Preparation method of edible propolis ethanol extract egg white protein composite gel

Abstract

The application provides a preparation method of edible propolis ethanol extract egg white protein composite gel, and relates to the technical field of gel preparation; the method comprises the following steps: crushing crude propolis, extracting and preparing propolis ethanol extract freeze-dried powder by using anhydrous ethanol, preparing egg white protein freeze-dried powder by using egg white, then dissolving and mixing the two kinds of freeze-dried powder, and under the action of a high-pressure pulse electric field, water bath heating is induced to form propolis ethanol extract egg white protein composite gel; the edible propolis ethanol extract egg white protein composite gel prepared by the method improves the gelation, water retention and the like of egg white protein; compared with single egg white protein gel, the hardness can be increased by 2.66 times, the elasticity can be increased by 33.8%, the water retention can be increased by 16.9%, the structure is more compact and smooth, the water retention of natural egg white protein gel is effectively improved, water loss of food in processing is avoided, the texture characteristics and flavor of food are further improved, meanwhile, the edible gel is endowed with good biological functional characteristics, and has a good development prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of novel composite gel preparation, and in particular relates to a method for preparing an edible propolis alcohol extract and egg white protein composite gel. Background Art

[0002] Egg white protein is a natural substance commonly used in traditional Chinese minced meat products. It possesses numerous functional properties, such as gelling, foaming, and emulsifying properties. It is often used as a gel filler and cross-linking agent during processing to enhance the gelling properties and impart flavor to minced meat products (e.g., meatballs, sausages, minced fish, and egg tofu). Egg white protein can also be used as a fat substitute, mimicking the mouthfeel of fat without altering the original quality of the food, reducing fat intake and mitigating the risk of cardiovascular disease. It is commonly used in dairy products, sauces, ice cream, and cream.

[0003] Gelation of protein is one of the primary means of imparting a desirable texture to food. When an egg white protein solution is heated or otherwise treated, the coiled, folded peptide chains within the protein gradually unfold and transform into extended, small peptide chains. Through β-pleating, these small peptide chains form insoluble aggregates of larger molecules. Hydrophobic interactions and disulfide bonds then allow some of these small peptide chains to react with each other. When the solution's viscosity reaches the critical gel point, a three-dimensional, ordered protein network, known as a gel network, is generated. This structure traps water and other components to form an egg white protein gel. This structure can absorb water, improving the water retention and tenderness of meat, as well as flavors to enhance meat flavor and improve food cohesion. Therefore, the gel properties of minced meat products determine the product's texture, water retention, and color. However, the stability and gel properties of egg white protein gels themselves are not ideal, resulting in suboptimal taste and flavor. Starch is often used to improve the viscosity of minced meat gels, but this can alter the original texture and natural flavor of the minced meat. In order to increase the added value of egg white protein gel and expand its application range, using special technology to prepare egg white protein gel products to replace fat or starch is an important direction for developing new meat products and one of the important solutions for developing healthy meat products.

[0004] High-voltage pulsed electric field processing (HPVP) involves placing liquid food as an electrolyte in a container. A high-voltage current is then passed through two insulated discharge electrodes, generating an electric pulse effect on the target food. This method not only produces new properties distinct from heat treatment but also preserves nutrients, such as vitamins. HPVP is primarily used for cold sterilization of liquid foods or as an aid in the extraction of functional ingredients, achieving excellent results. Research has shown that varying the parameters of the HPVP can alter the non-covalent bonds (such as hydrogen, ionic, and hydrophobic bonds) in proteins, thereby modifying their function and structure.

[0005] The main biologically active ingredients of propolis are polyphenols, including flavonoids, phenolic acids and their ester derivatives, which are easily soluble in polar solvents such as ethanol. Therefore, propolis alcohol extracts contain a large amount of antioxidant active substances, such as phenols and flavonoids.

