Preparation method of low-viscosity protein particles

Abstract

The invention discloses a preparation method of low-viscosity protein particles, and belongs to the field of protein processing. According to the invention, disulfide bonds in protein molecules are broken by using a reducing agent, so that the protein is unfolded and internal hydrophobic groups are exposed, and meanwhile, the unfolded protein is promoted to aggregate and recombine under the synergistic driving of hydrophobic interaction and hydrogen-bond interaction by using heating treatment, so that low-viscosity protein particles are formed; 10% (w / v) of a redissolved high-protein solution has better fluidity and lower viscosity, and the viscosity value of a high-concentration solution is close to 0.1 Pa.S when the shearing rate is 100s <-1 >. The low-viscosity protein particles prepared by the method effectively solve the problem of overhigh viscosity of a high-concentration system solution, and can be widely applied to high-protein foods, such as high-protein beverages, sports nourishment, infant foods, healthy meal replacement and other products.

Description

Technical Field

[0001] The invention belongs to the field of protein processing, and in particular relates to a method for preparing low-viscosity protein particles. Background Art

[0002] With the improvement of residents' living standards and the enhancement of healthy eating awareness, high-protein food products are gradually favored by consumers, and the market demand is increasing. However, high protein concentration tends to increase the density of protein molecules in the solution. Some proteins with easy aggregation properties, such as myofibrillar proteins, are prone to intermolecular interactions to form larger aggregates, which increases the viscosity of high-concentration solution systems. The rheology of the solution is a key physical property that is mainly screened in the early extraction and later deep processing stages of proteins, because the downstream processing and manufacturing steps (such as filtration, pumping and canning) and the use of the final product are greatly affected by the viscosity of the solution. This will not only increase the cost of industrialized food processing, but also limit the refined deep processing of high-value products. Therefore, developing a modification method that can reduce protein viscosity is an urgent problem to be solved in the current food processing field and pharmaceutical preparation industry.

[0003] At present, methods to reduce protein viscosity usually require high-power equipment such as ultrasound and high-pressure microfluidization homogenization; or additives such as TG enzyme and citrate need to be added for decalcification, which is a relatively cumbersome process. Summary of the invention

[0004] [Technical issues]

[0005] The present invention aims to solve the problem that the existing method for reducing protein viscosity requires high-power equipment and complicated steps.

[0006] [Technical solution]

[0007] In order to solve the above problems, the present invention provides a method for preparing low-viscosity protein particles, which is dedicated to solving the problem of excessive viscosity of protein products under high concentration conditions. A reducing agent is used to break the internal disulfide bonds of the protein molecules, so that the protein unfolds and exposes the internal hydrophobic groups. At the same time, heating treatment is used to promote the aggregation and reorganization of unfolded proteins under the synergistic driving of hydrophobic interactions and hydrogen bonds to form low-viscosity protein particles.

[0008] The present invention provides a method for preparing low-viscosity protein particles, comprising the following steps:

[0009] (1) dispersing protein powder in water to obtain a protein suspension, and adjusting the pH to 6 to 9;

[0010] (2) dissolving a reducing agent in a solvent to obtain a reducing agent solution, and adjusting the pH of the solution to a value of 6 to 9;

[0011] (3) mixing the reducing agent solution obtained in step (2) and the protein suspension obtained in step (1), and then performing heat treatment to obtain a modified protein suspension;

[0012] (4) adjusting the pH of the modified protein suspension obtained in step (3) to the isoelectric point and then centrifuging to obtain a protein precipitate;

[0013] (5) washing the protein precipitate obtained in step (4) with water and placing it in water, and adjusting the pH to 6 to 9 to obtain a primary modified protein solution;

[0014] (6) centrifuging the initially modified protein solution obtained in step (5) and taking the supernatant to obtain a modified protein solution;

[0015] (7) Drying the modified protein solution obtained in step (6) to obtain low-viscosity protein particles.

