A method for improving composite meat protein gel properties based on modified pea protein

Abstract

The present invention discloses a method for improving the properties of composite meat protein gel based on modified pea protein, which belongs to the technical field of food protein processing. The present invention fully dissolves natural pea protein or pH-shifted modified pea protein to obtain a protein solution; then adds a disulfide bond cleaving agent to the protein solution, dissolves and mixes the solution to obtain modified pea protein. The present invention is based on the disulfide bond cleavage treatment of pea protein to dissociate acidic and basic subunits and expose more internal groups, thereby achieving the improvement of the solubility and functionality of pea protein itself. In addition, both natural pea protein and modified pea protein with disulfide bond cleavage can enhance the structural properties of low-salt meat protein gel under the action of cross-linking enzymes, develop low-salt meat protein gel with good gelation and network structure, and provide solutions and technical support for the quality deterioration of meat products under various circumstances.

Description

Technical Field

[0001] The present invention relates to a method for improving the properties of composite meat protein gel based on modified pea protein, belonging to the technical field of food protein processing, and particularly relates to plant protein modification and application thereof in meat protein gel products. Background Art

[0002] Meat and meat products are an important source of high-quality protein in the human diet. Within skeletal muscle, proteins are primarily divided into three categories: water-soluble sarcoplasmic proteins (approximately 30%), salt-soluble myofibrillar proteins (approximately 60%), and alkali-soluble matrix proteins (less than 10%). Myofibrillar proteins play a crucial role in the texture of meat products. During processing, myofibrillar proteins dissolve from muscle tissue (meat), emulsify oils and fats, and unfold during heating, forming a viscoelastic gel that imparts the elasticity and juiciness of meat products. This process is easily affected by various factors, such as the quality of the raw meat, the amount of added salt and phosphate, the heating temperature, and the heating time. Consequently, the texture of meat products is susceptible to various defects. For example, reducing the amount of salt and phosphate can hinder the dissolution of myofibrillar proteins, significantly reducing gel strength and causing quality defects such as oiliness, looseness, and roughness in meat products. Even though low-salt and low-phosphate meat products are beneficial to human health in many ways, meat products with poor textural properties are still not accepted by consumers.

[0003] Although animal protein has a higher digestibility and a more balanced amino acid composition, plant protein has received increasing attention due to people's increased awareness of environmental protection and the development and utilization of sustainable resources. Pea protein, in particular, has the characteristics of low cost, high nutritional value and low allergenicity (compared with soy and peanut proteins), and is becoming an emerging product in the plant protein industry. As a substitute for soy protein, pea protein and soy protein have homology and are mainly composed of pea globulin (7S) and legumin (11S). Their amino acid composition and subunit structure are similar to β-soybean conglobulin and soybean globulin in soy protein. In the meat industry, pea protein is a natural food additive and functional enhancer. Its advantage is that it is highly safe and conforms to the concept of "clean label".

[0004] Despite this, the functional properties of pea protein in food processing are far inferior to those of soy protein and (or) meat protein. Adding natural pea protein to meat gels and meat products not only fails to improve gel strength, but may even hinder the network formation of meat protein. Summary of the Invention

[0005] To solve the above problems, the present invention uses a reducing agent to cleave the disulfide bonds of natural pea protein and modified pea protein treated with pH shift, and strengthens its interaction with meat protein under the action of a cross-linking enzyme, thereby developing a composite meat protein gel with structural reinforcement properties, providing technical support for situations where low salt, low phosphate, etc. lead to a decrease in the texture properties of meat protein gel.

[0006] Taking pea protein as an example, the present invention provides a disulfide bond cleavage method applicable to other plant proteins to open the tight structure of plant proteins and improve processing characteristics.

[0007] The first object of the present invention is to provide a method for preparing modified pea protein, comprising the following steps:

[0008] (1) fully dissolving natural pea protein or pH-shifted modified pea protein to obtain a protein solution;

[0009] (2) Add a disulfide bond cleaving agent to the protein solution, dissolve it, and mix it for reaction.