[0006] Therefore, in order to improve the gelling and water retention properties of egg white protein, further improve the texture characteristics of minced meat products, and at the same time solve the problem of poor taste and flavor of using only egg white protein, and meet consumers' multiple demands for food taste, nutrition and safety, the present invention provides a method for preparing an edible propolis alcohol extract egg white protein composite gel. Summary of the Invention

[0007] In order to improve the viscosity, emulsification and water retention of egg white protein, further improve the texture characteristics of minced meat products, effectively reduce the amount of egg white protein used, and impart certain biological functional properties to the gel, the present invention provides a method for preparing an edible propolis alcohol extract and egg white protein composite gel, the method comprising the following steps:

[0008] 1. Preparation of Propolis Ethanol Extract (EEP)

[0009] The crude propolis is crushed in a blender, mixed with anhydrous ethanol according to a ratio, the mixed solution is stirred uniformly at 20-25° C. using a magnetic stirrer, filtered with filter paper, concentrated using a vacuum rotary evaporator at 50-60° C., and the concentrate is collected and freeze-dried to obtain the propolis alcohol extract;

[0010] 2. Preparation of Egg White Protein Freeze-dried Powder (EWP)

[0011] After shelling fresh eggs, separate the yolk, egg white membrane and ligament, and remove impurities in the egg white liquid; use a high-speed disperser to stir and disperse the egg white to obtain a natural and uniform egg white liquid, freeze-dry it, and refrigerate it for future use;

[0012] 3. Preparation of Propolis Alcohol Extract-Egg White Protein (EEP-EWP) Composite Gel

[0013] The egg white protein freeze-dried powder was dissolved in deionized water to prepare an egg white protein solution, and the propolis alcohol extract was added while stirring. The pH was adjusted with citric acid. Under the action of a high-voltage pulsed electric field, the propolis alcohol extract-egg white protein mixture was heated in a water bath to induce gel formation, and then immediately cooled in an ice water bath, sealed, and refrigerated for storage.

[0014] Preferably, the fresh eggs are chicken eggs or duck eggs;

[0015] Preferably, the mixing ratio of propolis to anhydrous ethanol is 1-3:8, the speed of the magnetic stirrer is 150-200 rpm, the stirring time is 2-3 days, and the concentration time is 2-3 days;

[0016] Preferably, the speed of the high-speed disperser is 2000-4000 r / min, and the stirring and dispersing time is 3-6 min;

[0017] Preferably, the mass concentration of the egg white protein solution is 8% to 12%, and the amount of the propolis alcohol extract added is 3% to 12% by mass volume ratio;

[0018] Preferably, the pH is adjusted to 6.0-9.0;

[0019] Preferably, the intensity of the high-voltage pulse electric field is 20-40 kV / cm, and the number of pulses is 2-8;

[0020] Preferably, the water bath temperature is 80-100°C, the time is 30-40 min, the sealing is sealed with plastic wrap or a plastic cover, and the refrigeration temperature is 4-6°C.

[0021] Experimental methods

[0022] Texture testing——

[0023] The hardness and elasticity of the gel samples were measured using a texture analyzer. The following parameters were used: a flat-bottomed cylindrical probe (P / 50), a downward pressure distance of 10.0 mm, a time of 5 seconds, a triggering force of 0.049 N, and pre-, test-, and post-measurement speeds of 5.0, 2.0, and 2.0 mm / s, respectively.

[0024] Determination of dynamic rheology——

[0025] The rheometer was first used to perform a strain sweep at 1 Hz and 0.01-10% strain to determine the linear viscoelastic region (LVR). This was followed by a frequency sweep from 0.1 to 25 Hz at a strain amplitude of 0.1%, with three replicate measurements.

[0026] Determination of water holding capacity——

[0027] Weigh 15 g of gel sample and place it in a centrifuge tube. Centrifuge it at 6000×g for 20 minutes. Weigh the mass of the gel before and after centrifugation. The water holding capacity (WHC) of the gel is the percentage of the mass of the gel before and after centrifugation. Each sample is measured three times in parallel and the average value is taken.