[0016] In a specific embodiment of the present invention, in step (1), the protein is animal myofibrillar protein or plant protein; the animal myofibrillar protein is aquatic myofibrillar protein or livestock and poultry myofibrillar protein, the aquatic myofibrillar protein is one or more of cod protein, scallop protein, sea bass protein, rainbow trout protein, and northern pike protein; the livestock and poultry myofibrillar protein is one or more of chicken protein, pork protein, beef protein, milk protein, and serum protein; the plant protein is one or more of soy protein, pea protein, and rice protein.

[0017] In a specific embodiment of the present invention, in step (1), the addition ratio of protein powder and water is 0.2-2 g:100 mL.

[0018] In a specific embodiment of the present invention, in step (2), the solvent is one of water, PBS buffer, HBSS buffer, and Tris-HCl buffer, and the pH of the solvent is 6-9.

[0019] In a specific embodiment of the present invention, in step (2), the reducing agent is one or more of sodium pyrosulfite, sodium sulfite, potassium pyrosulfite, cysteine, and reduced glutathione.

[0020] In a specific embodiment of the present invention, in step (2), the concentration of the reducing agent in the reducing agent solution is 25-200 mM.

[0021] In a specific embodiment of the present invention, in step (3), the volume ratio of the reducing agent solution to the protein suspension is 1:19.

[0022] In a specific embodiment of the present invention, in step (3), the heat treatment parameters are: temperature of 60 to 120° C., and heating time of 5 to 120 min.

[0023] In a specific embodiment of the present invention, in step (4), the pH is adjusted to 3-5, the centrifugal force is 5000-15000g, the centrifugal time is 5-20min, and the centrifugal temperature is 2-10°C.

[0024] In a specific embodiment of the present invention, in step (6), the centrifugal force is 6000-18000 g, the centrifugal time is 10-40 min, and the centrifugal temperature is 2-10°C.

[0025] In a specific embodiment of the present invention, in step (7), the drying method is vacuum freeze drying or spray drying; the parameters of vacuum freeze drying are: temperature of -80 to -30°C, vacuum degree of 0.01Pa to 50Pa, and drying time of 24 to 72h; the parameters of spray drying are: inlet air temperature of 175 to 195°C, and outlet air temperature of 80 to 100°C.

[0026] The present invention provides low-viscosity protein particles prepared by the method described above.

[0027] The present invention also provides application of the low-viscosity protein particles described above in the food field.

[0028] In a specific embodiment of the present invention, the low-viscosity protein particles are used in high-protein beverages, sports nutrition products, infant food, and healthy meal replacements.

[0029] [Beneficial Effects]

[0030] (1) The present invention uses a reducing agent to break the internal disulfide bonds of protein molecules, allowing the protein to unfold and expose the internal hydrophobic groups. At the same time, heat treatment is used to promote the aggregation and reorganization of unfolded proteins under the synergistic driving of hydrophobic interactions and hydrogen bonds to form low-viscosity protein particles.

[0031] (2) The present invention develops a simple, convenient and low-cost protein modification method, and low-viscosity protein particles can be prepared without the assistance of high-power equipment. The low-viscosity protein particles prepared by this method can be used in the development of high-protein beverages, sports nutrition products, infant food, healthy meal replacements and other products to meet consumers' demand for high-protein healthy diets.

[0032] (3) The method of the present invention modifies cod myofibrillar protein in animal protein to obtain low-viscosity cod protein. The high-protein solution 10% (w / v) after reconstitution has better fluidity and lower viscosity than the natural cod protein solution. -1 The viscosity of the high concentration solution is close to 0.1 Pa·S. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The cold field scanning electron microscope observation results of the low viscosity cod protein particles and the natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1;

[0034] Figure 2 The appearance pictures of the cod protein solution samples obtained in Examples 1 to 4 and Comparative Example 1 of the present invention;

[0035] Figure 3 The rheological properties of the cod protein solution samples obtained in Examples 1 to 4 and Comparative Example 1 of the present invention: the change of elastic modulus with frequency;

[0036] Figure 4 The rheological properties of the codfish protein solution samples obtained in Examples 1 to 4 and Comparative Example 1 of the present invention: the change of the viscosity modulus with frequency;