[0010] In one embodiment of the present invention, the preparation process of the pH-shifted modified pea protein is specifically as follows: dispersing the pea protein in water, adjusting the pH of the protein solution to 11-13 with an alkaline solution at room temperature; after standing for 30-90 minutes, adding acid for neutralization, solid-liquid separation, and collecting the precipitate to obtain the pH-shifted modified pea protein.

[0011] In one embodiment of the present invention, the protein solution is adjusted to pH 12 with 2M sodium hydroxide, and the residence time is 30 min.

[0012] In one embodiment of the present invention, a 2M hydrochloric acid solution is used for acid neutralization.

[0013] In one embodiment of the present invention, the disulfide bond cleaving agent in step (2) is cysteine, and the added amount is 0.1-8 mM / g (based on protein amount), preferably 1-7 mM / g protein, and more preferably 1-5 mM / g protein.

[0014] In one embodiment of the present invention, the mixing reaction in step (2) is carried out at room temperature and for 10 minutes.

[0015] The second object of the present invention is to provide a modified pea protein prepared based on the above method.

[0016] A third object of the present invention is to provide a processing method for improving the gel strength of a low-salt composite meat protein gel using the modified pea protein, comprising the following steps:

[0017] 1) dissolving the myofibrillar protein and the modified pea protein in phosphate buffer to prepare a mixed protein solution;

[0018] 2) adding a cross-linking enzyme to the mixed protein solution;

[0019] 3) heating the mixed protein solution to form a composite protein gel.

[0020] In one embodiment of the present invention, the mass fraction of myofibrillar protein in the mixed protein solution is 3.0%.

[0021] In one embodiment of the present invention, the modified pea protein content in the mixed protein solution is 0.75%.

[0022] In one embodiment of the present invention, the modified pea protein is added to the myofibrillar protein in a mass ratio of 5 to 50%.

[0023] In one embodiment of the present invention, the amount of cross-linking enzyme added is 1-8% of the total mass of protein in the product, and specifically 5% can be selected.

[0024] In one embodiment of the present invention, the cross-linking enzyme is transglutaminase, and the added amount is 6.5 IU / (g protein).

[0025] In one embodiment of the present invention, the enzymatic activity of the cross-linking enzyme is 130 IU / g.

[0026] The fourth object of the present invention is to provide a low-salt composite meat protein gel prepared by the aforementioned processing method.

[0027] The present invention also provides application of the low-salt composite meat protein gel in food, functional food and health care products.

[0028] For example, the plant protein and its components prepared by the present invention can be used as additives to improve product processing performance defects, or as nutritional supplements to improve product nutritional properties.

[0029] The present invention also provides the use of the low-salt composite meat protein gel prepared by the aforementioned processing method as food.

[0030] For example, various meat products, minced meat gel products, emulsified minced meat products or plant meat products can be obtained by using the low-salt meat protein composite gel prepared by the present invention as raw materials or models.

[0031] The fifth object of the present invention is to provide the application of the processing method of the aforementioned meat protein composite gel in the production of other healthy meat products.

[0032] For example, low-phosphate meat products produced by the processing method of the meat protein composite gel of the present invention, or other healthy meat products for controlling certain harmful components in the product.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention proposes for the first time that cysteine-induced disulfide bond cleavage treatment dissociates the acid-base subunits of natural pea protein, causing the protein structure to unfold and the disulfide bond conformation to shift to the unstable TGT type. When a low-salt meat protein composite gel is prepared together with a cross-linking enzyme, the gel strength is significantly increased from 0.26N (containing natural pea protein) to 0.34N (containing natural pea protein treated with 1.7mM / g cysteine) and 0.30N (containing natural pea protein treated with 3.3mM / g cysteine), without causing a decrease in gel elasticity.

