A sodium alginate-leafy grass protein double-network gel bead embedded with postbiotics and a preparation method thereof

Abstract

This invention discloses a sodium alginate-leafwort protein dual-network gel bead with encapsulated metagenic elements and its preparation method. The method includes: dissolving sodium alginate and leafwort protein to obtain a composite solution; adjusting the pH to neutral, adding laccase to perform a cross-linking reaction, and obtaining an enzyme-cross-linked composite solution after inactivation; adding a metagenic element and mixing thoroughly; and solidifying the mixture by dropping it into a coagulation bath containing calcium ions. This invention utilizes laccase to catalyze the formation of dityrosine covalent bonds from tyrosine residues in leafwort protein, combined with ionic cross-linking between calcium ions and sodium alginate, to construct a stable dual-network structure. The resulting gel beads exhibit significantly improved mechanical properties, a dense and uniform internal structure, high encapsulation rate of metagenic elements, low release rate in simulated gastric juice, and targeted release in simulated intestinal juice. This invention is environmentally friendly and uses a mild process, making it widely applicable to the development of functional foods, health products, and pharmaceutical preparations.

Description

Technical Field

[0001] This invention relates to the technical field of hydrogel composites and applications, specifically to a sodium alginate-leafwort protein dual-network gel bead with encapsulated post-genetic agents and its preparation method. Background Technology

[0002] Postbiotics are an emerging concept in the functional food field, referring to preparations of inactive microorganisms and their components that provide health benefits to the host. The International Society for the Study of Probiotics and Prebiotics (ISAPP) defines "postbiotics" as metabolites or cell lysates of probiotics. Recent research indicates that the health benefits of probiotics do not entirely depend on the live bacteria themselves; their metabolites and bacterial components can also be key drivers of health-promoting effects. With increasing public awareness of gut health, the probiotic, prebiotic, and postbiotic industries have shown enormous market potential. Compared to traditional live probiotics, postbiotics do not contain live bacteria, offering greater safety and making them particularly suitable for individuals with weakened immune systems (such as newborns and the elderly). Furthermore, postbiotics exhibit higher stability and safety during processing, storage, and transportation, without the potential risks of bacteremia, thus gaining widespread attention in the functional food and pharmaceutical fields in recent years. Postbiotics possess various biological activities, including modulating host immunity, improving intestinal barrier function, and antagonizing pathogens.

[0003] Hydrogels are hydrophilic biopolymers with a three-dimensional network structure. While insoluble in water, hydrogels possess strong hydrophilicity due to the presence of numerous hydrophilic groups (such as hydroxyl and carboxyl groups). Their three-dimensional network structure allows them to maintain a solid shape even after absorbing large amounts of water and swelling. The porous structure of hydrogels allows for the inclusion of drugs and functional active ingredients within a matrix. Furthermore, their high water content, soft texture, good biocompatibility, and excellent mechanical properties make them well-suited for the encapsulation and controlled release of bioactive substances or drugs.

[0004] Sodium alginate (SA) is a natural anionic polysaccharide abundant in seaweed. Due to its thickening, emulsifying, and gelling properties, as well as its advantages such as low cost, low cytotoxicity, and biodegradability, it has become a preferred natural polysaccharide, widely used in the food, medical, and pharmaceutical industries. In the presence of divalent ions, the carboxyl groups of sodium alginate can interact with calcium ions under extremely mild conditions through electrostatic interactions to form a three-dimensional network structure with a unique "egg-box" structure. Therefore, sodium alginate can be easily transformed into a reversible hydrogel.

[0005] Traditional protein production methods cannot meet the future needs of human life in terms of quantity, quality, and sustainable supply. Therefore, large-scale, low-cost, sustainable, and high-quality protein production and application urgently require innovation and development. In 2021, leafy greens were listed as a new food ingredient by the National Health Commission of my country. With a protein content as high as 36%, it is also known as "protein grass." Leafy green protein has a balanced amino acid composition and is rich in aromatic amino acids such as tyrosine. This structural characteristic endows it with good hydrophilicity and potential cross-linking activity.

[0006] Laccase is a copper-containing polyphenol oxidase that uses only oxygen as a cosubstrate and water as its sole byproduct, representing a green and mild reaction process. Laccase specifically catalyzes the oxidation of tyrosine residues to generate reactive free radicals, which can then bind together to form stable dityrosine covalent cross-links. This catalytic mechanism is highly compatible with the tyrosine-rich structural characteristics of leafy plants. The formation of robust covalent cross-links within and between molecules through these dityrosine bonds significantly enhances the rigidity and stability of the network and may strengthen the interactions between the protein and polysaccharide interfaces.