[0037] Figure 5 The rheological properties of the cod protein solution samples obtained in Examples 1 to 4 and Comparative Example 1 of the present invention: the change of surface viscosity with shear rate;

[0038] Figure 6 The dynamic light scattering measurement results of the low-viscosity cod protein particles and the natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1;

[0039] Figure 7 The endogenous fluorescence spectra of the low-viscosity cod protein particles and the natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1 are measured;

[0040] Figure 8 The maximum fluorescence intensity and corresponding wavelength analysis results of the low-viscosity cod protein particles and natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1;

[0041] Fig. 9 The circular dichroism spectra of the low-viscosity cod protein particles and the natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1 are measured;

[0042] Fig.10 The relative content analysis results of the secondary structures of the low-viscosity cod protein particles and the natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1 are shown;

[0043] Fig.11 The zeta potential measurement results of the low-viscosity cod protein particles and the natural cod protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1;

[0044] Fig.12 These are the measurement results of the total thiol content of the low-viscosity codfish protein particles and the natural codfish protein particles obtained in Examples 1 to 4 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0045] Example 1

[0046] (1) dispersing natural cod protein powder in deionized water to prepare a protein suspension with a concentration of 0.5% (the addition ratio of protein powder to water is 0.5 g: 100 mL), and adjusting the pH to 7 at room temperature;

[0047] (2) dissolving sodium metabisulfite in deionized water (pH 7) to obtain a reducing agent solution with a concentration of 25 mM, and adjusting the pH to 7 at room temperature;

[0048] (3) adding the reducing agent solution to the protein suspension obtained in step (1) at a volume ratio of 1:19, mixing, and immediately heating at 100° C. for 30 min to obtain a modified protein suspension;

[0049] (4) adjusting the pH of the modified protein suspension obtained in step (3) to the isoelectric point (pH 4.5) and centrifuging at a centrifugal force of 10,000 g, a centrifugal time of 5 min, and a centrifugal temperature of 4° C. to obtain a protein precipitate;

[0050] (5) washing the protein precipitate with water and resuspending it in water, adjusting the pH to 7 at room temperature to obtain a primary modified protein solution;

[0051] (6) centrifuging the initially modified protein solution at a centrifugal force of 13,000 g, a centrifugal time of 20 min, and a centrifugal temperature of 4° C., and taking the supernatant to obtain the modified protein solution;

[0052] (7) The modified protein solution was subjected to vacuum freeze drying at a temperature of -30°C, a vacuum degree of 1 Pa, and a drying time of 24 h to obtain a low-viscosity codfish protein powder.

[0053] Example 2

[0054] The preparation method is consistent with that of Example 1, except that in step (2), the concentration of the reducing agent solution is 50 mM.

[0055] Example 3

[0056] The preparation method is consistent with that of Example 1, except that in step (2), the concentration of the reducing agent solution is 100 mM.

[0057] Example 4

[0058] The preparation method is consistent with that of Example 1, except that in step (2), the concentration of the reducing agent solution is 200 mM.

[0059] Comparative Example 1

[0060] Natural cod protein powder was prepared into a protein solution with a concentration of 0.1 mg / mL using deionized water.

[0061] The low-viscosity cod protein powder of Examples 1 to 4 was prepared into a protein solution with a concentration of 0.1 mg / mL; the protein solution was frozen under liquid nitrogen at -196°C and then cut under vacuum conditions; the surface of the obtained sample was sprayed with gold-palladium alloy for 60 seconds at a current of 10 mA, and scanning imaging was performed at -145°C and an accelerating voltage of 10 kV to obtain the microscopic morphology of the low-viscosity cod protein particles.

[0062] After freezing the protein solution of Comparative Example 1 with liquid nitrogen, the protein sample was cut under vacuum, and the sample surface was sprayed with gold-palladium alloy at a current of 10 mA for 60 s. Scanning imaging was performed at -145° C. and an accelerating voltage of 10 kV to obtain the microscopic morphology of natural protein particles.