[0035] (2) Compared with natural pea protein, the present invention uses cysteine-induced disulfide bond cleavage treatment to more significantly improve the functional properties of pea protein modified by pH shift. After cysteine ​​treatment, the solubility of the modified pea protein increased significantly (from 67.52% to 71.94%), the surface hydrophobicity increased significantly, the internal groups of the molecule were exposed, and the disulfide bond conformation changed to the unstable tgt type. When the low-salt meat protein composite gel was prepared together with the cross-linking enzyme, the gel strength was significantly increased from 0.31N (containing modified pea protein) to 0.38N (containing modified pea protein treated with 1.7mM / g cysteine); and the gel elasticity increased significantly; the network structure of the gel became more dense. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0037] in:

[0038] Figure 1 The subunit composition changes of the pea proteins prepared in Control Examples 1 and 2 and Examples 1 to 8;

[0039] Figure 2 The solubility changes of pea proteins prepared in Control Examples 1 and 2 and Examples 1 to 8;

[0040] Figure 3 The surface hydrophobicity changes of pea proteins prepared in Control Examples 1 and 2 and Examples 1 to 8;

[0041] Figure 4 The intrinsic fluorescence spectrum changes of the pea proteins prepared in Control Examples 1 and 2 and Examples 1 to 8;

[0042] Figure 5 The disulfide bond conformational changes of pea proteins prepared in Control Examples 1 and 2 and Examples 1, 2, 5, and 6;

[0043] Figure 6 The changes in gel strength of the composite meat protein gels prepared in Control Examples 3 and 4 and Examples 9 to 12;

[0044] Figure 7 The elastic modulus changes of the composite meat protein gels prepared in Control Examples 3 and 4 and Examples 9 to 12;

[0045] Figure 8 The microstructural changes of the composite meat protein gels prepared in Control Example 4 and Examples 10 and 12 are shown. DETAILED DESCRIPTION

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0048] The testing and characterization methods involved in the embodiments and comparative examples of the present invention are as follows:

[0049] Determination of pea protein subunit composition: Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 5% stacking gel and a 12% separating gel. 25 μL of protein solution (2 mg / mL, diluted in loading buffer) was added to each well, and electrophoresis was performed in constant voltage mode. The sample was passed through the stacking gel at a voltage of 80 V and the separating gel at a voltage of 120 V. After electrophoresis, the samples were stained with Coomassie Brilliant Blue G-250 to visualize protein bands.

[0050] Pea protein solubility determination: A pea protein solution was diluted to a protein concentration of 10 mg / mL and centrifuged at 8,000 g for 15 minutes. The protein concentration in the supernatant was determined by UV absorption. Solubility (%) was expressed as the percentage of the protein concentration in the supernatant relative to the total protein concentration before centrifugation.

[0051] Determination of pea protein surface hydrophobicity: 5 mL of pea protein solutions at concentrations of 0.05, 0.10, 0.15, 0.20, and 0.25 mg / mL were added to 40 μL of 8 mM ANS-NH4 (1-anilinonaphthalene-8-sulfonic acid ammonium salt) and mixed thoroughly. Fluorescence intensity was recorded using a fluorescence spectrometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. Surface hydrophobicity was calculated from the slope of the linear regression of fluorescence intensity and protein concentration.

[0052] Intrinsic fluorescence spectroscopy: After centrifugation at 8000 g, the supernatant of the pea protein solution was diluted with 10 mM phosphate buffer (pH 7.0) to a protein concentration of 0.2 mg / mL. Fluorescence spectrometer scanning was performed using the following scanning conditions: excitation wavelength of 280 nm, scan speed of 600 nm / min, emission wavelength of 300-450 nm, and slit width of 5 nm. The blank sample was the phosphate buffer solution described above.

[0053] Identification of disulfide bond conformation: Raman spectrum of pea protein solution (100 mg / mL) was obtained in the range of 200-1800 cm -1 The acquisition time was 40 s and the scans were repeated 5 times. The spectral range of 470-580 cm was recorded using a Savitzky-Golay filter. -1 The spectra in the interval were smoothed and fitted to analyze the changes in the three basic conformations. The three Gaussian components peaked at 510, 525, and 540 cm-1, corresponding to the ggg (gauche-gauche-gauche), tgg (trans-gauche-gauche), and tgt (trans-gauche-trans) conformations, respectively.

