Preparation method and application of myofibrillar protein hydrosol

Abstract

The invention provides a preparation method and application of myofibrillar protein hydrosol, and the method comprises the following steps: uniformly dispersing myofibrillar protein in water to obtain a myofibrillar protein suspension, mixing the myofibrillar protein suspension with protein glutaminase to obtain a mixed solution A, mixing the mixed solution A with a glucan aqueous solution to carry out glycosylation reaction to obtain a mixed solution B, and carrying out freeze drying to obtain the myofibrillar protein hydrosol. And cooling in an ice-water bath, and storing at 4 DEG C to obtain the myofibrillar protein hydrosol. The myofibrillar protein hydrosol is used for preparing protein beverages and nasal feeding nutrient solutions. The method for preparing the myofibrillar protein hydrosol with excellent dispersity and storage stability at lower production cost under the condition that professional large-scale instruments and equipment are not needed is used for preparing protein beverages and nasal feeding nutrient solutions.

Description

Technical Field

[0001] This invention belongs to the field of protein processing technology, specifically relating to a method for preparing myofibrillar protein hydrosol and its application. Background Technology

[0002] Muscle is widely recognized as a high-quality protein source due to its balanced amino acid profile, including all essential and non-essential amino acids, as well as its high biological value, excellent digestibility, and low allergenicity. However, traditional meat products are relatively tough, and their main protein (myofibrillar protein) is insoluble in water and cannot be stably and uniformly dispersed. This characteristic is particularly unfriendly to the elderly, infants, and especially patients with swallowing difficulties. The World Health Organization (WHO) has included dysphagia in the 10th edition of the International Classification of Diseases. Dou Zulin et al., at the 7th European Conference on Dysphagia and the 1st World Forum on Dysphagia, stated that the prevalence of dysphagia in the elderly is as high as 11.4%–33.7%. Against this backdrop, developing an easily swallowable fluid-like myofibrillar protein hydrosol has significant practical application implications and commercial potential. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing myofibrillar protein hydrosol and its application, which addresses the shortcomings of the prior art. This method prepares myofibrillar protein hydrosol with excellent dispersibility and storage stability at a lower production cost without the need for specialized large-scale instruments and equipment, and can be used in the preparation of protein beverages and nasal feeding solutions.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing myofibrillar protein hydrosol, the method being as follows: S1. Disperse myofibrillar protein evenly in water to obtain a myofibrillar protein suspension; S2. Mix the myofibrillar protein suspension obtained in S1 with protein glutaminase and stir to obtain mixture A. S3. Mix the mixed solution A obtained in S2 with the dextran aqueous solution and stir in a water bath at 37 ℃ to carry out the glycosylation reaction to obtain mixed solution B. S4. Cool the mixed solution B obtained in S3 in an ice-water bath to obtain a sol. S5. The sol obtained in S4 is stored at a temperature of 4 ℃ to obtain myofibrillar protein hydrosol. Preferably, the concentration of the myofibrillar protein suspension in S1 is 40 mg / mL; Preferably, the mass of the added protein glutaminase in S2 is 3.2% of the protein mass; Preferably, the concentration of the dextran aqueous solution in S3 is 20 mg / mL, and the molecular weight of the dextran in the dextran aqueous solution is 70 kDa; Preferably, the volume ratio of the mixed solution A and the dextran aqueous solution in S3 is 2:1; Preferably, the glycosylation reaction in S4 takes 8 hours; The present invention also provides the application of the myofibrillar protein hydrosol prepared by the above preparation method, wherein the myofibrillar protein hydrosol is used in the preparation of protein beverages and nasogastric feeding solutions.

