Ginkgo biloba protein isolate / hyaluronic acid composite gel and its preparation method and application
Through the complexation of ginkgo protein isolate and hyaluronic acid, a composite gel is formed, which solves the problems of low solubility of ginkgo protein in water and poor gel performance, and realizes the texture, water-holding, swelling and Significant improvement in thermal stability.
Patent Information
- Application Number
- CN202310358617.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-06
- Publication Date
- 2025-02-25
- Estimated Expiration
- 2043-04-06
AI Technical Summary
In the prior art, the solubility of ginkgo protein in water is low, resulting in poor gel performance, limiting its widespread use in the food industry.
By complexing ginkgo protein isolate with hyaluronic acid, the high solubility and hydrophilicity of hyaluronic acid are used to form ginkgo protein isolate/hyaluronic acid composite gel through heat induction.
It significantly improves the texture, water-holding, swelling and thermal stability of the gel, imparts functionality and diversity to the composite gel, and solves the problem of poor performance of Ginkgo Protein Gel.
Smart Images
Figure CN116355427B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of gel materials, and in particular to a ginkgo fruit protein isolate / hyaluronic acid composite gel and a preparation method and application thereof. Background Art
[0002] Ginkgo biloba L. has a long history of being widely planted and used in China. Its fruit is rich in protein components, ranging from 10% to 15%. The amino acid composition and proportion of the protein are reasonable, and the content of essential amino acids is much higher than the high-quality protein standard recommended by FAO / WHO. Ginkgo biloba protein does not contain limiting amino acids and is rich in amino acids such as methionine and cysteine, which are generally absent in plant-based proteins. In addition, it is rich in glutamic acid, aspartic acid and arginine. It is a relatively high-quality food protein with good edible value and significant potential for food development and utilization. However, due to the low solubility of ginkgo biloba protein in water, their functions are poor, especially the gelation is poor. Therefore, its widespread use in the food industry is limited. The gelation property is an important functional property of food protein. The gelation behavior and rheological properties of protein are the decisive factors for the formation of the unique texture, sensory and flavor of certain foods. In-depth research on the gel properties of ginkgo protein can promote the development of the ginkgo protein industry and effectively improve the utilization value of ginkgo resources. Summary of the invention
[0003] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention aims to provide a ginkgo biloba protein isolate / hyaluronic acid composite gel with excellent gel properties, and the present invention also provides a preparation method and application of the ginkgo biloba protein isolate / hyaluronic acid composite gel.
[0004] Technical solution: The ginkgo biloba protein isolate / hyaluronic acid composite gel of the present invention is formed by heat-induction of ginkgo biloba protein isolate and hyaluronic acid.
[0005] Furthermore, the molecular weight of the hyaluronic acid is 50-1500 kDa.
[0006] Furthermore, the mass ratio of the ginkgo biloba protein isolate to hyaluronic acid is 120:0.1-5.
[0007] The preparation method of the Ginkgo biloba protein isolate / hyaluronic acid composite gel of the present invention comprises the following steps:
[0008] (1) preparing a Ginkgo biloba protein isolate stock solution;
[0009] (2) preparing hyaluronic acid stock solution;
[0010] (3) mixing the Ginkgo biloba protein isolate stock solution and the hyaluronic acid stock solution, cooling after heat induction, and storing at low temperature to form a Ginkgo biloba protein isolate / hyaluronic acid composite gel.
[0011] Furthermore, in step (1), the preparation method of the ginkgo nut protein isolate is as follows: fresh ginkgo nuts are shelled and peeled, dried, ground into powder, and sieved to obtain ginkgo nut powder; the ginkgo nut powder is mixed with n-hexane and defatted to obtain defatted ginkgo nut powder; and the ginkgo nut protein isolate is extracted from the defatted ginkgo nut powder by an alkali dissolution and acid precipitation method to obtain ginkgo nut protein isolate powder.