[0063] Cold field scanning electron microscopy results are as follows Figure 1 The results showed that natural cod protein was a typical spherical morphology with a particle diameter of approximately 100 nm, while low-viscosity cod protein particles were atypical spherical morphology, indicating that cod protein underwent varying degrees of unfolding and aggregation after the disulfide bonds were broken by the reducing agent. Moreover, as the concentration of the reducing agent increased, the aggregation behavior of the protein sample became more obvious, and the size of the recombinant cod protein particles formed was larger.

[0064] The low-viscosity cod protein powder and natural cod protein powder obtained in Examples 1 to 4 were prepared into a protein solution with a concentration of 10% (w / v), and placed in a 20 mL sample vial. The sample appearance was observed in a tilted state ( Figure 2 ). The rheological properties of the above protein solution samples were measured using a rheometer. The shear rate was set at 0.1 to 100 s -1 , the storage modulus and loss modulus of the sample are obtained as follows Figure 3 and Figure 4 As shown, the viscosity change of the sample is Figure 5 As shown, viscosity was then used as the dependent variable and shear rate as the independent variable to fit the Power law (Ostwald-deWaele) rheological model τ = Kγ n In the equation, shear stress (Pa) is represented by τ, and shear rate (s -1 ) is represented by γ, the unit of the consistency coefficient K is Pa·S, and the fluidity index is represented by n. The fluidity indexes of the low viscosity codfish protein solution sample and the codfish protein sample are obtained, as shown in Table 1.

[0065] Table 1 Fluidity index of low viscosity codfish protein solution

[0066]

[0067] Different letters in the table represent significant differences between samples (p<0.05)

[0068] Figure 2 It can be seen from the appearance picture that the low-viscosity cod protein solution samples are all transparent solutions with good fluidity after tilting, while the natural cod protein solution sample group in the control group has obvious gelation. Figure 3 and Figure 4 The modulus-frequency scanning curves of low-viscosity cod protein solutions (10%, w / v) prepared by different reducing agent concentrations combined with preheating are shown. In all modified samples, the values ​​of elastic modulus and viscous modulus increased with the increase of sodium metabisulfite concentration, but were much lower than those of natural cod protein solutions, indicating that the modified low-protein solutions were more fluid and thinner than the control group. Similarly, the viscosity of the modified cod protein solutions was much lower than that of the natural cod protein solution ( Figure 5 ), but with the increase of sodium metabisulfite concentration, the viscosity of the protein solution showed an upward trend. The modified cod protein particles obtained under the modification conditions of sodium metabisulfite concentration of 1.25mM and heating at 100℃ had the lowest solution viscosity. -1 The viscosity of the high concentration solution (10%, w / v) was close to 0.1 Pa·S. In addition, the fluidity index of all modified groups was closer to 1 than that of the control group (Table 1), indicating that the fluidity of the solution of the modified group was improved compared with that of the control group.

[0069] The modified cod protein powder and natural cod protein powder of Examples 1 to 4 were prepared into a 1.0 mg / mL solution with deionized water and filtered through a 0.88 μm water filter membrane for dynamic light scattering measurement. The particle size distribution results of the modified cod protein and natural cod protein were obtained by scattering at a fixed scattering angle of 90°.

[0070] Dynamic light scattering measurement results show that ( Figure 6 ), with the increase of reducing agent concentration, the particle size distribution of low viscosity cod protein particles first becomes smaller and then larger than that of natural cod protein particles. Combined with the results of cold field scanning electron microscopy, this is because the lower reducing agent concentration causes the protein to unfold to a certain extent, exposing some hydrophobic groups, and under heating conditions, the hydrophobic force drives the reorganization to form smaller protein aggregates. Higher reducing agent concentrations make the protein disaggregate more fully, thus triggering more violent aggregation behavior and forming larger aggregates.

