Method for preparing gel delivery epigallocatechin gallate by compounding soybean protein isolate with dialdehyde starch to overcome isoelectric point

Abstract

The invention discloses a method for preparing gel for delivering epigallocatechin gallate (EGCG) by utilizing soybean protein isolate compounded dialdehyde starch to overcome isoelectric points, the prepared gel can efficiently carry EGCG, and the method belongs to the field of food processing. The method comprises the following steps: respectively dispersing SPI and DAS with the concentration of 1% in ultrapure water, stirring, adjusting the pH value, and hydrating to obtain a soybean protein isolate solution and a dialdehyde starch solution; sPI and DAS are mixed according to the ratio of 4: 1, 2: 1, 1: 1, 1: 2 and 1: 4 respectively, stirring is conducted for 2 h in the alkaline environment (pH = 8.0), then the mixture is adjusted to be faintly acid (pH = 6.0), EGCG with the mass 1 / 30 of that of protein is added, and stirring is conducted again for 1 h. Respectively adjusting the pH value of the obtained mixture to 4.0 and 4.5, standing for 24 hours, and centrifuging to obtain the EGCG-carrying composite gel. The SPI-DAS gel prepared by the method is free of any additive, the obtained composite gel is more environment-friendly and safer and has higher stability, and the bioavailability of EGCG (epigallocatechin gallate) can be improved when the composite gel is used for carrying EGCG (epigallocatechin gallate). The method is simple in process and environmentally friendly, the application difficulty of SPI at the isoelectric point is solved to a certain extent, and the application range of the soybean protein isolate in the food industry is widened.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology and relates to a method for preparing gel delivery of epigallocatechin gallate by using soybean protein isolate combined with dialdehyde starch to overcome the isoelectric point. Background Technology

[0002] Soy protein isolate (SPI) is an important food ingredient and a recognized complete plant protein. Its composition includes acidic amino acids and their amides, basic amino acids, polar and nonpolar amino acids, and cysteine, resulting in a structure with many highly reactive groups and thus excellent performance in molecular interactions. Gel properties, as a key functional characteristic directly reflecting SPI molecular interactions, further broaden the application prospects and scope of SPI. However, unmodified SPI gels often exhibit poor mechanical strength and stability during food processing and storage. Oxidation of polysaccharides is a more effective means to enhance protein-polysaccharide interactions, thereby improving the texture and properties of protein-polysaccharide composite gels. Oxidized polysaccharides can form Schiff base covalent bonds with proteins, rather than hydrogen bonds. Compared to non-covalent interactions, covalent interactions can preserve the protein's conformation to the greatest extent, ensuring that the original excellent functional properties are not affected and compensating for the shortcomings of single-protein gels.

[0003] Starch is a natural polysaccharide derived from plants, composed of amylose and amylopectin. Natural starch is often limited in practical applications and production due to its poor water solubility and stability. Therefore, four basic modification methods have been developed to adapt to production systems: physical, chemical, enzymatic, and genetic. All modification methods target the three highly reactive hydroxyl groups (C2, C3, and C6) on the starch molecule. Among these, chemical modification, by altering or introducing new functional groups without changing the natural properties of starch, has become the most commonly used method. Starch oxidation is one of the most important methods. Commonly used oxidants include permanganate, nitrogen dioxide, sodium hypochlorite, periodate, and chromate. Sodium periodate, as a special selective oxidant, can target C2 and C3 to obtain dialdehyde starch (DAS), which has applications in the food, paper, and pharmaceutical fields.

[0004] Epigallocatechin-3-gallate (EGCG) is the most abundant bioactive compound among catechins and a typical flavonoid-3-ol phenolic substance, showing great potential in antitumor, antiviral, and modulating treatment of various chronic diseases. However, its high instability, such as its easy degradation under light and heat conditions, leads to low bioavailability, severely limiting its clinical translation and practical application. Furthermore, self-assembly, chelation, and partial oxidation of EGCG under physiological conditions may reduce its bioactivity during gastrointestinal digestion. Therefore, researchers have developed various methods to improve the bioavailability and stability of EGCG.

