Pea protein isolate composite emulsion, preparation method thereof and application of pea protein isolate composite emulsion in emulsified meat balls

Abstract

The invention belongs to the technical field of processing and production of emulsified meat products, and particularly relates to pea protein isolate composite emulsion, a preparation method thereof and application of the pea protein isolate composite emulsion in emulsified meat balls. The pea protein isolate is modified by adopting arginine modification and quercetin grafting technologies to prepare a high-performance plant protein-based emulsifier, and the high-performance plant protein-based emulsifier is mixed with myofibrillar protein to prepare the composite emulsion. The emulsion partially replaces animal fat in traditional emulsified meat balls, the content of saturated fatty acid in the product is remarkably reduced, meanwhile, generation of grease oxidation products is effectively inhibited, and the oxidation stability of the product in the storage period is prolonged. Besides, the emulsified meat balls added with the composite emulsion are excellent in taste, juiciness, flavor and the like, have better texture characteristics and oxidation resistance, meet the requirements of modern consumers for low-fat healthy diet and have high market application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of emulsified meat product processing and production, and provides a preparation method of a modified pea protein isolate, particularly relating to the synthesis of a pea protein isolate-arginine-quercetin covalent complex and the application of the prepared modified pea protein isolate-myofibrillar protein emulsion in an emulsified pork meatball system. Background Art

[0002] Traditional meat products typically contain between 20% and 30% fat. However, excessive fat intake can negatively impact human health, with the incidence of related chronic diseases such as obesity, hypertension, hyperlipidemia, and certain types of cancer increasing. As people's focus on healthy eating continues to grow, they are seeking healthier alternatives to reduce their intake of animal fat, thereby mitigating health risks such as cardiovascular disease. Consumers are increasingly concerned about the nutritional content and sources of food, and are showing a growing interest in low-fat options. Low-fat ground meat products and imitation meat products represent development trends and innovation directions in the meat products industry.

[0003] However, vegetable oils are high in unsaturated fatty acids, which can easily lead to oxidative rancidity during processing and storage. Oxidative rancidity in emulsion-based meat emulsions can directly impact product quality, severely impacting shelf life and consumer adoption. Therefore, a processing method is needed to address the high fat content and susceptibility to oxidation of emulsified pork meatballs while maintaining product quality. Summary of the Invention

[0004] In order to reduce the saturated fatty acid content in minced meat products, the present invention considers using vegetable oil to prepare a pre-emulsion to partially replace animal fat to prepare improved emulsified pork meatballs. After the animal fat in the product is replaced by the composite pre-emulsion, the saturated fatty acid content is significantly reduced, which meets the needs of modern consumers for a low-fat and healthy diet. The present invention uses arginine modification and quercetin grafting technology to modify pea protein isolate to prepare a high-performance plant protein-based emulsifier. The arginine-modified pea protein isolate-quercetin-myofibrillar protein composite emulsion is applied to emulsified minced meat products, which can effectively exert the antioxidant properties of quercetin to inhibit the formation of oil oxidation products, maintain the oxidative stability of the product during storage, extend the shelf life of the product, and reduce the economic losses of enterprises caused by rapid product deterioration.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for preparing a pea protein isolate composite emulsion comprises the following steps:

[0007] The myofibrillar protein dilution and the modified pea protein isolate dilution are mixed and stirred, and vegetable oil is added and homogenized to obtain a pea protein isolate composite emulsion;

[0008] The preparation method of the modified pea protein isolate comprises: adding arginine to a pea protein isolate solution, magnetically stirring, fully hydrating the solution, adding a quercetin solution, fully mixing the solution, adding laccase, reacting the solution at room temperature, heating to inactivate the enzyme after the reaction, cooling the solution, and dialyzing the solution to obtain a pea protein isolate-arginine-quercetin covalent complex; and freeze-drying the solution to obtain the modified pea protein isolate.

[0009] Furthermore, the concentration of the myofibrillar protein diluent is 1.5-2.5wt%, the concentration of the modified pea protein isolate diluent is 0.8-1.2wt%, and the mass ratio of the myofibrillar protein diluent to the modified pea protein isolate diluent is 1:1; the vegetable oil is soybean oil, and the volume percentage of the vegetable oil in the pea protein isolate composite emulsion is 15-25%.