[0028] Microstructure (SEM) observation——

[0029] Freeze-dried samples of the composite gel were cut with a razor blade and mounted on a SEM column with double-sided conductive tape. Gold was then applied using a Model IB-3 ion sputtering apparatus. The samples were then observed under a scanning electron microscope (Hitachi S-4800, Japan) at a magnification of 4000x and an accelerating voltage of 3 kV. Images were acquired using XT Microscope Control software. Beneficial effects

[0030] The composite gel prepared by the present invention combines two edible natural raw materials, propolis alcohol extract and egg white protein, with high-voltage pulsed electric field synergistic thermal induction gelation technology. The structure is denser and smoother. Moreover, compared with single egg white protein gel, the gel hardness can be increased by 2.66 times, the elasticity can be increased by 33.8%, and the water holding capacity can be improved by 16.9% (p<0.05). The reason may be that the high-voltage pulsed electric field changes the spatial structure of egg white protein, causing more hydrophobic areas to be exposed, which is manifested as an increase in the hydrophobicity of the protein surface; at the same time, the propolis alcohol extract causes the spatial conformation of egg white protein to change during the high-voltage pulsed electric field synergistic thermal induction, and the active thiol groups of the protein macromolecular texture are The number of flavonoids and whey protein decreases, and oxidation forms disulfide bonds, forming hydrophobic interactions with the non-polar aromatic rings in the propolis alcohol extract. Furthermore, the hydroxyl groups in the propolis alcohol extract can form hydrogen bonds with various polar groups on the side chains of egg white proteins. Given the charge in the propolis alcohol extract, electrostatic interactions may occur with groups with opposite charges on the protein surface. These changes cause the molecular chains of the composite gel to align more orderly during heating, leading to a higher degree of cross-linking of the protein molecules, thus forming a dense and stable gel network structure. This structural change was further verified by dynamic rheological testing and scanning electron microscopy, thereby improving the gel strength, elasticity, chewiness, and water retention of egg white proteins. Studies have shown that hydrogen bonds and van der Waals forces are the primary factors maintaining the stability of the flavonoid-whey protein complex. The action of a high-voltage pulsed electric field can denature whey protein, while reducing the number of active sulfhydryl groups in the protein macromolecules, leading to oxidation and the formation of disulfide bonds. The improved water retention of the gel may also be due to the fact that after the electric field induces polarization of protein molecules, the disulfide bonds and hydrogen bonds are broken, the protein molecules are partially unfolded, and the interaction with water is enhanced; at the same time, the electric field causes water molecules to aggregate, shortening the distance between water molecules, allowing free water to fill the vicinity of the protein amino acid side chains and become bound water, thereby improving water retention.

[0031] While reducing the amount of egg white protein used, the composite gel effectively improves the water retention of the egg white protein gel, avoids water loss in the food system during processing, directly improves the food texture characteristics, and solves the problem of poor taste and flavor of natural egg white protein gel preparations in meat products. In addition, the cholesterol-lowering and lipid metabolism-enhancing effects of egg white protein, as well as the large amount of antioxidant active substances in the propolis alcohol extract, such as phenols and flavonoids, are not easily oxidized in the composite gel, which can improve the environmental stability of the composite gel. All of these give the natural edible propolis alcohol extract-egg white protein composite gel good biological functional properties.