[0054] Extraction of myofibrillar protein: Myofibrillar protein was extracted from the longissimus dorsi muscle of the pig. The muscle was minced and homogenized in 4 volumes of phosphate buffer (containing 0.1M sodium chloride, 2mM magnesium chloride, 10mM sodium dihydrogen phosphate and 1mM EGTA, pH = 7). The resulting homogenate was centrifuged at 2000g for 15 minutes. Under the same homogenization and centrifugation conditions, the myofibrillar protein was separated from the precipitate by washing twice with the same phosphate buffer and then twice with 0.1M sodium chloride. In the final washing step, the myofibrillar protein precipitate was suspended in 0.1M NaCl and the pH was adjusted to 6.25, followed by centrifugation. The protein content was determined using the biuret method.

[0055] Preparation of composite protein gels: Use a plastic pipette to pipette 5.0 g of the composite protein solution to be tested into a small glass vial (16 mm inner diameter). Heat in a water bath from 23°C to 75°C at a heating rate of 1°C / min. Immediately after heating, cool the sample in an ice-water mixture for 30 minutes, then refrigerate at 4°C overnight to produce the corresponding protein gels for the following gel performance measurements. Prior to measurement, remove the gel sample from the refrigerator and allow it to equilibrate at room temperature for 2 hours.

[0056] Gel strength: Gel strength was measured using a texture analyzer. The pre-test speed was 2 mm / s; the test speed was 1 mm / s; and the post-test speed was 5 mm / s. The probe diameter was 12.7 mm. Gel strength was defined as the maximum pressure during the downward compression.

[0057] Gel rheology: The protein complex solution was placed in a 30 mm diameter parallel plate fixture with a 1.0 mm gap. The heating program was from 23°C to 75°C and then cooled to 23°C. Both the heating and cooling rates were 5°C / min. Dynamic measurements were taken every 30 seconds, controlled at a maximum strain of 0.01 and a frequency of 0.1 Hz.

[0058] Gel microstructure determination: Gel samples were fixed with 2.5% glutaraldehyde solution, frozen in liquid nitrogen, and dried under vacuum. The dried samples were gold-spray coated and imaged using a scanning electron microscope at 3 kV.

[0059] The natural pea protein solution involved in the embodiment of the present invention refers to: the dehulled pea powder after being crushed by a mill is mixed with deionized water at a material-liquid ratio of 1:10 (w / v), the pH value is adjusted to 8.0 with 2M NaOH, and after stirring for 2 hours, it is centrifuged at 6000g speed for 30 minutes at 4°C, and the supernatant after centrifugation is adjusted to pH 4.5 with 2M HCl. Centrifuge at 3300g speed for 30 minutes at 4°C. Wash the precipitate twice with deionized water, take the precipitate and disperse it in water and adjust the pH to 7.0 with 2M NaOH. After freeze-drying, natural pea protein is obtained and stored at 4°C for use. A certain amount of natural pea protein is dispersed in water to prepare the corresponding natural pea protein solution.

[0060] The pH-shifted modified pea protein solution described in the embodiments of the present invention is prepared by titrating 5% of the natural pea protein solution from Control Example 1 with 2M NaOH to a pH of 12, maintaining the solution at room temperature for 30 minutes, and then neutralizing the solution with 2M HCl to a pH of 7.0. The pH-shifted modified pea protein is freeze-dried and stored at 4°C until use. A certain amount of the pH-shifted modified pea protein is dispersed in water to prepare a corresponding pH-shifted modified pea protein solution.

[0061] Example 1 Natural pea protein treated with 1.7 mM / g cysteine

[0062] 1.7 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to a 3% natural pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a natural pea protein solution treated with 1.7 mM / g cysteine.