[0007] However, while hydrogels offer certain advantages in encapsulating and delivering active substances, their mechanical properties, stability, and functional regulation capabilities are often limited. For example, monopolysaccharide hydrogels often exhibit poor mechanical properties, loose internal structures, and insufficient water-holding capacity. Therefore, improving the mechanical properties and structural stability of hydrogels to achieve efficient encapsulation and targeted release of metabiotics is a pressing technical problem to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a sodium alginate-leafgrass protein dual-network gel bead for encapsulating metagenes and its preparation method, so as to overcome the defects of poor mechanical properties and loose structure of single sodium alginate hydrogel, and achieve efficient encapsulation of metagenes and targeted release in the gastrointestinal environment.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing sodium alginate-leafwort protein dual-network gel beads with encapsulated post-biotics, comprising the following steps:

[0010] Step 1: Dissolve sodium alginate and leafy green protein in a solvent to obtain a composite solution;

[0011] Step 2: Adjust the pH of the composite solution to neutral, add laccase to carry out the cross-linking reaction, and inactivate the laccase after the reaction is completed to obtain the enzyme cross-linked composite solution;

[0012] Step 3: Add postbiotic to the enzyme cross-linking complex solution, mix well, and obtain the bacterial loading mixture;

[0013] Step 4: Drop the bacterial mixture into a coagulation bath containing divalent metal ions to solidify and form gel beads.

[0014] Preferably, in step one, the mass fraction of sodium alginate is 2% to 4%, and the mass fraction of leafwort protein is 0.25% to 2%.

[0015] Preferably, the sodium alginate has a mass fraction of 3%, and the leafwort protein has a mass fraction of 0.5%.

[0016] Preferably, in step two, the amount of laccase added is 1–3 mg / mL of the composite solution, the cross-linking reaction time is 1–3 hours, and the reaction temperature is 35–45°C.

[0017] Preferably, the amount of laccase added is 2 mg / mL, the cross-linking time is 2 hours, and the reaction temperature is 40℃.

[0018] Preferably, in step two, the conditions for inactivating laccase are: heating in a water bath at 90–100°C for 5–15 minutes.

[0019] Preferably, in step three, the amount of post-genetic agent added does not exceed 1% (w / v), and the mixing method is magnetic stirring for 20 to 40 minutes.

[0020] Preferably, in step four, the coagulation bath is a CaCl2 solution with a concentration of 2% to 4%, the solidification time is 20 to 40 minutes, and the dropping distance is 10 to 20 cm.

[0021] The present invention also provides a sodium alginate-leafwort protein dual-network gel bead with encapsulated post-genetic agent prepared by the above method, characterized in that the gel bead contains dityrosine covalent bonds formed by laccase catalysis of tyrosine residues in leafwort protein, and a sodium alginate network formed by cross-linking sodium alginate with calcium ions.

[0022] Preferably, the gel beads are used in the preparation of functional foods or health products.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention utilizes laccase to catalyze the formation of dityrosine covalent bonds from tyrosine residues in leafwort protein, constructing a protein cross-linking network. Simultaneously, it leverages calcium ions to form an "eggbox" structured ionic cross-linking network with the carboxyl groups of sodium alginate. The synergistic effect of these two networks significantly enhances the hardness, elasticity, and storage modulus of the gel beads compared to single sodium alginate gels or composite gels without laccase cross-linking. The dual-network structure effectively reduces the porosity of the gel beads, making their internal network denser and more regular, which improves the encapsulation efficiency of metagenics and delays their release in the acidic environment of the stomach. The dense internal network effectively prevents the outward diffusion of metagenics, resulting in a significantly higher encapsulation rate than gel beads prepared using other plant proteins (such as peanut protein, soy protein, and pea protein).

[0025] The gel beads prepared by this invention have a low release rate in simulated gastric fluid but can achieve rapid and complete release in simulated intestinal fluid, exhibiting good intestinal targeted delivery characteristics, and are suitable for oral functional foods or pharmaceutical preparations.