[0032] The present invention can be easily combined with food, can be used as a preferred fat substitute, starch substitute, etc., can significantly improve the quality of food, and also has certain practical significance for people's healthy life. It has a promising future development prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The composite gel prepared by adding different amounts of propolis alcohol extract under the action of high voltage pulse electric field

[0034] Figure 2 The following is the dynamic rheological diagram of the composite gel prepared with different amounts of propolis alcohol extract under the action of a high-voltage pulsed electric field (Note: EWP is propolis alcohol extract, EEP is egg white protein freeze-dried powder, Frequency is frequency, G' is storage modulus, and G" is loss modulus)

[0035] Figure 3 SEM images of composite gels prepared with different amounts of propolis alcohol extract added under the action of a high-voltage pulsed electric field (Note: A to E are composite gels with 0%, 3%, 6%, 9%, and 12% propolis alcohol extract added, respectively) DETAILED DESCRIPTION

[0036] In order to better explain the present invention and facilitate understanding, the present invention is described in detail through specific embodiments. In the present invention, the crude propolis raw material comes from Bozhou Hongbaotang Trading Co., Ltd., and the fresh egg raw material comes from Hefei Feidong Hongwei Farm.

[0037] Example 1 The purpose of the present invention is to provide a method for preparing an edible propolis alcohol extract egg white protein composite gel, comprising the following steps:

[0038] 1. Preparation of propolis alcohol extract

[0039] The crude propolis was crushed in a blender and mixed with anhydrous ethanol in a ratio of 1:8. The mixed solution was shaken at 150 rpm at 25°C for 2 days using a magnetic stirrer, filtered with filter paper, and concentrated using a vacuum rotary evaporator at 50°C for 3 days. The concentrate was then collected and freeze-dried.

[0040] 2. Preparation of egg white protein freeze-dried powder

[0041] After shelling fresh eggs, separate the yolk, albumen membrane, and ligament, and remove impurities from the egg white solution. Use a high-speed disperser at 3000 rpm for 6 minutes to disperse the egg white to obtain a uniform egg white solution. Freeze-dry and store at 4°C until ready for use.

[0042] 3. Preparation of propolis alcohol extract and egg white protein composite gel

[0043] Lyophilized egg white protein powder was dissolved in deionized water to obtain an 8% egg white protein solution. Then, 3% propolis alcohol extract (w / v, calculated based on the volume of the egg white protein solution) was added while stirring. The pH was adjusted to 8.0 with citric acid. The propolis alcohol extract-egg white protein mixture was exposed to a high-voltage pulsed electric field at 30 kV / cm with 6 pulses. Simultaneously, the mixture was heated in a 90°C water bath for 30 minutes to induce gel formation. After heating, the mixture was immediately cooled in an ice-water bath. The beaker was sealed with plastic wrap and stored at 4°C until further use.

[0044] Comparative tests were conducted according to different addition amounts of propolis alcohol extract. Other experimental conditions were the same. A control group and an experimental group were set up. The blank control was the egg white protein gel treated with heat induction alone. Control group 1 was the heat-induced egg white protein gel treated with high-voltage pulse electric field without adding propolis alcohol extract. Control group 2 was the heat-induced composite gel treated with 6% propolis alcohol extract without high-voltage pulse electric field. Figure 1 .

[0045] Experimental groups Propolis alcohol extract added High-voltage pulse electric field treatment Composite gel hardness Composite gel elasticity Composite gel water holding capacity Blank control 0% no 113.22g 0.65g 82.16% Control group 1 0% yes 184.53g 0.72g 85.95% Control group 2 6% no 179.74g 0.70g 84.37% Experimental Group 1 3% yes 225.92g 0.75g 91.66% Experimental Group 2 6% yes 414.05g 0.87g 96.05% Experimental Group 3 9% yes 288.96g 0.79g 93.49% Experimental Group 4 12% yes 297.64g 0.80g 91.64% result