[0063] The obtained solution system is freeze-dried to obtain the modified protein product.

[0064] Example 2: Natural pea protein treated with 3.3 mM / g cysteine

[0065] 3.3 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to a 3% natural pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a natural pea protein solution treated with 3.3 mM / g cysteine.

[0066] The obtained solution system is freeze-dried to obtain the modified protein product.

[0067] Example 3: Natural pea protein treated with 5.0 mM / g cysteine

[0068] 5.0 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to a 3% natural pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a natural pea protein solution treated with 5.0 mM / g cysteine.

[0069] The obtained solution system is freeze-dried to obtain the modified protein product.

[0070] Example 4: Natural pea protein treated with 6.6 mM / g cysteine

[0071] 6.6 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to a 3% natural pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a natural pea protein solution treated with 6.6 mM / g cysteine.

[0072] The obtained solution system is freeze-dried to obtain the modified protein product.

[0073] Example 5 Modified pea protein treated with 1.7 mM / g cysteine

[0074] 1.7 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to the 3% pH-shifted modified pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a pH-shifted modified pea protein solution treated with 1.7 mM / g cysteine.

[0075] The obtained solution system is freeze-dried to obtain the modified protein product.

[0076] Example 6 Modified pea protein treated with 3.3 mM / g cysteine

[0077] 3.3 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to the 3% pH-shifted modified pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a pH-shifted modified pea protein solution treated with 3.3 mM / g cysteine.

[0078] The obtained solution system is freeze-dried to obtain the modified protein product.

[0079] Example 7 Modified pea protein treated with 5.0 mM / g cysteine

[0080] 5.0 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to the 3% pH-shifted modified pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a pH-shifted modified pea protein solution treated with 5.0 mM / g cysteine.

[0081] The obtained solution system is freeze-dried to obtain the modified protein product.

[0082] Example 8 Modified pea protein treated with 6.6 mM / g cysteine

[0083] 6.6 mM / g cysteine ​​(calculated based on the dry weight of pea protein) was added to the 3% pH-shifted modified pea protein solution and the mixture was stirred continuously at room temperature for 10 minutes to obtain a pH-shifted modified pea protein solution treated with 6.6 mM / g cysteine.

[0084] The obtained solution system is freeze-dried to obtain the modified protein product.

[0085] Control Example 1 Natural pea protein

[0086] Dehulled pea flour, ground in a mill, was mixed with deionized water at a 1:10 (w / v) ratio. The pH was adjusted to 8.0 with 2M NaOH. After stirring for 2 hours, the mixture was centrifuged at 6000g for 30 minutes at 4°C. The pH of the supernatant was adjusted to 4.5 with 2M HCl. The supernatant was then centrifuged at 3300g for 30 minutes at 4°C. The precipitate was washed twice with deionized water, dispersed in water, and the pH was adjusted to 7.0 with 2M NaOH.

[0087] After freeze-drying, store at 4°C for future use.

[0088] Control Example 2 pH Shift Modified Pea Protein

[0089] A 5% solution of the natural pea protein in Control Example 1 was titrated with 2M NaOH to a pH of 12, maintained at room temperature for 30 minutes, and then neutralized with 2M HCl to a pH of 7.0. The solution was freeze-dried and stored at 4°C for later use.

[0090] Comparative analysis of Control Example 1 and Examples 1 to 4 shows that the acid-base subunits of the natural pea protein treated with cysteine ​​in the present invention gradually dissociate with the increase of cysteine ​​treatment concentration compared with the untreated natural pea protein (see Figure 1 ), solubility increases under certain conditions (see Figure 2 ), the tryptophan group inside the molecule is exposed outward (see Figure 4 ), the disulfide bond conformation changes from ggg type and tgg type to tgt type (please refer to Figure 5 ). This shows that the cysteine ​​treatment used in the present invention can cleave the disulfide bonds in natural pea protein, promote structural unfolding, expose more internal groups, and facilitate the functional properties in different food systems.