[0005] This invention utilizes SPI and DAS as raw materials to prepare a gel at the isoelectric point of SPI and transport EGCG, effectively improving the bioaccessibility and stability of EGCG. This helps to develop more stable small molecule nutrient carriers and, to some extent, solves the application difficulties of SPI. It is of great significance for expanding the development of SPI in plant protein-based gels. Summary of the Invention

[0006] The purpose of this invention is to prepare a gel at the isoelectric point of SPI and DAS using SPI as raw materials and to transport EGCG, so that the composite gel has better properties while carrying the maximum amount of EGCG and improving the bioavailability and stability of EGCG.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solution: A method for preparing a gel delivery system for epigallocatechin gallate using soybean protein isolate combined with dialdehyde starch to overcome the isoelectric point includes the following steps: Step 1: Disperse 1% SPI and DAS separately in ultrapure water and stir at 700 r / min for 2 h to ensure complete dissolution. Then, place at 4℃ for 12 h to allow the protein to fully hydrate. Prepare complex solutions with SPI to DAS mass ratios of 4:1, 2:1, 1:1, 1:2, and 1:4. Adjust the pH to 8.0 with 0.1 M NaOH and stir at room temperature for 2 h to ensure complete reaction. Step 2: Take the SPI:DAS samples with ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 after stirring, adjust the pH to 6.0, add 1 / 30 of the SPI mass of EGCG powder to each sample, stir in the absence of air for 1 h, then adjust the pH to 4.0 and 4.5 respectively, let stand for 24 h, and then incubate at 6,000 × 10⁻⁶. g Centrifuge for 20 min and remove the supernatant to obtain a gel sample.

[0008] The optimal preparation parameters for the EGCG-loaded SPI-DAS composite gel are: SPI∶DAS=1∶2, pH 4.5.

[0009] This invention uses food-grade SPI and DAS as raw materials to prepare composite gels, which can avoid the addition of other modifying agents and effectively improve the stability and safety of composite gels.

[0010] The production process used in this invention is simple, low-cost, and environmentally friendly, which is conducive to industrial production.

[0011] The method of this invention utilizes SPI-DAS to prepare composite gels. The resulting composite gels exhibit excellent performance and can efficiently transport EGCG, effectively improving the bioaccessibility and stability of EGCG. Attached Figure Description

[0012] Appendix Figure 1 Mechanism experimental diagram of the present invention; Appendix Figure 2 The photostability of EGCG in the composite gel was obtained by this method; Appendix Figure 3 The thermal stability of EGCG in the composite gel was obtained by this method; Appendix Figure 4 The freeze-thaw stability of EGCG in the composite gel was obtained by this method; Appendix Figure 5 The storage stability of EGCG in the composite gel was obtained by this method; Appendix Figure 6 The encapsulation efficiency and drug loading of EGCG in the composite gel were obtained by this method. Appendix Figure 7 The bioavailability of EGCG in the composite gel was obtained by this method; Appendix Figure 8 The kinetic release curve of EGCG in the composite gel was obtained by this method. Detailed Implementation

[0013] The present invention will now be described in more detail with reference to the accompanying drawings and specific embodiments. Implementation Cases

[0014] This implementation example demonstrates the preparation of the SPI-DAS composite gel carrying EGAG according to the following steps: 1% SPI and DAS were dispersed separately in ultrapure water and stirred at 700 r / min for 2 h to ensure complete dissolution. The solutions were then placed at 4 °C for 12 h to allow complete protein hydration. Complex solutions were prepared with SPI and DAS mass ratios of 4:1, 2:1, 1:1, 1:2, and 1:4. The pH was adjusted to 8.0 with 0.1 M NaOH and stirred at room temperature for 2 h to ensure complete reaction.

[0015] Take the SPI:DAS samples with ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 after stirring, adjust the pH to 6.0 with 0.1 M HCl, add 1 / 30 of the SPI mass of EGCG powder to each sample, stir in the absence of air for 1 h, then adjust the pH to 4.0 and 4.5 respectively, let stand for 24 h, and then incubate at 6,000 × 10⁻⁶. g Centrifuge for 20 min, remove supernatant to obtain gel samples. Gel samples with different proportions were named S4D1, S2D1, S1D1, S1D2, and S1D4, and divided into two groups according to different pH values.