[0012] Furthermore, the preparation method of the above-mentioned ginkgo nut protein isolate is specifically as follows: fresh ginkgo nuts without corruption or deterioration are selected, the ginkgo nuts are shelled and peeled to obtain ginkgo seeds, dried in an oven at 40-60°C for 50-60 hours, ground into powder, sieved through an 80-mesh sieve to obtain ginkgo nut powder, and defatted the ginkgo nut powder 2-4 times using n-hexane, wherein the mass volume ratio of the ginkgo nut powder to the n-hexane is 1:8-10kg / L; and the ginkgo nut protein isolate is extracted by an alkali dissolution and acid precipitation method: the dried defatted ginkgo nut powder is dispersed in deionized water to obtain a mixed solution, wherein the mass volume ratio of the defatted ginkgo nut powder to the deionized water is 1:10-12kg / L, and the mixed solution is precipitated with 1mol / LNaOH solution. The pH value is adjusted to 9.0, stirred at room temperature for 30-40 minutes, then centrifuged at 4-6°C and 10000g for 15-20 minutes, the supernatant is taken, the pH value thereof is adjusted to 4.4 with 1mol / L HCl solution, centrifuged at 4-6°C and 10000g for 15-20 minutes, the precipitate is dispersed in water, the pH value thereof is adjusted to 7.0 with 1mol / L NaOH solution, and stored in a refrigerator at 4-6°C after freeze-drying; if the concentration of the NaOH solution and the HCl solution used in the alkali dissolution and acid precipitation method is too low, the usage amount will be too large and unfavorable for operation, and if the concentration is too high, the protein will precipitate with a granular feel, therefore, the concentration of the NaOH solution and the HCl solution is preferably 1mol / L.
[0013] Furthermore, the preparation method of the ginkgo nut protein isolate stock solution is: dissolving the ginkgo nut protein isolate powder in water, stirring for 3-6 hours and then keeping it at 2-4°C for 12-24 hours to obtain a completely hydrated ginkgo nut protein isolate stock solution with a concentration of 180g / L.
[0014] Furthermore, in step (2), the concentration of the hyaluronic acid stock solution is 6-15 g / L, when the molecular weight of hyaluronic acid is 50-1000 kDa, the concentration of the hyaluronic acid stock solution is 12-15 g / L, and when the molecular weight of hyaluronic acid is 1000-1500 kDa, the concentration of the hyaluronic acid stock solution is 6-9 g / L.
[0015] Furthermore, the preparation method of the hyaluronic acid stock solution is: dissolving hyaluronic acid powder in water, stirring for 3-6 hours, and then keeping it at 2-4° C. for 12-24 hours to obtain a completely hydrated hyaluronic acid stock solution.
[0016] Furthermore, in step (3), the pH of the ginkgo nut protein isolate stock solution and the hyaluronic acid stock solution are adjusted to 6.0 before mixing; in the mixed solution, the concentration of the ginkgo nut protein isolate is 100-140 g / L, and the concentration of the hyaluronic acid is 0.1-5 g / L; the conditions for the heat induction are: water bath, 90-95° C., 30-40 minutes, and the conditions for the low-temperature storage are: 2-4° C., 12-24 hours.
[0017] Invention principle: Hyaluronic acid is a natural linear polysaccharide, a glycosaminoglycan formed by repeated alternation of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine disaccharide, with a molecular weight of 10 3 -10 6 Da varies greatly, and the molecular formula is (C 14 H 20 NO 11 Na) n . Hyaluronic acid is a hydrophilic macromolecule with -OH and -COOH functional groups, so it has high solubility in water and can form a highly viscous solution. It can capture water much larger than its own weight and form a gel, showing unique rheological properties. After being compounded with Ginkgo biloba protein isolate, it can change the hydration characteristics of the composite solution. Hyaluronic acid can combine with Ginkgo biloba protein isolate through electrostatic interaction, hydrophobic interaction and hydrogen bonding to form a continuous gel network composed of aggregates and porous structures, thereby improving the mechanical properties and microstructure of the protein gel. As the molecular weight of hyaluronic acid increases, the non-covalent interaction between Ginkgo biloba protein isolate and hyaluronic acid is enhanced, and the functionality of the protein gel is improved.
[0018] In the present invention, ginkgo biloba protein isolate and hyaluronic acid are compounded, and the ginkgo biloba protein isolate is modified by hyaluronic acid, which can change the various structures and properties of the gel, such as mechanical properties, hydration properties, microstructure and thermal properties, thereby giving the composite gel functionality and diversity.
[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the preparation method of the ginkgo fruit protein isolate / hyaluronic acid composite gel adopted by the present invention can improve the texture, water retention, swelling and thermal stability of the gel, and a variety of gels can be produced by selecting hyaluronic acid with different molecular weights and concentrations, thereby avoiding the waste of ginkgo resources; in addition, the preparation method of the present invention has good safety, no environmental pollution, simple process, no need for complex equipment, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The rheological diagrams of the gel solutions prepared in Examples 1 to 11 of the present invention and Comparative Example 1;
[0021] Figure 2 The texture characteristics of the gels prepared in Examples 1 to 11 of the present invention and Comparative Example 1 are shown;
[0022] Figure 3 The actual pictures of the gels prepared in Examples 1 to 11 of the present invention and Comparative Example 1;
[0023] Figure 4 The scanning electron microscope images of the gels prepared in Examples 1 to 11 of the present invention and Comparative Example 1;
[0024] Figure 5 The water holding capacity and swelling capacity of the gels prepared in Examples 1 to 11 of the present invention and Comparative Example 1;
[0025] Figure 6 This is a thermal property diagram of the gels prepared in Examples 1 to 11 of the present invention and Comparative Example 1. DETAILED DESCRIPTION
[0026] The present invention is further described below in conjunction with specific embodiments and drawings.