[0091] Comparative analysis of Control Example 2 and Examples 5 to 8 shows that the pH-shifted modified pea protein treated with cysteine ​​in the present invention has completely dissociated acidic and basic subunits at lower concentrations of cysteine ​​than the modified pea protein treated with pH shift alone (see Figure 1 ), solubility and surface hydrophobicity are significantly improved (please refer to Figure 2 and Figure 3 ), the tryptophan group inside the molecule is exposed outward (see Figure 4 ), the disulfide bond conformation mainly changes from ggg type to tgt type (please refer to Figure 5 ). This shows that the cysteine ​​treatment used in the present invention can produce a synergistic effect with the pH shift treatment, further opening up the molecular structure of the pea protein and significantly improving the functional properties of the modified pea protein.

[0092] The solubility and hydrophobicity results of the products obtained in the specific examples and comparative examples are shown in Table 1.

[0093] Table 1

[0094]

[0095]

[0096] Results analysis: Combining Control Example 1 (native protein), Example 2 (native protein + cysteine), Control Example 2 (native protein + pH shift), and Example 6 (native protein + pH shift + cysteine), cysteine ​​treatment alone did not show a significant performance improvement. However, simultaneous cysteine ​​modification and pH shift modification treatment showed a significant performance improvement compared to pH shift modification alone. This shows that there is a synergistic effect between the two modification methods.

[0097] Example 9 Composite meat protein gel containing natural pea protein treated with 1.7 mM / g cysteine

[0098] A natural pea protein solution treated with 1.7 mM / g cysteine ​​was mixed with a myofibrillar protein solution in a phosphate buffer solution (0.05 M sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system, pH 6.25) containing 0.3 M NaCl to prepare a mixed protein solution with a meat protein mass fraction of 3.0% and a pea protein mass fraction of 0.75%. An enzyme preparation (130 IU / g) was added to the solution at a concentration of 5% of the total protein mass, and the mixture was uniformly mixed. After incubation at 4°C for 2 hours, a composite protein solution sample was formed. A composite protein gel was prepared according to the composite protein gel preparation method described above.

[0099] The natural pea protein solution treated with 1.7 mM / g cysteine ​​was referred to Example 1.

[0100] Example 10: Composite meat protein gel containing pH-shifted modified pea protein treated with 1.7 mM / g cysteine

[0101] A pH-shifted, modified pea protein solution treated with 1.7 mM / g cysteine ​​was mixed with a myofibrillar protein solution in a phosphate buffer (0.05 M sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system, pH 6.25) containing 0.3 M NaCl to create a mixed protein solution with a myofibrillar protein mass fraction of 3.0% and a pea protein mass fraction of 0.75%. An enzyme preparation (130 IU / g) was added to the solution at a concentration of 5% of the total protein mass, and the mixture was uniformly mixed. The solution was then incubated at 4°C for 2 hours to form a composite protein solution sample. A composite protein gel was then prepared according to the composite protein gel preparation method described above.

[0102] The pH-shifted modified pea protein solution treated with 1.7 mM / g cysteine ​​was prepared as described in Example 5.

[0103] Example 11 Composite meat protein gel containing natural pea protein treated with 3.3 mM / g cysteine

[0104] A 3.3 mM / g cysteine-treated natural pea protein solution and a myofibrillar protein solution were mixed in a phosphate buffer solution (0.05 M sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system, pH 6.25) containing 0.3 M NaCl to prepare a mixed protein solution with a meat protein mass fraction of 3.0% and a pea protein mass fraction of 0.75%. An enzyme preparation (130 IU / g) was added to the solution at a concentration of % by weight of the total protein, mixed evenly, and allowed to stand at 4°C for 2 hours to form a composite protein solution sample. A composite protein gel was prepared according to the composite protein gel preparation method described above.

[0105] The natural pea protein solution treated with 3.3 mM / g cysteine ​​was referred to Example 2.