[0005] Compared with the prior art, the present invention has the following advantages: This invention combines enzymatic deamidation and glycosylation methods to prepare myofibrillar protein hydrosols without requiring large-scale specialized equipment. Glycosylation induces structural relaxation of myosin molecules, thereby exposing previously buried glutamine and asparagine residues and enhancing their accessibility to deamidase recognition and catalysis. Enzymatic deamidation increases the surface negative charge of myosin, enhancing its affinity for reducing sugars. Furthermore, deamidation exposes other masked sugar-binding sites (e.g., the ε-amino group in lysine or the guanidin group in arginine), further promoting the glycosylation modification of myofibrillar proteins. The resulting hydrosol exhibits high nutritional value, good stability and dispersibility, uniform structure, and high solubility and turbidity. As a novel meat product, this product can be formulated with various flavors to become a convenient protein beverage for everyday consumers, or used as a nutrient carrier and nutritional supplement for the elderly, infants, and patients with swallowing difficulties. It also has the potential to be used as a nasogastric feeding solution for patients who have lost the ability to swallow. The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0006] Figure 1 This is a graph showing the effect of different preparation methods on (A) microstructure, (B) particle size distribution, (C) average particle size, and (D) appearance of myofibrillar protein hydrosol in Examples 1 and 1-3 of the present invention. Figure 2 This is a graph showing the effect of different preparation methods on (A) deamidation degree, (B) solubility, (C) turbidity, (D) potential, (E) intrinsic fluorescence intensity, and (F) surface hydrophobicity of myofibrillar protein hydrosol in Example 1 and Comparative Examples 1-3 of the present invention. Figure 3 This is a graph showing the influence of different preparation methods on the myofibrillar protein hydrosol in Example 1 and Comparative Examples 1-3 of the present invention, specifically on (A) frequency scanning results and (B) apparent viscosity measurement results. Detailed Implementation Example

[0007] The method for preparing myofibrillar protein hydrosol in this embodiment is as follows: S1. Disperse myofibrillar protein evenly in water to obtain a myofibrillar protein suspension with a concentration of 40 mg / mL. S2. The myofibrillar protein suspension obtained in S1 is mixed with protein glutaminase at a protein content of 3.2% to obtain mixture A. S3. Mix the mixed solution A obtained in S2 with a dextran aqueous solution with a concentration of 40 mg / mL, seal the top to isolate oxygen, and stir in a water bath at a temperature of 37 ℃ for 8 h to carry out the glycosylation reaction to obtain mixed solution B; the molecular weight of dextran is 70 kDa. S4. The mixed solution B obtained in S3 is placed in an ice-water bath and cooled to 0-4 ℃ to terminate the glycosylation reaction and obtain a sol. S5. The sol obtained in S4 is stored overnight at a temperature of 4 ℃ to obtain myofibrillar protein hydrosol. The myofibrillar protein hydrosol prepared in this embodiment contains 3.2±0.76 g of protein glutaminase per 100 g of protein, has a protein solubility of 78.92±2.64%, a turbidity of 0.30±0.01, a significantly reduced viscosity exhibiting shear-thinning behavior, and no obvious stratification was observed after storage at 4 ℃ for 7 days.

[0008] Comparative Example 1 The preparation method of the myofibrillar protein hydrosol in this comparative example differs from that in Example 1 in that steps S2, S3, and S4 are omitted, i.e., the addition of protein glutaminase and glycosylation treatment and termination of reaction are not performed. The remaining preparation methods are consistent with those in Example 1.

[0009] Comparative Example 2 The preparation method of the myofibrillar protein hydrosol in this comparative example differs from that in Example 1 in that step S2, protein glutaminase addition, is omitted; the remaining preparation methods are the same as those in Example 1.

[0010] Comparative Example 3 The preparation method of myofibrillar protein hydrosol in this comparative example differs from that in Example 1 in that the glycosylation treatment and termination reaction in step S3 are not performed, while the rest of the preparation method is the same as that in Example 1. The preparation method of myofibrillar protein hydrosol in this comparative example S1. Disperse myofibrillar protein evenly in water to obtain a myofibrillar protein suspension with a concentration of 40 mg / mL. S2. Mix the mixed solution A obtained in S1 with a 20 mg / mL dextran aqueous solution, seal the top to isolate oxygen, and stir in a water bath at 37°C for 8 h to carry out the glycosylation reaction, to obtain mixed solution B; the molecular weight of dextran is 70 kDa. S3. Mix the mixed solution A obtained in S1 with protein glutaminase containing 3.2% protein to obtain mixed solution C; S4. Mix the mixed solution B obtained in S2 with protein glutaminase at a protein content of 3.2% and place it in an ice-water bath to cool to 0-4 ℃ to terminate the glycosylation reaction and obtain a sol. S5. The sol obtained in S4 is stored overnight at a temperature of 4 °C to obtain myofibrillar protein hydrosol. Example This embodiment is a test experiment of the myofibrillar protein hydrosol prepared in Example 1 and Comparative Examples 1-3.