[0026] Using leafy grass as a novel plant protein source ensures sustainable availability; the laccase-catalyzed reaction uses oxygen as an auxiliary substrate and water as a byproduct, making it green and environmentally friendly; the entire preparation process does not require high temperature and high pressure, making it suitable for industrial production.

[0027] The method of the present invention is not only applicable to the encapsulation of postbiotics, but can also be extended to the delivery of other active substances (such as probiotics, peptides, vitamins, etc.), and can also be used in cosmetics, pharmaceuticals and other fields. Attached Figure Description

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0029] Figure 1 This is a flowchart illustrating the preparation process of the sodium alginate-leafgrass protein dual-network gel beads with encapsulated post-biotics according to the present invention.

[0030] Figure 2 The results show the test results of the textural properties of different gel samples in Examples 1, 2, and 3.

[0031] Figure 3 The results show the test results of the rheological properties of different gel samples in Examples 1, 2, and 3.

[0032] Figure 4 The results show the test results of the porosity of different gel samples in Examples 1, 2, and 3.

[0033] Figure 5 The images show the Fourier transform infrared (FTIR) spectra of different gel samples in Examples 1, 2, and 3.

[0034] Figure 6The images show the scanning electron microscope (SEM) microstructures of different gel samples from Examples 1, 2, and 3.

[0035] Figure 7 The images show the fluorescence spectra of laccase before and after cross-linking in Examples 2 and 3.

[0036] Figure 8 The results show the test results of the encapsulation efficiency of post-genetic agents by gel beads prepared from different proteins in Example 3. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0038] Test method:

[0039] (1) Determination of textural properties: The hardness and elasticity of the gel samples were determined using a textural analyzer. The test mode was TPA compression mode, the probe was a cylindrical probe, the test temperature was 25℃, and the deformation was set to 40%.

[0040] (2) Rheological property determination: The dynamic viscoelasticity of the gel sample was determined using a rheometer, and the changes in storage modulus (G′) and loss modulus (G″) with angular frequency were recorded. The test adopted frequency scanning mode, with a scanning frequency range of 0.1 to 10 Hz, a fixed deformation of 0.1%, and a test temperature of 25 ℃.

[0041] (3) Porosity determination: After freeze-drying the gel sample for 48 hours, weigh it (W1) and measure its volume (V). Immerse the freeze-dried sample in anhydrous ethanol for 12 hours, remove it, blot off the surface ethanol, and weigh it again (W2). Calculate the porosity (P) using the following formula:

[0042]

[0043] Wherein, W1 is the weight of the freeze-dried hydrogel (g); W2 is the weight of the hydrogel after soaking in ethanol (g); V is the volume of the hydrogel; ρ is the density of anhydrous ethanol; V is the volume of the hydrogel.

[0044] (4) Fourier transform infrared spectroscopy (FTIR) analysis: The freeze-dried sample was mixed with potassium bromide at a mass ratio of 1:100, ground, and compressed into tablets. The tablets were then heated at 500–4000 cm⁻¹. -1 Transmittance was measured within the wavenumber range to analyze changes in functional groups and interaction forces.

[0045] (5) Microstructure observation: The cross-sectional morphology of the freeze-dried gel sample was observed using field emission scanning electron microscopy. After gold sputtering, the sample was observed under an electron accelerating voltage of 3 kV.

[0046] (6) Dityrosine fluorescence spectroscopy determination: Based on the fluorescence characteristics of dityrosine, fluorescence spectroscopy was used to monitor the laccase crosslinking reaction. The sample was diluted to 0.1 mg / mL, the excitation wavelength was set to 310 nm, and the emission spectrum was scanned in the range of 290–500 nm. The excitation and emission slit widths were both set to 5 nm.

[0047] (7) Determination of post-genetic encapsulation efficiency: The encapsulation efficiency of the gel beads for the post-genetic was determined by ultraviolet spectrophotometry. The absorbance of the CaCl2 solution after curing the gel beads was measured at 600 nm (A2); an equal amount of blank gel beads without post-genetic encapsulation was taken, and an equal volume of CaCl2 solution and an equal amount of post-genetic were added, and the absorbance was measured (A1). The encapsulation efficiency was calculated according to the following formula:

[0048] EE (%) = 100 - ×100

[0049] (8) Determination of release rate of simulated gastrointestinal digestion in vitro: The gastrointestinal environment was simulated by sequential incubation. Simulated gastric juice: containing 2.0 g / L NaCl, pH 2.0±0.1, with 3.2 mg / mL pepsin added. Simulated intestinal juice: containing 6.8 g / L KH2PO4, pH 6.8±0.1, with 10 mg / mL pancreatin and 3 mg / mL bile salt added. The gel beads containing the postbiotic were placed in preheated simulated gastric juice and incubated at 37℃ and 100 rpm for 2 hours with shaking; then the pH was adjusted to 6.8, an equal volume of simulated intestinal juice was added, and incubation was continued for 4 hours with shaking, and the cumulative release rate of the postbiotic was measured.