[0027] Embodiment 1: The preparation method of the Ginkgo biloba protein isolate / hyaluronic acid composite gel of the present invention comprises the following steps:
[0028] (1) Preparation of Ginkgo Biloba Protein Isolate Stock Solution: Select fresh Ginkgo Biloba without corruption or deterioration, peel and remove the Ginkgo Biloba seeds, dry them in an oven at 40°C for 50 hours, grind them into powder, sieve through an 80-mesh sieve to obtain Ginkgo Biloba powder, defatted the Ginkgo Biloba powder three times with n-hexane, the mass volume ratio of Ginkgo Biloba powder to n-hexane being 1:8 kg / L; extract Ginkgo Biloba Protein Isolate Solution by Alkaline Dissolution and Acid Precipitation Method: Disperse the dried defatted Ginkgo Biloba powder in deionized water to obtain a mixed solution, the mass volume ratio of defatted Ginkgo Biloba powder to deionized water being 1:10 kg / L, adjust the pH of the mixed solution to 9.0 with 1 mol / L NaOH, stir at room temperature for 30 minutes, centrifuge at 4°C and 10,000 g for 15 minutes, take the supernatant, adjust the pH to 4.4 with 1 mol / L HCl, centrifuge at 4°C and 10,000 g for 15 minutes, take the precipitate, disperse it in water, and The pH value was adjusted to 7.0 by adding NaOH, and the mixture was freeze-dried and stored in a refrigerator at 4°C to obtain a ginkgo nut protein isolate powder; the ginkgo nut protein isolate powder was dissolved in water, stirred for 4 hours, and then stored at 4°C for 12 hours to obtain a fully hydrated ginkgo nut protein isolate stock solution with a concentration of 180 g / L;
[0029] (2) preparing a hyaluronic acid stock solution: dissolving hyaluronic acid powder with a molecular weight of 50 kDa in water, stirring for 4 hours, and then storing at 4° C. for 12 hours to obtain a low molecular weight hyaluronic acid stock solution with a concentration of 15 g / L;
[0030] (3) The pH of the ginkgo biloba protein isolate stock solution and the hyaluronic acid stock solution were adjusted to 6.0, and then mixed and stirred to obtain a mixed solution, and deionized water was added to adjust the concentration of hyaluronic acid in the mixed solution to 1 g / L and the concentration of the ginkgo biloba protein isolate to 120 g / L; the mixed solution was heated at 90° C. in a constant temperature water bath for 30 minutes, then naturally cooled to room temperature, and then stored at 4° C. for 12 hours to form a ginkgo biloba protein isolate / hyaluronic acid composite gel.
[0031] Example 2: The difference from Example 1 is that in step (2) and step (3), the concentration of hyaluronic acid in the mixed solution is 3 g / L.
[0032] Example 3: The difference from Example 1 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 5 g / L.
[0033] Example 4: The difference from Example 1 is that in step (2), the molecular weight of the hyaluronic acid is 500 kDa, and a medium molecular weight hyaluronic acid stock solution with a concentration of 15 g / L is obtained; in step (3), the concentration of hyaluronic acid in the mixed solution is 1 g / L.
[0034] Example 5: The difference from Example 4 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 3 g / L.
[0035] Example 6: The difference from Example 4 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 5 g / L.
[0036] Example 7: The difference from Example 4 is that in step (2), the molecular weight of the hyaluronic acid is 1500 kDa, and a high molecular weight hyaluronic acid stock solution with a concentration of 9 g / L is obtained; in step (3), the concentration of hyaluronic acid in the mixed solution is 0.1 g / L.
[0037] Example 8: The difference from Example 7 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 0.3 g / L.
[0038] Example 9: The difference from Example 7 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 0.5 g / L.
[0039] Example 10: The difference from Example 4 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 1 g / L.
[0040] Example 11: The difference from Example 4 is that in step (3), the concentration of hyaluronic acid in the mixed solution is 3 g / L.
[0041] Comparative Example 1: Ginkgo biloba protein isolate gel.