[0071] The low-viscosity cod protein particles and natural cod protein particles of Examples 1 to 4 were prepared into a 1.0 mg / mL solution with deionized water, and measured using a F-2700 fluorescence spectrophotometer. The emission spectrum in the wavelength range of 300-500 nm was obtained at an excitation wavelength of 280 nm, and the slits for excitation and emission were set to 5 nm. The determination of the intrinsic fluorescence spectrum revealed the effect of the modification method of reductive cleavage combined with preheating treatment on the tertiary structure of cod protein. Figure 7 , 8 As shown in the figure, the maximum fluorescence intensity of low-viscosity cod protein was significantly reduced compared with the control group, indicating that the tryptophan residues buried inside the molecule were reburied inside the recombinant protein particles during the protein unfolding-aggregation process. This shows that after the reducing agent destroys the disulfide bonds of the protein, more hydrophobic sites are exposed, causing stronger hydrophobic interactions. This is a protein aggregation reorganization caused by hydrophobic interactions, thereby achieving the effect of consuming active groups and reducing viscosity.

[0072] The cod protein particles and natural cod protein particles obtained in Examples 1 to 4 were prepared into a 1.0 mg / mL solution with deionized water, and then the solution was diluted to 0.2 mg / mL for measurement. The secondary structure of the low-viscosity protein was measured using a J-1500 circular dichroism spectrometer. The wavelength range of the measurement was set to 190-300 nm, the optical path of the quartz cuvette was 0.1 cm, the scanning speed was 100 nm / min, and the average scanning was 3 times. The secondary structure information of the low-viscosity protein was analyzed using the Yang'Reference model in the Protein Secondary Structure Estimation software, and the secondary structure ratios of different protein samples were calculated. The secondary structure information of natural cod protein was analyzed to obtain the secondary structure information of different aggregates.

[0073] like Fig. 9 As shown, natural cod protein has double negative peaks at 208nm and 222nm, indicating that cod protein is rich in α-helical structure. The circular dichroism spectrum of low-viscosity cod protein shows that the extreme values ​​of the negative peaks at 208nm and 222nm are reduced and the wavelengths are blue-shifted, indicating that the secondary structure of the protein has changed and the α-helical structure has been lost. Fig.10 The results showed that the secondary structure of natural cod protein was composed of 37.90% α-helix, 31.25% β-fold, 1.20% β-turn, and 29.65% random coil. The secondary structure of low-viscosity cod protein (10mM+100℃ combined treatment) was composed of 24.80% α-helix, 43.00% β-fold, 0% β-turn, and 32.30% random coil. The results confirmed the transformation of the secondary structure from α-helix to β-fold and random coil during the formation of low-viscosity cod protein.

[0074] The codfish protein particles and natural codfish protein particles of Examples 1 to 4 were prepared into a 1.0 mg / mL solution with deionized water, and then the solution was diluted to 0.20 mg / mL for measurement. The Zeta potential was measured using a 3000HSA laser particle size potential analyzer, and the potential was calculated using the Henry equation.

[0075] like Fig.11 The results showed that the absolute value of the Zeta potential of the modified cod protein was greater than that of the natural cod protein, indicating that the protein had more charged groups on the surface, higher stability and greater intermolecular repulsion.

[0076] The low-viscosity cod protein particles of Examples 1 to 4 were configured into a 5.0 mg / mL protein solution with deionized water, and 2 mL of the diluted low-viscosity cod protein solution was taken and added to 6 mL of Tris-Gly buffer (0.086 M Tris, 0.09 M glycine, 4.0 mM ethylenediaminetetraacetic acid, 8.0 M urea) to obtain a low-viscosity cod protein solution with a concentration of 0.6 mg / mL. Then 80 μL of Ellman's reagent solution (4 mg / mL DTNB dissolved in Tris-Gly buffer) was added to the above protein solution (0.6 mg / mL), and the absorbance value (A1) at 412 nm was recorded after culturing in the dark for 25 min. The test samples without protein solution and Ellman's reagent were used as controls (A2) and negative values ​​(A3), respectively. Data was determined according to the following formula:

[0077]

[0078] Where 13,600 is the molar absorptivity of Ellman's reagent (M -1 cm -1 ), b is the optical path length of the cuvette, which is 0.5 cm, and c is the protein concentration (mg / mL).

