Preparation method of heat-resistant and aggregating egg white protein microgel particles and application thereof

Abstract

The application discloses a preparation method of heat-resistant and anti-aggregation egg white protein microgel particles, which comprises the following steps: 1) adjusting the pH of separated egg white to neutral by acid water, stirring, centrifuging, and collecting supernatant; 2) diluting the collected supernatant to an egg white protein solution with a protein concentration of 1-5% by water, then adding an anionic polysaccharide solution with a concentration of 1-3%, and heating at 70-100 DEG C for 10-60 minutes to realize phase separation, and obtaining an egg white protein microgel suspension after cooling; and 3) centrifuging the microgel suspension and cleaning with water to obtain egg white protein microgel particles. The egg white protein particles are prepared by using the heat treatment-protein polysaccharide phase separation method, which is beneficial to preventing the re-aggregation of the egg white protein particles under high-temperature conditions, and has a better application prospect in the preparation of egg white protein beverages.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food and beverage, and relates to a preparation method of egg white protein microgel particles with heat resistance and application thereof. BACKGROUND

[0002] Egg white is rich in various proteins and has high nutritional value. As a large egg producing country, China still mainly sells fresh eggs, and the processing of poultry eggs accounts for only 0.2% of the output of poultry eggs, which is far behind the advanced level in the world. Therefore, the development of egg white protein beverages can solve the problem of weak egg industry in China. However, heat treatment as a necessary operation unit in the beverage processing process can cause denaturation and aggregation of proteins, which seriously affects the quality of the product. How to obtain egg white protein with heat resistance is a difficult problem that is focused on in the field.

[0003] Egg white protein is rich in covalent bonds such as disulfide bonds, and can form gel blocks that are not easily destroyed under heating conditions. In order to improve the heat resistance of egg white protein, the prior art usually uses a method of heating combined with mechanical crushing to make it into micron-sized aggregates. Common mechanical crushing methods include shearing, high-pressure homogenization, micro-jet, etc. For example, CN 105660983 A uses a heat treatment-mechanical shearing method to prepare stable egg protein aggregate particles. However, mechanical crushing not only depends on special equipment and high energy input, but also has high production cost. Moreover, the protein aggregates prepared by mechanical crushing can achieve temporary stability, but they are still essentially a heat-unstable system and will still re-aggregate under high temperature conditions. Therefore, the protein particles prepared by mechanical crushing are still not suitable for the heat treatment process of beverages.

[0004] Therefore, the present application proposes a method of preparing protein particles by heat treatment-protein polysaccharide phase separation, which makes the protein hydrophobic group involute to form a structure with certain rigidity and relative order and disperse in the polysaccharide continuous phase to achieve gelation. The prepared microgel particles have a narrow size distribution and excellent heat resistance, which can meet the production needs of various egg white protein beverages. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of egg white protein microgel particles and its application in the production of egg white protein beverages, aiming to solve the technical problem of poor heat resistance of egg white protein leading to the decline in beverage quality.

[0006] The present application is realized by the following technical solutions:

[0007] A preparation method of egg white protein microgel particles with heat resistance, comprising the following steps:

[0008] 1) Adjust the pH of the separated egg white to neutral with acid water, stir and centrifuge, and collect the supernatant;

[0009] 2) dilute the collected supernatant with water to an egg white protein solution with a protein concentration of 1-5%, then add a solution of anionic polysaccharide with a concentration of 1-3% (mass), heat at 70-100°C for 10-60 minutes to achieve phase separation and gelation, and obtain an egg white protein microgel suspension after cooling;

[0010] 3) centrifuge the microgel suspension and wash with water to obtain egg white protein microgel particles.

[0011] Preferably, the anionic polysaccharide includes sodium alginate, carboxymethyl cellulose, high-methoxyl pectin, and the like.

[0012] Preferably, the concentration of the anionic polysaccharide solution is 1.5-2.5%, more preferably 2%.

[0013] Preferably, the volume ratio of the egg white protein solution to the anionic polysaccharide solution is 1:1-4.

[0014] A black tea beverage containing black tea liquid and egg white protein microgel particles prepared by the method described above.

[0015] Further, the black tea beverage further contains beta cyclodextrin, sucrose, erythritol, essence, guar gum.

[0016] A method for making the black tea beverage, comprising the steps of: dissolving the egg white protein microgel particles, beta cyclodextrin, sucrose, erythritol, essence, guar gum with water, then mixing with black tea liquid, sterilizing at high temperature, and then filling to obtain the finished product.