[0016] Gel samples were stored under natural light and ultraviolet light for 8 h. Using free EGCG as a control, the EGCG retention rate in the treated samples was determined, representing the photostability of EGCG in gel embedding. Simulated storage temperatures of 25℃, processing temperatures of 45℃ and 65℃, and pasteurization temperature of 85℃ were applied. Samples were placed in water baths at these temperatures for 2 h. Using free EGCG as a control, the EGCG retention rate in the treated samples was determined, representing the thermal stability of EGCG in gel embedding. 2.0 g of gel was placed in a sample vial and stored at room temperature for 7 days. The EGCG content in the composite gel was determined, representing the storage stability of EGCG in the composite gel. The EGCG-loaded composite gel was placed in a 25 mL beaker and stored at -20℃ for 12 h to freeze it, followed by thawing at room temperature. Three freeze-thaw cycles were performed, and the EGCG content in the composite gel was determined after each freeze-thaw cycle, representing the freeze-thaw stability of EGCG.

[0017] The supernatant after gel preparation was collected, and the fluorescence intensity was measured at excitation wavelengths of 275 nm and 350 nm-450 nm. The EGCG concentration in the supernatant was calculated by comparing the EGCG fluorescence intensity with the concentration standard curve to determine the amount of EGCG delivered by the gel. The encapsulation efficiency and drug loading were calculated using the following formulas.

[0018]

[0019] Gastric stage: The sample (5 mL) was mixed with simulated gastric juice (SGF) (15 mL). The pH of the mixture was then adjusted to 2.0 with HCl, and the mixture was shaken for 2 h at 100 rpm and 37°C using a constant temperature water bath shaker.

[0020] Small Intestinal Stage: The pH of the solution (20 mL) from the gastric stage was adjusted to 7.0 and then mixed with simulated intestinal fluid (SIF). The pH-adjusted solution from the gastric stage was mixed with SIF at a 1:1 (v / v) ratio. The resulting mixture was maintained at pH 7.0 by adding NaOH (0.1 mol / L). Intestinal digestion was performed at 37°C, with the mixture continuously shaken at 120 rpm / min for 2 h using a constant-temperature water bath shaker.

[0021] Measurement of EGCG content: Centrifuge 5 mL of digestion solution (1,000 × 10⁻⁶) g (5 min), take the supernatant, measure the absorbance at 275 nm, and calculate its content according to the standard curve.

[0022] After the in vitro simulated digestion is complete, a certain amount of digestive fluid is taken and subjected to 8,000 × g Centrifuge for 30 min and collect the transparent intermediate layer formed in the centrifuge tube as the "micelle phase". Record the absorbance at 445 nm to determine the EGCG content.

[0023] In the formula, C I It refers to the content of riboflavin in the micelle phase, C M It refers to the EGCG content in the chyme after complete digestion in the small intestine.

Claims

1. A method for preparing a gel delivery system for epigallocatechin gallate using soybean protein isolate combined with dialdehyde starch to overcome the isoelectric point, comprising the following steps: Step 1: Disperse 1% SPI and DAS separately in ultrapure water and stir at 700 r / min for 2 h to ensure complete dissolution. Then, place at 4℃ for 12 h to allow the protein to fully hydrate. Prepare complex solutions with SPI to DAS mass ratios of 4:1, 2:1, 1:1, 1:2, and 1:

4. Adjust the pH to 8.0 with 0.1 M NaOH and stir at room temperature for 2 h to ensure complete reaction. Step 2: Take the SPI:DAS samples with ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 after stirring, adjust the pH to 6.0, add 1 / 30 of the SPI mass of EGCG powder to each sample, stir in the absence of air for 1 h, then adjust the pH to 4.0 and 4.5 respectively, let stand for 24 h, and then incubate at 6,000 × 10⁻⁶. g Centrifuge for 20 min and remove the supernatant to obtain a gel sample.

2. The optimal preparation parameters for the EGCG-loaded SPI-DAS composite gel are: SPI∶DAS=1∶2, pH 4.

5.

3. This invention uses food-grade SPI and DAS as raw materials to prepare composite gels, which can avoid the addition of other modifying agents and effectively improve the stability and safety of composite gels.

4. The production process used in this invention is simple, low-cost, and environmentally friendly, which is conducive to industrial production.

5. The method of the present invention utilizes SPI-DAS to prepare composite gels. The resulting composite gels have good performance and can efficiently transport EGCG, effectively improving the bioaccessibility and stability of EGCG.

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