[0046] As shown in Table 1, compared with the control group, the strength and water holding capacity of the composite gel obtained by heat induction of propolis alcohol extract and egg white protein under the synergistic effect of the pulsed electric field in the experimental group were significantly improved, especially in experimental group 2, the gel hardness increased by 2.66 times, the elasticity increased by 33.8%, and the water holding capacity increased by 16.9% (p < 0.05); the reason may be that the high-voltage pulsed electric field changed the spatial structure of egg white protein, causing more hydrophobic areas to be exposed, which is manifested as an increase in the hydrophobicity of the protein surface; at the same time, the propolis alcohol extract caused the spatial conformation of egg white protein to change during heat induction. This increases the surface hydrophobicity and surface thiol content of the protein, forming hydrophobic interactions with the non-polar aromatic rings in the propolis alcohol extract. Furthermore, the hydroxyl groups in the propolis alcohol extract can form hydrogen bonds with various polar groups on the side chains of egg white proteins. Given the charge of the propolis alcohol extract, electrostatic interactions may occur with the oppositely charged groups on the protein surface. These changes cause the molecular chains of the composite gel to align more orderly during heating, increasing the degree of cross-linking of the protein molecules and forming a dense and stable gel network structure. This, in turn, improves the gel strength, elasticity, chewiness, and water retention of the egg white protein. Studies have shown that hydrogen bonds and van der Waals forces are the primary factors maintaining the stability of the flavonoid-whey protein complex. The high-voltage pulsed electric field can denature whey proteins, while reducing the number of active thiol groups in the protein macromolecules, leading to oxidation and the formation of disulfide bonds. The improved water retention of the gel may also be due to the fact that after the electric field induces polarization of protein molecules, the disulfide bonds and hydrogen bonds are broken, the protein molecules are partially unfolded, and the interaction with water is enhanced; at the same time, the electric field causes water molecules to aggregate, shortening the distance between water molecules, allowing free water to fill the vicinity of the protein amino acid side chains and become bound water, thereby improving water retention.

[0047] Depend on Figure 2 The G' and G'' of the composite gel first increase and then decrease with increasing propolis alcohol extract content. Both the storage modulus G' and loss modulus G'' reach their highest values ​​when the propolis alcohol extract content ranges from 3% to 10%, with G' > G''. Therefore, a 6% propolis alcohol extract content results in the most elastic gel-like structure. Studies have shown that when the storage modulus (G') of the aggregates is higher than the loss modulus (G''), it indicates a highly elastic gel-like structure. Furthermore, high-voltage pulsed electric field and heat-induced treatments unfold protein molecules, forming a complex structure in the presence of propolis alcohol extract, promoting the reorganization of protein cross-links and increasing the modulus.

[0048] Depend on Figure 3 Scanning electron microscopy images of the composite gels with different addition amounts of propolis alcohol extracts showed that the composite gel samples with propolis addition amounts ranging from 3% to 10% displayed a highly cross-linked protein matrix and a complex network structure, with a smoother, more uniform and dense structure.

[0049] Example 2: 1. Preparation of Propolis Ethanol Extract

[0050] The crude propolis was crushed in a blender and mixed with anhydrous ethanol in a ratio of 2:8. The mixed solution was shaken at 180 rpm at 20°C for 3 days using a magnetic stirrer, filtered with filter paper, and concentrated using a vacuum rotary evaporator at 55°C for 3 days. The concentrate was then collected and freeze-dried.

[0051] 2. Preparation of egg white protein freeze-dried powder

[0052] After shelling fresh eggs, separate the yolk, albumen membrane, and ligament, and remove impurities from the egg white solution. Use a high-speed disperser at 2000 rpm for 6 minutes to disperse the egg white to obtain a uniform egg white solution. Freeze-dry and store at 4°C until ready for use.

[0053] 3. Preparation of propolis alcohol extract and egg white protein composite gel

[0054] Lyophilized egg white protein powder was dissolved in deionized water to obtain an 8% egg white protein solution. Then, 6% propolis alcohol extract (w / v, calculated based on the volume of the egg white protein solution) was added while stirring. The pH was adjusted to 6.0 with citric acid. The propolis alcohol extract and egg white protein mixture was exposed to a high-voltage pulsed electric field at 20 kV / cm with 8 pulses. Simultaneously, the mixture was heated in an 80°C water bath for 40 minutes to induce gel formation. After heating, the mixture was immediately cooled in an ice-water bath, sealed with a plastic cap, and stored at 5°C until ready for use.