[0017] The present application has the following beneficial effects:

[0018] (1) The present application uses a heat treatment-protein polysaccharide phase separation method to prepare protein particles, which is different from the existing heat treatment-mechanical crushing method. First, the present application disperses the rigid structure of the protein in the polysaccharide continuous phase and performs gelation treatment, and the obtained is a microgelized protein particle. Second, the microgelized protein particle is more stable than the mechanically crushed protein particle, especially under high temperature conditions, which is more conducive to preventing the protein particles from re-aggregating, and therefore has better application prospects in egg white protein beverage production.

[0019] (2) The polysaccharide used in the present application is a natural product and does not contain any other chemical reagents, which will not cause any safety hazards to the human body. The black tea beverage produced has the advantages of stable color, no precipitation, good flavor and taste, rich in nutritional value, etc.

[0020] (3) The present application is easy to operate, does not depend on expensive equipment and high energy input, has low cost, and is suitable for industrialized production.BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : Fluorescence micrographs (top) and optical micrographs (bottom) of microgel particles prepared using different polysaccharides, where A represents chitosan; B represents konjac glucomannan; C represents locust bean gum; D represents carboxymethylcellulose (low viscosity); E represents carboxymethylcellulose (high viscosity); F represents high methoxyl pectin.

[0022] Figure 2 : Fluorescence micrographs (top) and optical micrographs (bottom) of microgel particles prepared using different concentrations of polysaccharides.

[0023] Figure 3 : Particle size distribution of microgel particles prepared using different concentrations of polysaccharides.

[0024] Figure 4 : Comparison of appearance of heat-induced aggregation of microgel particles prepared using different concentrations of polysaccharides, left is the suspension before heat treatment at 100°C, right is the suspension after heat treatment at 100°C and inversion, EWP represents the untreated egg white protein group; HEWP represents the heat-treated egg white protein group.

[0025] Figure 5 : Comparison of particle size distribution of heat-induced aggregation of microgel particles prepared using different concentrations of polysaccharides, the two curves in the figure represent the particle size distribution before and after heat treatment of the suspension at 100°C.

[0026] Figure 6 : Comparison of rheological diagrams of heat-induced aggregation of microgel particles prepared using different concentrations of polysaccharides.

[0027] Figure 7 : Comparison of appearance of heat-induced aggregation of microgel particles prepared by the present application and mechanically sheared egg white protein particles.

[0028] Figure 8 : Comparison of optical micrographs of heat-induced aggregation of microgel particles prepared by the present application and mechanically sheared egg white protein particles.

[0029] Figure 9 : Appearance of black tea beverage made using egg white protein microgel particles. DETAILED DESCRIPTION DETAILED DESCRIPTION

[0030] In order to better understand the present application, the present application will be further described below in conjunction with examples, but the embodiments of the present application are not limited thereto.

[0031] Example 1: Preparation of egg white protein microgel particles

[0032] 1. Effect of polysaccharide type on microgel particle formation

[0033] In the preparation of egg white protein microgel particles, the effects of different polysaccharides (charge, viscosity, structure) on the formation of microgel particles were investigated respectively to screen the suitable polysaccharide types for the preparation of microgel particles.

[0034] (1) Pretreatment of egg white: After washing the egg, the egg white was separated and 1-2 times the volume of water was added. The pH was adjusted to 7.0 with citric acid, and after stirring for 30 min, the pH was again adjusted to 7.0. The insoluble proteins were removed by centrifugation at 5500 g for 30 min at room temperature, and the supernatant was collected as the egg white protein solution. The protein concentration of the solution was determined by the biuret method.

[0035] (2) According to the results of protein concentration determination, the egg white protein solution obtained in step (1) was diluted to a protein concentration of 4%; the concentrations of chitosan, konjac glucomannan, locust bean gum, carboxymethyl cellulose (low viscosity), carboxymethyl cellulose (high viscosity), and high methoxyl pectin were 2.0%, 1.0%, 2.0%, 3.0%, 2.0%, and 8.0%, respectively; the egg white protein solution and the polysaccharide solution were mixed in a volume ratio of 1:1, heated in a 90°C water bath for 30 min, cooled to room temperature, and stored at 4°C for 12 h to obtain an egg white protein microgel suspension.

[0036] (3) The microgel suspension obtained in step (2) was centrifuged at 10000 g for 30 min to remove free polysaccharides and proteins. The particles were redispersed with deionized water, and the supernatant was removed by centrifugation. This washing step was repeated 3 times, and then the particles were stored at 4°C for measurement.

[0037] Fluorescence microscopy and optical microscopy observation: FITC was used as a dye to stain the proteins in the microgel suspension obtained in step (2), and an inverted fluorescence microscope was used to observe the microstructure. The Optikam B3 digital camera (OPTIKA, Italy) was used to perform optical microscopy on the microgel suspension obtained in step (2).

[0038] Particle size and Zeta potential detection: The particle size distribution of the particles obtained in step (3) was determined using a laser particle size distribution instrument (MS2000), and the charge of the particles was determined using a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, U.K).