[0106] Example 12: Composite meat protein gel containing pH-shifted modified pea protein treated with 3.3 mM / g cysteine

[0107] A pH-shifted, modified pea protein solution treated with 3.3 mM / g cysteine ​​was mixed with a myofibrillar protein solution in a phosphate buffer (0.05 M sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system, pH 6.25) containing 0.3 M NaCl to create a mixed protein solution with a myofibrillar protein mass fraction of 3.0% and a pea protein mass fraction of 0.75%. An enzyme preparation (130 IU / g) was added to the solution at a concentration of 5% of the total protein mass, mixed evenly, and incubated at 4°C for 2 hours to form a composite protein solution sample. A composite protein gel was then prepared according to the composite protein gel preparation method described above.

[0108] The pH-shifted modified pea protein solution treated with 3.3 mM / g cysteine ​​was referred to Example 6.

[0109] Comparative Example 3: Composite meat protein gel containing natural pea protein

[0110] A natural pea protein solution and a myofibrillar protein solution were mixed in a phosphate buffer solution (0.05M sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system, pH 6.25) containing 0.3M NaCl to prepare a mixed protein solution with a myofibrillar protein mass fraction of 3.0% and a pea protein mass fraction of 0.75%. An enzyme preparation (130 IU / g) was added to the solution at a concentration of 5% of the total protein mass, and the mixture was uniformly mixed. The solution was incubated at 4°C for 2 hours to form a composite protein solution sample. A composite protein gel was then prepared according to the composite protein gel preparation method described above.

[0111] Natural pea protein refers to Control Example 1.

[0112] Comparative Example 4: Composite meat protein gel containing pH-shifted modified pea protein

[0113] A pH-shifted pea protein solution and a myofibrillar protein solution were mixed in a phosphate buffer solution (0.05M sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system, pH 6.25) containing 0.3M NaCl to prepare a mixed protein solution with a myofibrillar protein mass fraction of 3.0% and a pea protein mass fraction of 0.75%. An enzyme preparation (130 IU / g) was added to the solution at a concentration of 5% of the total protein mass, and the mixture was uniformly mixed. After incubation at 4°C for 2 hours, a composite protein solution sample was formed. A composite protein gel was then prepared according to the composite protein gel preparation method described above.

[0114] The pH shift modified pea protein refers to Control Example 2.

[0115] The gel hardness results of the composite meat protein gels obtained in Examples 9-12 and Comparative Examples 3-4 are shown in Table 2.

[0116] Table 2

[0117]

[0118]

[0119] Comparative analysis of Control Example 3 and Examples 9 and 11 shows that the composite meat protein gel containing natural pea protein treated with cysteine ​​in the present invention has a slightly increased gel strength of more than 15% compared to the composite meat protein gel containing natural pea protein not treated with cysteine ​​(e.g. Figure 6 ), the gel elasticity was not negatively affected (as shown Figure 7 This indicates that the cysteine ​​treatment used in the present invention can enhance the structural strengthening effect of natural pea protein in the meat protein gel.

[0120] Comparative analysis of Control Example 4 and Example 10 shows that the composite meat protein gel containing pH-shifted modified pea protein treated with cysteine ​​in the present invention has a significantly increased gel strength of nearly 19% compared to the composite meat protein gel containing modified pea protein treated only with pH shift (Example 10, as shown in FIG. Figure 6 As shown), the elastic modulus is significantly improved (as shown Figure 7 As shown), the microstructure of the gel is characterized by being smaller and more branched (see Figure 8 ). This shows that the cysteine ​​treatment used in the present invention can further enhance the structural strengthening effect of the modified pea protein in the meat protein gel.

[0121] Effects and functions of the embodiment:

[0122] The disulfide-linked AB subunits in the 11S protein partially dissociate to form acidic subunits (A) and basic subunits (B), which then reassemble through disulfide bonds into soluble macromolecular aggregates. Pea protein with these structural characteristics acts as a highly effective emulsifier and structural filler in various meat products, successfully binding water and fat / oil to create a firm texture. These functional properties are particularly important for improving the textural defects of meat protein gels and meat products.