[0011] Experiment 1 Myofibrillar protein hydrosols prepared using different processes in Examples 1 and Comparative Examples 1-3 were centrifuged at 10000 g and 4 °C for 10 min using a refrigerated centrifuge, and the resulting supernatant was diluted 50-fold. 10 μL aliquots of the diluted sample were mixed with 90 μL of ammonia analysis reagent and incubated at room temperature in the dark for 15 min. Fluorescence intensity was measured at excitation and emission wavelengths of 360 nm and 450 nm, respectively. Ammonia concentration was quantified using a standard curve (0–1 mmol / L) generated using 1 mM NH4Cl solution. To determine total releasable ammonia, MP solution was mixed with 6 M HCl and incubated at 95 °C for 4 h. The degree of deamidation was calculated using the following formula: Deamidation degree (%) = Ammonia released during deamidation [mmol / L] / Total ammonia released [mmol / L] × 100% like Figure 2 As shown in (A), the degree of deamidation in Comparative Example 3 was increased compared to Comparative Example 1, indicating that glutamine residues were efficiently and specifically converted into glutamate residues and the release of ammonia during protein glutaminase treatment. Example 1 showed the highest degree of deamidation, suggesting that glycosylation can promote the deamidation process. This phenomenon may be due to glycosylation-induced protein unfolding, which exposes buried glutamine and asparagine residues, thereby enhancing their accessibility to deamidases.

[0012] Experiment 2 The myofibrillar protein hydrosols prepared using different processes in Example 1 and Comparative Examples 1-3 were diluted with deionized water to a concentration of 2 mg / mL. The hydrosols were centrifuged at 5000 g and 4 °C for 10 min using a refrigerated centrifuge. The protein concentration before and after centrifugation was determined using the Biuret method. The final solubility result was calculated using the following formula: Solubility (%) = (Supernatant protein concentration / Original protein concentration) × 100 like Figure 2 As shown in (B), the solubility of native myofibrillar protein in Comparative Example 1 was very low (2.79%), mainly because myosin is the main protein in myofibrillar proteins, with periodically distributed tails and clusters carrying different electrostatic charges. This leads to the spontaneous aggregation of the protein into bipolar helical filaments under low-salt conditions. The solubility of myofibrillar protein in Comparative Examples 2 and 3 was significantly increased, reaching 46.82% and 31.87%, respectively. This improvement may be due to the addition of protein glutaminase to convert glutamine to glutamate, thereby introducing additional hydrophilic and negatively charged residues. Dextran coupling removes positively charged lysine residues, and its hydrophilic portion enhances hydration by increasing the interaction between the conjugate and water molecules, promoting the dispersion of myofibrillar protein in aqueous solution. It is worth mentioning that the combination of the two methods further significantly improved the solubility, with Example 1 being significantly better than Comparative Examples 2 and 3, and the solubility of myofibrillar protein in Example 1 being 78.92 ± 2.64%. Glycosylation unfolds the protein structure, transforming the α-helix into other structures and exposing internal protein groups to the protein surface. Meanwhile, deamidase-induced conversion of neutral amino acids into negatively charged residues increases in intermolecular electrostatic repulsion, disrupting protein aggregates and exposing additional reaction sites. This promotes enhanced reactions between myofibrillar proteins and dextran, significantly improving grafting efficiency.

[0013] Experiment 3 The myofibrillar protein hydrosols prepared using different processes in Examples 1 and Comparative Examples 1-3 were diluted with deionized water to a concentration of 1 mg / mL. The absorbance of the samples at 600 nm was measured using a spectrophotometer and used as the turbidity of the samples. Figure 2(C) The turbidity of Comparative Examples 1-3 and Example 1 were 0.94±0.03, 0.43±0.02, 0.57±0.02, and 0.30±0.01, respectively. Turbidity and solubility were generally negatively correlated, especially the turbidity of Comparative Example 2 and Example 1, which were quite low, at 0.43 and 0.30, respectively. This was mainly because protein glutaminase caused the myofibrillar protein double helix structure to unfold, and myosin lost its self-assembled rod-like structure. Glycosylation also slightly reduced the turbidity because the polyhydroxy sugar molecules added by glycosylation increased the hydrophilicity of the protein. The improvement in solubility and turbidity made the product appearance more uniform, which is beneficial to the future formulation and development of this product and will not have a significant impact on consumers' sensory experience.