[0039] As Figure 1As shown from the morphological observation results, the cationic polysaccharide chitosan is positively charged and strongly interacts with the negatively charged egg white protein to produce insoluble complex particles by electrostatic attraction. The anionic polysaccharide group (carboxymethyl cellulose, high methoxyl pectin) and the protein mixed system undergo phase separation and form the desired microgel particles. The anionic polysaccharide (carboxymethyl cellulose, high methoxyl pectin) and the egg white protein are both negatively charged under neutral conditions. When the protein and the polysaccharide carry the same charge, thermal incompatibility occurs due to the repulsive force between the two polymers and the different affinities for the solvent, resulting in phase separation of the protein-rich and polysaccharide-rich phases. Since the polysaccharide has a larger molecular weight and can occupy more space, it serves as the continuous phase in this system, while the protein-rich domains are dispersed in the polysaccharide continuous phase to form tiny protein droplets. After heating, the dispersed phase rich in egg white protein gels, forming microgel particles. The mixed system of the neutral polysaccharide konjac glucomannan and locust bean gum and the protein undergoes phase separation due to thermodynamic incompatibility, but the neutral polysaccharide group forms larger protein gel particles. It is possible that the neutral polysaccharide is not charged and has a larger molecular weight, so the protein and the polysaccharide will further undergo emptying interaction, so that the proteins close to each other completely exclude the polysaccharide, causing further crosslinking between the protein aggregates to form larger gel particles.

[0040] From the particle size and Zeta potential detection results in Table 1, the particles formed by the anionic polysaccharide have smaller particle sizes and more uniform distribution, and the larger absolute value of the Zeta potential indicates better particle stability.

[0041] Therefore, anionic polysaccharides are selected to prepare microgel particles with egg white protein.

[0042] Table 1 Particle size and Zeta potential of different egg white protein

[0043]

[0044] Note: Heat-treated egg white protein (HEWP): The product obtained by mixing the pretreated egg white solution according to step (1) with the same volume of water and heating at 90°C.

[0045] 2. Effect of polysaccharide concentration on microgel particle formation and thermal aggregation resistance

[0046] The effect of different polysaccharide concentrations on microgel particle formation and thermal aggregation resistance was further investigated to determine the optimal process conditions for preparing heat-stable microgel particles.

[0047] Steps (1) and (3) are the same as above.

[0048] In step (2), the egg white protein solution obtained in step (1) is diluted to a protein concentration of 4%, a carboxymethyl cellulose (high viscosity) concentration of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, or 3.0%, and the egg white solution and the polysaccharide solution are mixed at a volume ratio of 1:1, heated in a water bath at 90°C for 30 minutes, cooled to room temperature, and stored at 4°C for 12 hours to obtain an egg white protein microgel suspension.

[0049] As can be seen from the morphological observation and particle size determination results, when the sodium carboxymethyl cellulose concentration is less than 1.0%, egg white protein aggregates are formed, and microgels cannot be formed and phase separation cannot be achieved; when the CMC concentration is higher than 1.0%, the protein-polysaccharide mixture undergoes phase separation, and microgel particles begin to form. As can be seen from the results shown in Figure 2 ,when the CMC concentration is 2.0%, the particle size of the formed particles is smaller and the distribution is more uniform; when the CMC concentration is 3%, the microgel particle size increases due to the severe phase separation. Figure 3

[0050] Therefore, the optimal CMC concentration is 2.0%.

[0051] The egg white protein microgel particles obtained at different CMC concentrations are dispersed in water and diluted to a concentration of 5%, and then stirred uniformly to obtain a particle suspension. The suspension is heated at 100°C for 30 minutes, and the thermal stability of the suspension is observed by observing the appearance, particle size, and rheological properties of the suspension, and compared with the untreated egg white protein solution. As can be seen from the results shown in Figure 4 , Figure 5 , Figure 6 the microgel particles obtained (polysaccharide concentration of 1.5%, 2.0%, and 2.5%) still maintain good fluidity after heating, and the particle size and system viscosity do not change significantly. However, the EWP (untreated egg white protein group) and HEWP group (heat-treated egg white protein group) change significantly in appearance and fluidity after heating. In addition, the egg white protein aggregate group with a polysaccharide concentration of 0.5% and 1.0% also shows poor fluidity and large particle size, indicating that the particles aggregate after heating. When the polysaccharide concentration is 3%, although the appearance of the microgel does not change significantly, the particle size decreases significantly, indicating that the microgel aggregates.

[0052] Based on the above tests, it is finally determined that the anionic polysaccharide solution at a concentration of 1.5-2.5% is heated with the egg white protein solution. In this process, the mixed system undergoes phase separation, and the protein is dispersed in the polysaccharide continuous phase to form microgel particles with uniform particle size distribution, thereby improving the heat aggregation resistance of the egg white protein under high temperature conditions.