[0123] Disulfide bonds are usually formed by coupling two sulfhydryl groups, providing important intermolecular forces during the formation of meat protein gels, and are widely present in natural plant protein structures. Many plant proteins (such as pea protein, soy protein, oat protein, etc.) contain multiple disulfide bonds, which are responsible for maintaining the spatial conformation of proteins. Taking pea protein as an example, these disulfide bonds are widely present in 11S protein, and they still exist even after pH shift treatment. Therefore, the disulfide bonds in the undissociated AB subunits and the formed soluble aggregates may hinder the interaction between pea protein and myosin, limiting the formation of the gel network.

[0124] Many chemicals (such as β-mercaptoethanol, dithiothreitol, sodium sulfite, and cysteine) have the ability to trigger sulfhydryl and disulfide bond exchange reactions. Among them, cysteine, despite its relatively low redox capacity, is the most suitable for modifying dietary proteins due to safety considerations.

[0125] Unlike the pH shift and thermosound methods mentioned in the patent "A Modified Pea Protein and a Method for Preparing a Low-Phosphate Composite Protein Gel Therefrom," this invention's cysteine-induced disulfide bond cleavage of protein molecules represents a pioneering plant protein modification technology. This technology differs from the previously applied patent in both its modification principle and expected effects. Disulfide bond cleavage technology has proven to be highly effective, whether used alone or in conjunction with other modification methods.

Claims

1. A method for processing a low-salt composite meat protein gel, characterized in that: The steps include: (1) fully dissolving the pH-shifted modified pea protein to obtain a protein solution; (2) adding a disulfide bond cleaving agent to the protein solution, dissolving and mixing the solution to obtain a modified pea protein; the disulfide bond cleaving agent is cysteine; the amount of the disulfide bond cleaving agent added is 1 to 8 mM / g protein based on the protein mass; the mixing reaction is carried out at room temperature for 10 minutes; (3) dissolving the myofibrillar protein and the modified pea protein obtained after the reaction in step (2) in phosphate buffer to prepare a mixed protein solution; (4) adding a cross-linking enzyme to the mixed protein solution; the cross-linking enzyme is transglutaminase; (5) heating the mixed protein solution to form a composite meat protein gel; The preparation process of the pH-shifted modified pea protein is specifically as follows: dispersing the pea protein in water, adjusting the pH of the protein solution to 11-13 with an alkaline solution at room temperature; after leaving it for 30-90 minutes, adding acid for neutralization, solid-liquid separation, and collecting the precipitate to obtain the pH-shifted modified pea protein.

2. The method according to claim 1, characterized in that The added amount of the disulfide bond cleaving agent is 1-7 mM / g protein.

3. The method according to claim 1, characterized in that The added amount of the disulfide bond cleaving agent is 1-5 mM / g protein.

4. The method according to claim 1, wherein The modified pea protein after the reaction is added to the myofibrillar protein at a mass ratio of 5 to 50%.

5. The method according to claim 1, wherein The added amount of the cross-linking enzyme is 1-8% of the total mass of the protein.

6. The method according to claim 1, characterized in that In the mixed protein solution, the mass fraction of myofibrillar protein was 3.0%; the mass fraction of modified pea protein after the reaction was 0.75%.

7. The method according to claim 1, characterized in that The amount of cross-linking enzyme added was 6.5 IU / g protein.

8. The method according to claim 1, characterized in that The enzymatic activity of the cross-linking enzyme is 130 IU / g.

9. Low-salt composite meat protein gel prepared by the processing method according to any one of claims 1 to 8.

10. Use of the low-salt composite meat protein gel according to claim 9 in preparing food.

11. Use of the low-salt composite meat protein gel according to claim 9 in the preparation of functional foods.

12. Use of the low-salt composite meat protein gel according to claim 9 in the preparation of health products.

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