel network, directly exposed to ultraviolet light and high temperature, and rapidly degraded.

[0096] Comparative Example 2 (with very little gellan gum) performed the worst (encapsulation efficiency 18.75 ± 1.84%, high-temperature half-life only 15.46 h). The extremely low polysaccharide concentration could not support the solidification and molding of the system, and could only form a loose flocculent dispersion, which basically completely lost the physical encapsulation and barrier effect on anthocyanins, and its half-life was close to the decay rate of free anthocyanins.

[0097] Comparative Example 3 (excessive gellan gum) had an encapsulation efficiency of only 51.21 ± 2.43%. Excessive cross-linking of the system led to a sharp contraction of pores, and the strong "extrusion effect" forced a large amount of anthocyanins to aggregate and precipitate out and be exposed on the gel surface or in microcracks, making them extremely susceptible to photothermal damage.

[0098] Comparative Example 4 (without microwave assistance) resulted in insufficient protein defolding, low enzymatic hydrolysis efficiency, insufficient active sites for cross-linking, and a gel with high water content and loose structure, exhibiting significant physical leakage.

[0099] Comparative Example 5 (unfractionated peptides) resulted in disordered competitive binding between long and short chain peptides and polysaccharides, leading to a chaotic microporous structure. This heterogeneous network easily triggers anthocyanin "burst release," which in turn leads to a decrease in stability data.

[0100] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing anthocyanin-phycocyanin peptide gel, characterized in that, Includes the following steps: S1. Add phycocyanin to water to obtain a phycocyanin aqueous solution, and then microwave it. S2. Next, add biological enzymes to the phycocyanin aqueous solution for enzymatic hydrolysis, then inactivate the enzymes and centrifuge to obtain a phycocyanin peptide solution. S3. Use ultrafiltration technology to fractionate the phycocyanin peptide solution to different molecular weight ranges. S4. Add anthocyanins and gellan gum to the phycocyanin peptide solutions with different molecular weight ranges obtained in S3 and incubate them to obtain anthocyanin-phycocyanin peptide gel.

2. The method according to claim 1, characterized in that, In S1, the mass-to-volume ratio of phycocyanin to water is 1:4~8, g / mL; the microwave parameters are 100~600W, and the microwave treatment time is 5~30min.

3. The method according to claim 1, characterized in that, In S2, the bio-enzyme is one of papain, flavor protease, bromelain, or neutral protease.

4. The method according to claim 1, characterized in that, In S2, the amount of the added bio-enzyme is 1~10 units / mg, based on substrate mass.

5. The method according to claim 1, characterized in that, In S2, the enzymatic hydrolysis conditions are as follows: temperature 45~60℃, time 0.5~1.5h; the enzyme inactivation treatment conditions are as follows: temperature 95~100℃, time 10~20min.

6. The method according to claim 1, characterized in that, In S3, phycocyanin peptides of different molecular weight ranges include those with a molecular weight <10kDa, a molecular weight of 10kDa~30kDa, and a molecular weight >30kDa.

7. The method according to claim 1, characterized in that, In S4, the amount of gellan gum added is 5-20% of the phycocyanin protein content.

8. The method according to claim 1, characterized in that, In S4, the amount of anthocyanins added is 10-20% of the phycocyanin protein content.

9. The anthocyanin-phycocyanin peptide gel prepared by any one of the methods described in claims 1 to 8.

10. The application of the anthocyanin-phycocyanin peptide gel according to claim 9 in the fields of biomaterials, food, and cosmetics.

Images