[0014] Experiment 4 The myofibrillar protein hydrosols prepared by different processes in Example 1 and Comparative Examples 1-3 were placed into sample appearance bottles and stored in a light-protected cold storage at 4 ℃ for 7 days before observing the appearance of the samples. Figure 1 As shown in (D), corresponding to the turbidity results, different degrees of stratification were observed after storage in a cold storage at 4 °C for 7 days. Comparative Example 1 showed significant phase separation, characterized by a distinct upper aqueous layer and a large amount of white myofibrillar protein precipitate at the bottom, indicating poor water stability of myosin. The migration levels of Comparative Examples 2-3 and Example 1 were reduced to varying degrees. In particular, in Example 1, the solution was uniformly dispersed with almost no phase separation, indicating that the treatment method of the present invention significantly enhances the storage stability of the protein and can ensure that the product will not affect its shelf life due to spontaneous aggregation during storage.

[0015] Experiment 5 The myofibrillar protein hydrosols prepared by different processes in Example 1 and Comparative Examples 1-3 were diluted to a solution of 0.1 mg / ml, and the micron-sized particles were measured using a laser particle size analyzer. Figure 1As shown in (B, C), in low ionic strength solutions, periodically distributed charged clusters in the myosin tail attract each other and induce self-assembly. Therefore, Comparative Example 1 exhibits a significantly larger average particle size (d4,3), reaching 115.13 ± 3.85 μm. The particle size distribution shows a bimodal pattern, with the large particle peak attributed to myosin self-assembled aggregates, and the low particle peak due to monomers and oligomers within the myosin. In contrast, the average volume diameter of Comparative Example 2 was significantly reduced to 55.55 ± 1.98 μm, and the peak of large particles shifted to that of small particles in the particle size distribution. This indicates that the addition of dextran promoted the dissociation of myosin filament aggregates into soluble myosin oligomers. Simultaneously, we found that the particle size distributions of Comparative Example 2, Comparative Example 3, and Example 1 were significantly wider. This was mainly due to the steric hindrance introduced by the attached dextran and the enhanced electrostatic repulsion caused by the conversion of amide to carboxyl groups during deamidation. These effects collectively promoted the dissociation of coarse filaments into smaller aggregates (monomers, dimers, and oligomers), resulting in the smallest particle size in Example 1 (35.09 ± 11.99 μm), significantly lower than that of Comparative Example 3 (77.19 ± 1.87 μm). Smaller particle sizes contribute to a more delicate and uniform product texture and are also beneficial for digestion and utilization.

[0016] Experiment Six The myofibrillar protein hydrosols prepared by different processes in Examples 1 and Comparative Examples 1-3 were diluted to a solution of 0.1 mg / ml, and the micron-sized particle size and zeta potential were measured using a laser particle size analyzer and a particle size potential analyzer, respectively. Figure 2 (D) Due to the deprotonation of amino acids in myofibrillar proteins, all samples exhibited a negative surface charge at neutral pH. The potentials of Comparative Examples 2 and 3 were -12.75 ± 0.71 mV and -15.94 ± 2.08 mV, respectively, with absolute potentials higher than -7.8 mV of Comparative Example 1. The change in potential in Comparative Example 2 was due to the depletion of positively charged lysine residues induced by dextran. The change in potential in Comparative Example 3 was mainly attributed to the conversion of neutral glutamine to negatively charged glutamate by protein glutaminase. Example 1 exhibited the highest absolute negative potential at -27.94 ± 0.57 mV, superior to 12.75 ± 0.71 mV and -15.94 ± 2.08 mV of Comparative Examples 2 and 3, respectively. This is because glycosylation can induce structural loosening through steric hindrance, promoting the entry of deamidase into the substrate and facilitating the production of negatively charged glutamate and aspartic acid residues, while deamidation simultaneously exposes additional lysine residues to accelerate the depletion of positive charge. The resulting increase in absolute potential strengthens the electrostatic repulsion between particles, effectively overcoming electrostatic attraction, thereby minimizing aggregation and enhancing solubility; Generally, an increase in the absolute value of the zeta potential leads to increased electrostatic repulsion between proteins. This strong repulsion effectively disrupts electrostatic equilibrium, prevents unstable aggregation, and promotes the formation of a stable myofibrillar protein dispersion system. Furthermore, the increased potential inhibits electrostatic repulsion-dependent myosin tail polymerization, which is a potential mechanism for the stabilization of myofibrillar protein hydrosols.