[0042] Example 12: The gel materials obtained in Examples 1 to 11 and Comparative Example 1 were subjected to performance tests, including:
[0043] (1) Determination of viscosity: The viscosity of the gel solution was analyzed at room temperature using a MARS60 rheometer. A plate with a diameter of 40 mm and a gap of 1 mm was used for viscosity analysis. The sample volume was 500-800 μL and the shear rate was from 0.1 to 100 s. -1 .
[0044] (2) Texture property determination: The texture property of the gel was determined by uniaxial double compression test using TA-XT Plus Texture Analyzer, using a P / 0.5 cylindrical probe. The parameters were set as follows: 1 mm / s pre-test speed (speed before sample contact), 2 mm / s compression speed, 7 mm downward distance, and 5.0 g trigger force. The hardness, elasticity, cohesiveness, adhesiveness, chewiness, and resilience of each sample were determined.
[0045] (3) Appearance: Carefully take the gel sample out of the beaker and place it on a black screen to take a photo.
[0046] (4) Scanning electron microscopy: The gel was freeze-dried in vacuum and pretreated by KBr tableting. About 1 mg of freeze-dried gel sample was mixed with KBr at a mass ratio of 1:99 (g / g), ground into powder and pressed into tablets. The infrared spectrum of each sample was measured by VERTEX 80V infrared spectrometer. All samples were scanned between 4000 and 400 cm -1 The wave number range is 64 times, and the resolution is 4cm -1 .
[0047] (5) Determination of water holding capacity and swelling capacity:
[0048] Take 4g of gel and place it in a 50mL centrifuge tube and centrifuge at 3000×g for 20 minutes. Calculate the water holding capacity (WHC) according to the formula:
[0049]
[0050] m0 is the mass of the centrifuge tube; m1 is the mass of the gel and centrifuge tube before centrifugation; m2 is the mass of the gel and centrifuge tube after centrifugation.
[0051] Take a cylindrical gel of 8mm (diameter) × 5mm (height), weigh it, place it in water at room temperature for 30 minutes, take it out with tweezers, use filter paper to absorb the water on the surface of the gel, and weigh it again. The swelling ratio of the gel is calculated according to the formula:
[0052]
[0053] W1 is the initial weight of the gel, and W2 is the weight of the swollen gel.
[0054] (6) Determination of thermal stability: The gel sample was vacuum freeze-dried. The thermal stability of the gel was studied by thermogravimetric analysis. 5-8 mg of freeze-dried sample was placed in an alumina crucible and placed in a sample rack. The starting temperature was set to 20°C, the ending temperature was set to 600°C, the heating rate was set to 10°C / min, and the nitrogen flow rate was set to 20 mL / min. The instrument recorded the curve of the change of sample mass with time and temperature during the heating process.
[0055] The above test results are as follows Figure 1-Figure 6 As shown. Figure 1 As shown in the figure, the apparent viscosity of the Ginkgo biloba protein isolate / hyaluronic acid mixed solution decreases with increasing shear rate, indicating a classic shear-thinning non-Newtonian fluid behavior. The electrostatic repulsion between the negatively charged Ginkgo biloba protein isolate and the negatively charged hyaluronic acid affects the viscosity of the mixed solution. The addition of low molecular weight hyaluronic acid enhances the electrostatic repulsion and hinders the interaction between proteins, thereby reducing the viscosity. Medium molecular weight and high molecular weight hyaluronic acid can significantly increase the viscosity of the mixed solution, which may be due to the high viscosity of hyaluronic acid itself, thereby increasing the viscosity of the solution.
[0056] like Figure 2 As shown in the figure, the addition of low molecular weight hyaluronic acid has little effect on the hardness of the gel, while the addition of medium molecular weight and high molecular weight hyaluronic acid significantly increases the hardness of the gel. When 3g / L medium molecular weight hyaluronic acid and 0.5g / L high molecular weight hyaluronic acid are added, the hardness of the gel reaches the maximum, and then the hardness of the gel decreases slightly with the increase of concentration; low molecular weight hyaluronic acid can improve the elasticity of the gel, while medium molecular weight and high molecular weight hyaluronic acid will have a negative effect on the elasticity of the gel when the concentration is higher than 1g / L; hyaluronic acid with a concentration lower than 1g / L can improve the cohesion, viscosity, chewiness and recovery of the gel. In general, by adjusting the concentration and molecular weight of hyaluronic acid, a variety of ginkgo fruit protein isolate gels can be obtained. High molecular weight hyaluronic acid can significantly improve the texture characteristics of the gel, and the best effect is achieved at a concentration of 0.5g / L.
[0057] like Figure 3 As shown, the appearance of the Ginkgo biloba protein isolate / hyaluronic acid composite gel is opaque milky white, with a complete shape, and obvious bubbles can be observed on the surface of the colloid. The addition of hyaluronic acid has no obvious effect on the color of the gel, but will form some tiny pores on the surface of the gel.