[0053] Example 2: Further verification of the heat aggregation resistance of egg white protein microgel particles

[0054] 1. Preparation of test sample

[0055] (1) After washing the egg, the egg white was separated, the pH was adjusted to 7.0 with citric acid, stirred for 30 min, and then the pH was adjusted to 7.0 again. The suspension was centrifuged at 5500 g for 30 min at room temperature to remove insoluble proteins, and the supernatant was collected.

[0056] (2) The collected egg white protein solution was diluted to a protein concentration of 3%, and a carboxymethyl cellulose solution with a concentration of 1.5% was prepared. The egg white solution and the carboxymethyl cellulose solution were mixed in a volume ratio of 1:2, heated in a water bath at 85°C for 50 min, cooled to room temperature, and stored at 4°C for 12 h to obtain an egg white protein microgel suspension.

[0057] (3) The microgel suspension was centrifuged to remove free polysaccharides and proteins, and then washed with deionized water 3 times and stored at 4°C.

[0058] In addition, the following comparative examples were prepared according to the heat treatment-mechanical shearing method of CN 105660983 A:

[0059] The egg white protein solution with a protein concentration of 5% was adjusted to pH 3.5 with citric acid, heated in a water bath at 90°C for 30 min, and the formed colloid was placed at 4°C for one day, then high-speed sheared at 10000 rpm, followed by high-pressure homogenization, homogenization pressure was 10 MPa, and homogenization times was 2 times, to obtain protein particles.

[0060] The test sample and the comparative sample were adjusted to a concentration of 5%, heated in a water bath at 100°C for 30 min, and the appearance and optical micrograph of each sample before and after heat treatment were as shown in Figure 7 、 Figure 8 .

[0061] The appearance graph ( Figure 7 ) shows that the egg white protein particle system in the comparative example forms a large gel after heat treatment, which cannot flow after being inverted, and the egg white protein microgel particle system in the experimental example still maintains good flowability after heating at 100°C. The optical micrograph ( Figure 8 ) shows that the micro-particles formed in the comparative example have small and uniform particle sizes before heating, but form a dense gel structure after heating, indicating that the protein particles re-aggregate under high temperature conditions, while the experimental example maintains good micro-particle distribution before and after heat treatment, and the properties do not change significantly. Therefore, the egg white protein microgel particles prepared by the present application have better anti-thermal aggregation properties compared to the prior art.

[0062] Example 3: Preparation of black tea beverage using egg white protein microgel particles

[0063] Egg white protein microgel particles 4.0%

[0064] Black tea liquor 10%, 20%, 30%, 40%, 50%

[0065] Beta cyclodextrin 0.1%

[0066] A mixture of sucrose and erythritol in a ratio of 1:1 6%

[0067] Milk flavor 0.025%

[0068] Guar gum 0.10%

[0069] The balance is water.

[0070] The egg white protein microgel particles, beta cyclodextrin, sucrose, erythritol, flavor, guar gum are dissolved with water, then mixed with black tea liquor, sterilized at high temperature, aseptically filled to obtain the finished product, the product appearance is shown in Figure 9 , the black tea beverage does not occur protein aggregation phenomenon after high temperature sterilization, the product still maintains uniform and easy flowing appearance.

Claims

1. A method for preparing heat-aggregation resistant egg white protein microgel particles, characterized by It comprises the following steps: 1) Adjust the pH of the separated egg white to neutral with acid water, stir and centrifuge, and collect the supernatant; 2) Dilute the collected supernatant with water to an egg white protein solution with a protein concentration of 1-5%, then add a solution of anionic polysaccharide with a concentration of 1.5-2.5%, the volume ratio of the egg white protein solution to the anionic polysaccharide solution being 1:1-4, heat at 70-100℃ for 10-60 minutes to realize phase separation and gelation, and obtain an egg white protein microgel suspension after cooling; 3) Centrifuge the microgel suspension and wash with water to obtain egg white protein microgel particles, The anionic polysaccharide comprises sodium alginate, carboxymethyl cellulose, and high-methoxyl pectin.

2. A black tea beverage comprising black tea liquor and egg white protein microgel particles, characterised in that: The egg white protein microgel particles are prepared by the method of claim 1.

3. The black tea beverage according to claim 2, wherein: It also contains β-cyclodextrin, sucrose, erythritol, essence, and guar gum.

4. A method of making the black tea beverage of claim 2, characterized by: Dissolve the egg white protein microgel particles, β-cyclodextrin, sucrose, erythritol, essence, and guar gum in water, then mix with black tea liquid, sterilize at high temperature, and then fill to obtain the finished product.

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