[0079] The natural cod protein particles were prepared into a 5.0 mg / mL protein solution with deionized water, 2 mL of the diluted cod protein solution was taken and added to 6 mL of Tris-Gly buffer (0.086 M Tris, 0.09 M glycine, 4.0 mM ethylenediaminetetraacetic acid, 8.0 M urea) to obtain a cod protein solution with a concentration of 0.6 mg / mL, and then 80 μL of Ellman's reagent solution (4 mg / mL DTNB dissolved in Tris-Gly buffer) was added to the cod protein solution (0.6 mg / mL), and the absorbance value at 412 nm was recorded after culturing in the dark for 25 min (A1). The test samples without protein solution and Ellman's reagent were used as controls (A2) and negative values ​​(A3). The data was determined according to the following formula:

[0080]

[0081] Where 13,600 is the molar absorptivity of Ellman's reagent (M -1 cm -1 ), b is the optical path length of the cuvette, which is 0.5 cm, and c is the protein concentration (mg / mL).

[0082] The sulfhydryl group (-SH) and disulfide bond (-SS-) are often used as important indicators to measure the unfolding state of proteins. Fig.12 The results showed that the total thiol content of modified codfish protein was greater than that of natural codfish protein. This phenomenon was attributed to the effective cleavage of disulfide bonds within the protein by the reducing agent, which exposed the -SH groups within the molecule and led to an increase in the total thiol content.

[0083] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing low-viscosity protein particles, characterized in that: The following steps are involved: (1) dispersing protein powder in water to obtain a protein suspension, and adjusting the pH to 6 to 9; (2) dissolving a reducing agent in a solvent to obtain a reducing agent solution, and adjusting the pH of the solution to 6 to 9; (3) mixing the pH-adjusted protein suspension with a reducing agent solution, and then performing a heat treatment to obtain a modified protein suspension; (4) The modified protein suspension is adjusted to the isoelectric point and then centrifuged to obtain a protein precipitate. The protein precipitate is washed with water and placed in water, and the pH is adjusted to 6-9 to obtain an initial modified protein solution. The solution is centrifuged and the supernatant is taken to obtain a modified protein solution. Finally, the solution is dried to obtain low-viscosity protein particles.

2. The method for preparing low-viscosity protein particles according to claim 1, characterized in that: In step (1), the protein is animal myofibrillar protein or plant protein; the addition ratio of protein powder to water is 0.2-2 g:100 mL.

3. The method for preparing low-viscosity protein particles according to claim 1, characterized in that: In step (2), the solvent is one of water, PBS buffer, HBSS buffer, and Tris-HCl buffer, and the pH of the solvent is 6-9.

4. The method for preparing low-viscosity protein particles according to claim 1, characterized in that: In step (2), the reducing agent is one or more of sodium pyrosulfite, sodium sulfite, potassium pyrosulfite, cysteine, and reduced glutathione; and the concentration of the reducing agent in the reducing agent solution is 25 to 200 mM.

5. The method for preparing low-viscosity protein particles according to claim 1, characterized in that: In step (3), the volume ratio of the reducing agent solution to the protein suspension is 1:

19.

6. The method for preparing low-viscosity protein particles according to claim 1, characterized in that: In step (3), the parameters of the heat treatment are: temperature of 60 to 120° C., and heating time of 5 to 120 min.

7. The method for preparing low-viscosity protein particles according to claim 1, characterized in that: In step (4), the drying method is vacuum freeze drying or spray drying; The parameters of vacuum freeze drying are: temperature of -80 to -30°C, vacuum degree of 0.01Pa to 50Pa, and drying time of 24 to 72h; The parameters of spray drying are: inlet air temperature is 175~195℃, and outlet air temperature is 80~100℃.

8. Low-viscosity protein particles prepared by the method according to any one of claims 1 to 7.

9. Use of the low-viscosity protein particles according to claim 8 in the food field.

10. The use according to claim 9, characterized in that: The application of low viscosity protein particles in high protein beverages, sports nutrition products, infant food, and healthy meal replacements.

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