[0017] Experiment 7 Take 1 mL of each of the myofibrillar protein hydrosols prepared by different processes in Example 1 and Comparative Examples 1-3, mix them with a staining solution containing 1.0 g / L Nile blue, shake well, and let stand for 30 min. Place 5 μL of the stained sample onto a glass slide with coverslips. Observe the microstructure and distribution of the protein samples using a 40x objective confocal laser scanning microscope, and take photographs of typical fluorescence images. Figure 1 (A) Comparative Example 1 shows a large number of highly ordered aggregates. Comparative Examples 2 and 3 show a shift in individual myosin particles towards smaller sizes and a decrease in average particle size compared to Comparative Example 1, indicating that individual modification can partially inhibit myosin aggregation. The images from Example 1 show a more uniform distribution of small aggregates and short fibrils, mainly due to the steric hindrance introduced by the attached dextran and the enhanced electrostatic repulsion caused by the conversion of amide to carboxyl groups during deamidation. These effects collectively promote the dissociation of coarse filaments into smaller aggregates (monomers, dimers, and oligomers).

[0018] Experiment 8 The tertiary structure of myofibrillar protein hydrosols prepared using different processes in Examples 1 and Comparative Examples 1-3 was determined using a fluorescence spectrophotometer. Figure 2 Comparative Example 1 exhibited the lowest fluorescence intensity, attributed to its compact structure under low ionic strength conditions, which shielded tryptophan residues within the protein's hydrophobic core. Compared to Comparative Example 1, Comparative Examples 2 and 3 showed significantly increased fluorescence intensity, indicating structural unfolding and the resulting exposure of previously buried tryptophan residues. Notably, Example 1 exhibited the highest fluorescence intensity, suggesting that the combined treatment induced the most pronounced structural flexibility, shifting the tryptophan microenvironment to a hydrophilic state. like Figure 2Comparative Example F, Example 1, exhibits the lowest surface hydrophobicity, consistent with its tightly folded conformation. Comparative Examples 2, 3, and Example 1 show significantly increased surface hydrophobicity because the unfolded proteins expose previously buried hydrophobic residues, enhancing their interaction with the ANS probe. This unfolding also increases the accessibility of glutamine and asparagine residues, providing additional substrates for deamidases. Overall, the tertiary structural changes of this invention disrupt the original tight structure of proteins, resulting in a milder chewing texture and easier absorption by digestive enzymes in the body. This helps improve the digestibility and absorption of nutrients such as proteins, allowing the body to better utilize the nutrients in the product.