[0055] Example 3: 1. Preparation of propolis ethanol extract

[0056] The crude propolis was crushed in a blender and mixed with anhydrous ethanol in a ratio of 3:8. The mixed solution was shaken at 200 rpm at 23°C for 2 days using a magnetic stirrer, filtered with filter paper, and concentrated using a vacuum rotary evaporator at 60°C for 2 days. The concentrate was then collected and freeze-dried.

[0057] 2. Preparation of egg white protein freeze-dried powder

[0058] After shelling fresh eggs, separate the yolk, albumen membrane, and ligaments, and remove impurities from the egg white solution. Use a high-speed disperser at 4000 rpm for 6 minutes to disperse the egg white to obtain a uniform egg white solution. Freeze-dry and store at 6°C until ready for use.

[0059] 3. Preparation of propolis alcohol extract and egg white protein composite gel

[0060] Lyophilized egg white protein powder was dissolved in deionized water to obtain an 8% egg white protein solution. Then, 12% propolis alcohol extract (w / v, calculated based on the volume of the egg white protein solution) was added while stirring. The pH was adjusted to 9.0 with citric acid. The propolis alcohol extract and egg white protein mixture was exposed to a high-voltage pulsed electric field at 40 kV / cm with a pulse number of 2. Simultaneously, the mixture was heated in a 100°C water bath for 30 minutes to induce gel formation. After heating, the mixture was immediately cooled in an ice-water bath, sealed with a plastic cap, and stored at 6°C until ready for use.

Claims

1. A method for preparing an edible propolis alcohol extract and egg white protein composite gel, comprising the steps of: pulverizing crude propolis, mixing the crude propolis with anhydrous ethanol in a ratio of 1 to 3:8, stirring the mixture at 20 to 25° C. at a speed of 150 rpm to 200 rpm for 2 to 3 days, filtering, concentrating at 50 to 60° C., and freeze-drying the mixture to obtain the propolis alcohol extract; separating the egg yolk, egg white membrane, and ligament, removing impurities from the egg white solution, dispersing the egg white with high-speed stirring, and freeze-drying the mixture to obtain egg white protein freeze-dried powder; wherein: The method further comprises dissolving egg white protein freeze-dried powder in deionized water to prepare an egg white protein solution, adding propolis alcohol extract while stirring, adjusting the pH with citric acid, heating the propolis alcohol extract and egg white protein mixture in a water bath under the action of a high-voltage pulse electric field to induce gel formation, immediately cooling the mixture in an ice water bath, and sealing and refrigerating for storage.

2. The method for preparing the edible propolis alcohol extract egg white protein composite gel according to claim 1, wherein: The concentration time is 2 to 3 days.

3. The method for preparing the edible propolis alcohol extract egg white protein composite gel according to claim 1, wherein The high-speed stirring and dispersing speed is 2000-4000 r / min, and the stirring and dispersing time is 3-6 min.

4. The method for preparing the edible propolis alcohol extract egg white protein composite gel according to claim 1, wherein: The mass concentration of the egg white protein solution is 8% to 12%, and the added amount of the propolis alcohol extract is 3% to 12%.

5. The method for preparing the edible propolis alcohol extract and egg white protein composite gel according to claim 1, wherein: The pH is adjusted to 6.0-9.

0.

6. The method for preparing the edible propolis alcohol extract and egg white protein composite gel according to claim 1, wherein: The intensity of the high-voltage pulse electric field is 20-40 kV / cm, the number of pulses is 2-8, the water bath temperature is 80-100° C., the time is 30-40 min, the sealing is sealed with plastic wrap or a plastic cover, and the refrigeration temperature is 4-6° C.

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