[0058] like Figure 4 As shown in the figure, consistent with the appearance of the gel, after the addition of hyaluronic acid, some fine pores were formed inside the gel. The composite gel showed a continuous network composed of small aggregates and many small pores, whose diameter size was smaller than the spherical particles of the Ginkgo biloba protein isolate gel. This is due to the electrostatic repulsion between hyaluronic acid and Ginkgo biloba protein isolate. When high concentrations of hyaluronic acid were added, the microstructure of the gel showed a 3D network structure, which may be due to the cross-linking between molecules caused by excessive hyaluronic acid molecules.
[0059] like Figure 5 As shown in the figure, the addition of hyaluronic acid improves the water holding capacity of the gel. Hyaluronic acid contains a large number of carboxyl and hydroxyl groups, which allow it to form intermolecular and intramolecular hydrogen bonds in aqueous solution, cross-linking a network structure to improve the water holding capacity of the composite gel. Hyaluronic acid is hydrophilic and can bind water much larger than its own weight. With the increase of hyaluronic acid concentration, the swelling of the gel increases significantly. However, at higher concentrations, hyaluronic acid has a higher viscosity, causing the Ginkgo biloba protein isolate / hyaluronic acid complex to form small aggregates, reducing the pore size of the system and weakening the ability to absorb water.
[0060] like Figure 6 As shown, it can be observed that the gel has two main weight loss stages in the ranges of 30-150°C and 150-600°C. The first stage is mainly the loss of free water and bound water, and the second stage is the main weight loss stage of the gel, which is related to the thermal degradation behavior of protein and polysaccharide. With the increase of the molecular weight of hyaluronic acid, the maximum weight loss rate of the composite gel gradually decreases, indicating that the addition of hyaluronic acid enhances the thermal stability of the gel, and the best effect is achieved when 3g / L high molecular weight hyaluronic acid is added.
Claims
1. A Ginkgo biloba protein isolate / hyaluronic acid composite gel, characterized in that: The composite gel is formed by heat-induced ginkgo nut protein isolate and hyaluronic acid; the molecular weight of the hyaluronic acid is 50-1500 kDa; the mass ratio of the ginkgo nut protein isolate to the hyaluronic acid is 120:0.1-5; the preparation method of the ginkgo nut protein isolate comprises the following steps: shelling and peeling fresh ginkgo nut, drying, grinding into powder, and sieving to obtain ginkgo nut powder; mixing the ginkgo nut powder with n-hexane, and defatting to obtain defatted ginkgo nut powder; and extracting the ginkgo nut protein isolate from the defatted ginkgo nut powder by an alkali dissolution and acid precipitation method to obtain the ginkgo nut protein isolate powder.
2. A method for preparing the Ginkgo biloba protein isolate / hyaluronic acid composite gel according to claim 1, characterized in that: The following steps are involved: (1) Prepare Ginkgo biloba protein isolate stock solution; (2) preparing hyaluronic acid stock solution; (3) The Ginkgo biloba protein isolate stock solution and the hyaluronic acid stock solution are mixed and stirred to form a mixed solution, which is then cooled after heat induction and stored at low temperature to form a Ginkgo biloba protein isolate / hyaluronic acid composite gel.
3. The preparation method according to claim 2, characterized in that: In step (1), the preparation method of the ginkgo nut protein isolate is as follows: fresh ginkgo nuts are shelled and peeled, dried, ground into powder, and sieved to obtain ginkgo nut powder; the ginkgo nut powder is mixed with n-hexane and defatted to obtain defatted ginkgo nut powder; and the ginkgo nut protein isolate is extracted from the defatted ginkgo nut powder by an alkali dissolution and acid precipitation method to obtain ginkgo nut protein isolate powder.
4. The preparation method according to claim 2, characterized in that: In step (2), the concentration of the hyaluronic acid stock solution is 6-15 g / L.
5. The preparation method according to claim 2, characterized in that: In step (3), the pH of the ginkgo biloba protein isolate stock solution and the hyaluronic acid stock solution are adjusted to 6.0 before mixing.
6. The preparation method according to claim 2, characterized in that: In step (3), in the mixed solution, the concentration of ginkgo biloba protein isolate is 100-140 g / L, and the concentration of hyaluronic acid is 0.1-5 g / L.
7. The preparation method according to claim 2, characterized in that: In step (3), the conditions for heat induction are: water bath, 80-95°C, 20-40 minutes, and the conditions for low-temperature storage are: 2-4°C, 12-24 hours.