[0019] Experiment Nine The myofibrillar protein hydrosols prepared by different processes in Example 1 and Comparative Examples 1-3 were subjected to frequency scanning and apparent viscosity determination using a rheometer. The specific parameters are as follows: Frequency sweep: The correlation between storage modulus (G') and loss modulus (G'') and angular frequency (ω) in the range of 1-100 rad / s at 25 °C was determined. A 10% amplitude strain was applied to ensure that all dynamic measurements were performed within the linear viscoelastic range. Apparent viscosity: 1-100 s -1 The apparent viscosity was determined at the shear rate. like Figure 3(B) All samples exhibited significant shear-thinning behavior, characterized by a gradual decrease in viscosity with increasing shear rate. This phenomenon indicates the behavior of pseudoplastic fluids. Comparative Example 1 showed relatively high viscosity at low shear rates, indicating that rod-shaped myosin molecules tend to self-assemble in a low ionic strength environment, forming filamentous polymers with significant steric hindrance and strong intermolecular friction. However, with increasing shear rate, the disruption of protein chain interactions and the alignment of asymmetrically dispersed molecules along the shear plane reduced frictional resistance, leading to a significant decrease in viscosity. Comparative Example 2 showed a significantly lower apparent viscosity than Comparative Example 1, and the interaction between the myosin tail and head was weakened due to glycosylation. Example 3 showed a significantly reduced apparent viscosity, indicating enhanced electrostatic repulsion, which effectively inhibited myosin tail self-association. Among these, Example 1 showed the lowest apparent viscosity, indicating that protein glutaminase treatment combined with glycosylation can promote the maximum dissociation of myosin filaments into monomeric subunits. The release of monomeric subunits reduced the intermolecular contact area, weakened interactions, and reduced frictional resistance. Furthermore, changes in apparent viscosity between samples may reflect differences in net surface charge; in Example 1, the increased charge density enhanced the stability of small particles and minimized protein-protein interactions. From a taste perspective, lower viscosity makes the product smoother and lighter, reducing heaviness and stickiness, better meeting modern consumers' demand for a light texture. In terms of consumption, low-viscosity products have better flowability, making them easier to drink or pair with other foods, serving as a basis for the development of various flavored products. In the dynamic oscillation experiment, G' and G'' represent the elasticity and viscosity of the protein, respectively. Frequency scan results for each sample treated with each method are shown in the image below. Figure 3 As shown in (A). For each sample, both G' and G'' gradually increased with ω, indicating a small frequency dependence. G' consistently exceeded G'' in all treatment groups, indicating viscoelastic dispersion behavior. Specifically, the combined treatment of glycosylation and PG enzyme had a positive effect on the stability of MP solution. The main reason is the synergistic effect of the two, which reduced the degree of droplet aggregation and unfolded the protein structure. The G' value of Example 1 showed a parallel change throughout the range, classifying it as a weak gel. This was mainly because the hydroxyl groups introduced by glycosylation and the carboxyl groups introduced by deamidation provided more potential sites for hydrogen and ionic bonds, leading to local cross-linking between molecules and inducing the formation of a weak gel. In addition, the G' and G'' values ​​of Comparative Examples 2 and 3 were significantly lower than those of Comparative Example 1, which confirmed that the protein-protein interactions were inhibited after the Maillard reaction and the enzymatic deamidation reaction. This method prepares myofibrillar protein hydrosols with excellent dispersibility and storage stability at a lower production cost without requiring specialized large-scale instruments and equipment. The myofibrillar protein hydrosols prepared by this invention can be used in the preparation of protein beverages and nasal feeding solutions.

[0020] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a myofibrillar protein hydrosol, characterized in that, The method is as follows: S1: Disperse myofibrillar protein evenly in water to obtain a myofibrillar protein suspension; S2: Mix the myofibrillar protein suspension obtained in S1 with protein glutaminase and stir to obtain mixture A; S3: Mix the mixed solution A obtained in S2 with the dextran aqueous solution, and carry out the glycosylation reaction by stirring under water bath conditions at 37 ℃ to obtain mixed solution B; S4: Cool the mixed solution B obtained in S3 in an ice-water bath to obtain a sol; S5: The sol obtained in S4 is stored at a temperature of 4 ℃ to obtain myofibrillar protein hydrosol.

2. The method for preparing a myofibrillar protein hydrosol according to claim 1, characterized in that, The concentration of the myofibrillar protein suspension described in S1 is 40 mg / mL.

3. The method for preparing a myofibrillar protein hydrosol according to claim 1, characterized in that, The mass of the added protein glutaminase described in S2 is 3.2% of the protein mass.

4. The method for preparing a myofibrillar protein hydrosol according to claim 1, characterized in that, The concentration of the dextran aqueous solution in S3 is 20 mg / mL, and the molecular weight of the dextran in the dextran aqueous solution is 70 kDa.

5. The method for preparing a myofibrillar protein hydrosol according to claim 1, characterized in that, The volume ratio of the mixed solution A and the dextran aqueous solution in S3 is 2:

1.

6. The method for preparing a myofibrillar protein hydrosol according to claim 1, characterized in that, The glycosylation reaction described in S3 takes 8 hours.

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