[0050] All tests were repeated three times, and the results are expressed as mean ± standard deviation.

[0051] Example 1: Preparation of sodium alginate-leafwort protein dual-network gel (non-gel bead form)

[0052] This embodiment aims to investigate the effect of the amount of physalis protein added on gel properties.

[0053] A series of composite solutions containing 0%, 0.25%, 0.5%, 0.75%, 1%, and 2% protein from leafy greens were prepared, with the sodium alginate mass fraction fixed at 3%. The corresponding mass of powder was accurately weighed, mixed, and placed in a 50℃ constant-temperature magnetically stirred water bath. The mixture was stirred at 600 r / min for 2 hours, dissolved in deionized water, and prepared into 20 mL composite solutions.

[0054] Add 0.1% (w / v) gluconolactone (GDL) to the above composite solution, stir at room temperature for 15 minutes, then pour into a mold and let stand at 4°C for 12 hours to obtain sodium alginate-leaf grass protein double network hydrogel.

[0055] The obtained gel was subjected to texture, rheological, porosity, FTIR, and microstructure tests. The results are as follows: Figures 2-6 As shown, when the amount of edible grass protein added is 0.5%, the gel has the highest hardness, elasticity and storage modulus, the lowest porosity and the densest structure.

[0056] Example 2: Preparation of laccase-crosslinked sodium alginate-leafwort protein dual-network gel

[0057] This embodiment aims to investigate the effect of laccase crosslinking time on gel properties.

[0058] A 3% (w / v) sodium alginate solution and a 0.5% (w / v) leafminer protein solution were prepared, using the same dissolution method as in Example 1. The pH of the composite solution was adjusted to 7.0, and 2 mg / mL of laccase was added. The solutions were then crosslinked in a 40°C water bath for 0, 1, 1.5, 2, 2.5, and 3 hours, respectively. After crosslinking, the laccase was inactivated by heating in a 95°C water bath for 10 minutes.

[0059] Add 0.1% (w / v) GDL to the above system and stir at room temperature for 10 minutes. Then add 3% (w / v) calcium carbonate solution at a volume ratio of 1:5 (calcium carbonate solution: composite solution), continue stirring for 15 minutes, pour into a mold, and let stand at 4°C for 12 hours to obtain laccase-crosslinked sodium alginate-leafwort protein double network gel.

[0060] Test results are as follows Figures 2-7 As shown, the gel obtained by laccase cross-linking for 2 hours had the highest hardness and storage modulus, and the porosity was further reduced. FTIR confirmed the existence of hydrogen bonds, hydrophobic and electrostatic interactions between the protein and the polysaccharide, and fluorescence spectroscopy confirmed the formation of dityrosine covalent bonds.

[0061] The porosity measurement results were consistent with the microstructure observation results. Studies have shown that porosity and gel mechanical properties are generally negatively correlated, and reducing porosity helps improve the mechanical strength of the hydrogel. In Example 1, the pore size of the double-network gel with added leafwort protein was significantly smaller than that of the single sodium alginate gel; in Example 2, after further cross-linking with laccase, the pore size was further reduced, and the internal structure became more dense and uniform, which is consistent with the texture and rheological test results.

[0062] Example 3: Preparation and performance comparison of sodium alginate-leafwort protein dual-network gel beads encapsulated with postbiotics.

[0063] This embodiment aims to verify the encapsulation and release effect of the gel beads prepared by the method of the present invention on metagenics, and compare it with peanut protein, soy protein and pea protein.

[0064] Composite solutions containing 3% sodium alginate and 0.5% protein (leafgrass protein, peanut protein, soy protein, and pea protein) were prepared. Laccase cross-linking was performed according to the method in Example 2 (cross-linking time 2 hours). Then, 1% (w / v) of post-genetic agent was added to each system, and the mixture was magnetically stirred for 30 minutes to ensure homogeneity. The mixture was then sprayed dropwise into a 3% (w / v) CaCl2 solution using a syringe at a distance of 15 cm. After curing for 30 minutes, gel beads with embedded post-genetic agents were obtained.

[0065] Embedding rate determination: The amount of post-genetic residue in the solidified liquid was determined by ultraviolet spectrophotometry, and the embedding rate was calculated.

[0066] In vitro simulated release assay: The metabiotic was incubated sequentially in simulated gastric juice (pH 2.0, containing pepsin) for 2 hours and simulated intestinal juice (pH 6.8, containing pancreatic enzymes and bile salts) for 4 hours, and the cumulative release rate of the metabiotic was measured.

[0067] Laccase-crosslinked sodium alginate-leafwort protein gel beads showed the highest encapsulation rate (significantly higher than other protein groups), the lowest release rate in simulated gastric fluid (<20%), and a cumulative release rate of over 90% in simulated intestinal fluid after 4 hours, demonstrating the best targeted release performance.

[0068] Comparative example: Gel beads without laccase cross-linking

[0069] Using 3% sodium alginate and 0.5% leafwort protein as raw materials, the laccase cross-linking step was omitted, and the mixture was directly mixed and then a post-generic agent was added before being solidified by dripping CaCl2 solution. The resulting gel beads had poor mechanical properties, a significantly lower encapsulation rate than in Example 3, and a release rate exceeding 40% in simulated gastric juice, indicating poor targeted release effect.

[0070] This invention successfully prepared post-biotic gel beads with excellent mechanical properties, dense structure, high encapsulation efficiency, and intestinal-targeted release characteristics by compounding phytoesens with sodium alginate, synthesizing dityrosine covalent bonds using laccase cross-linking, and then forming a double network structure through calcium ion cross-linking. The optimal performance was achieved when the sodium alginate content was 3%, the phytoesens content was 0.5%, and the laccase cross-linking time was 2 hours.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing sodium alginate-leafwort protein dual-network gel beads with encapsulated post-biotics, characterized in that, Includes the following steps: Step 1: Dissolve sodium alginate and leafy green protein in a solvent to obtain a composite solution; Step 2: Adjust the pH of the composite solution to neutral, add laccase to carry out the cross-linking reaction, and inactivate the laccase after the reaction is completed to obtain the enzyme cross-linked composite solution; Step 3: Add postbiotic to the enzyme cross-linking complex solution, mix well, and obtain the bacterial loading mixture; Step 4: Drop the bacterial mixture into a coagulation bath containing divalent metal ions to solidify and form gel beads.

2. The preparation method according to claim 1, characterized in that, In step one, the mass fraction of sodium alginate is 2% to 4%, and the mass fraction of leafy green protein is 0.25% to 2%.

3. The preparation method according to claim 2, characterized in that, The sodium alginate has a mass fraction of 3%, and the leafy green protein has a mass fraction of 0.5%.

4. The preparation method according to claim 1, characterized in that, In step two, the amount of laccase added is 1-3 mg / mL of the composite solution, the cross-linking reaction time is 1-3 hours, and the reaction temperature is 35-45℃.

5. The preparation method according to claim 4, characterized in that, The amount of laccase added was 2 mg / mL, the cross-linking time was 2 hours, and the reaction temperature was 40℃.

6. The preparation method according to claim 1, characterized in that, In step two, the conditions for inactivating laccase are: heating in a water bath at 90–100°C for 5–15 minutes.

7. The preparation method according to claim 1, characterized in that, In step three, the amount of post-genetic agent added shall not exceed 1% (w / v), and the mixing method shall be magnetic stirring for 20 to 40 minutes.

8. The preparation method according to claim 1, characterized in that, In step four, the coagulation bath is a CaCl2 solution with a concentration of 2% to 4%, the solidification time is 20 to 40 minutes, and the drop distance is 10 to 20 cm.

9. A sodium alginate-leafwort protein dual-network gel bead with encapsulated post-biotics prepared by the method of any one of claims 1 to 8, characterized in that, The gel beads contain dityrosine covalent bonds formed by laccase catalysis of tyrosine residues in leafwort protein, and a sodium alginate network formed by cross-linking sodium alginate with calcium ions.

10. The gel beads according to claim 9, characterized in that, The gel beads are used in the preparation of functional foods or health products.

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