[0010] Furthermore, the concentration of the pea protein isolate is 25-35 mg / mL, the mass concentration ratio of the pea protein isolate to quercetin is 25-35:1, and the concentration of the arginine is 0.1-0.3%.

[0011] Furthermore, the final concentration of the laccase in the mixed solution is 1.0 U / mL, and the reaction time is 4-5 h.

[0012] Furthermore, the enzyme inactivation method includes: placing the reaction solution in an 80-85°C water bath and heating it for 2-3 minutes to inactivate the laccase; the dialysis method includes: placing the enzyme-inactivated and cooled reaction solution into a dialysis bag, and dialyzing it at 4-6°C using deionized water for 48-60 hours, with a molecular weight cutoff of 12-14 kDa, and replacing the deionized water every 6-8 hours.

[0013] Furthermore, the preparation method of the myofibrillar protein includes: removing fat and connective tissue from fresh pork tenderloin and cutting it into small pieces to obtain chopped pork tenderloin; mixing the chopped pork tenderloin with phosphate buffer, homogenizing, filtering, and centrifuging to obtain a precipitate; adding NaCl solution to the precipitate, homogenizing again, filtering, and centrifuging to obtain a precipitate; and finally adjusting the pH to 6-6.2, centrifuging to obtain a precipitate to obtain the myofibrillar protein.

[0014] The present invention also provides a pea protein isolate composite emulsion, which is prepared by the above-mentioned preparation method.

[0015] The present invention also provides application of the pea protein isolate composite emulsion in preparing emulsified meatballs.

[0016] Furthermore, the preparation method of the emulsified meatballs comprises the following steps: mincing the longissimus dorsi muscle of the pig, adding salt and ice water, stirring thoroughly, adding minced pork back fat, the above-mentioned pea protein isolate composite emulsion and ice water, continuing to stir, adding ice water again and stirring to obtain raw meat paste, kneading the raw meat paste into meatballs, cooking and cooling to obtain emulsified meatballs.

[0017] The present invention also provides the use of the pea protein isolate composite emulsion in improving the antioxidant property and taste of emulsified meatballs.

[0018] Beneficial effects

[0019] Emulsified pork meatballs were prepared by replacing part of the animal fat with the modified pea protein isolate-myofibrillar protein composite emulsion. These emulsified pork meatballs meet healthy, low-fat needs, exhibit good antioxidant properties, and excel in taste, juiciness, and flavor, demonstrating that the improved product formula is gaining wider consumer favor.

[0020] Experiments have also shown that the emulsified pork meatballs prepared by the method of the present invention effectively inhibit the formation of oil oxidation products, and the emulsified pork meatballs added with modified pea protein isolate composite pre-emulsion can maintain long-term good oxidative stability during low-temperature storage, and are favored by more consumers.

[0021] Emulsified pork meatballs prepared with a quercetin-containing vegetable oil pre-emulsion exhibited significantly lower TBARS values ​​during storage than the animal oil control group and other treatments without quercetin. Adding a composite emulsion prepared with a 0.2% Arg-modified pea protein isolate-quercetin covalent complex to the minced meat product significantly improved the antioxidant properties of the emulsified pork meatballs.

[0022] When animal fat was partially replaced with a vegetable oil pre-emulsion, sensory evaluation significantly improved. When animal fat was partially replaced with a composite emulsion prepared from modified pea protein isolate, the emulsified pork meatballs scored well in terms of texture, flavor, and juiciness, demonstrating high consumer acceptance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart for preparing the pea protein isolate-arginine-quercetin covalent complex of the present invention;

[0024] Figure 2 is the effect of arginine addition on the pH of PPI solution;

[0025] Figure 3 ABTS free radical scavenging ability (A) and DPPH free radical scavenging rate (B) of natural and modified pea protein isolate;

[0026] Figure 4The natural pea protein isolate and modified pea protein isolate were detected by infrared wavelength (1800-600cm -1 ) chemical structure changes within;

[0027] Figure 5 Free sulfhydryl content of composite emulsions prepared from natural and modified pea protein isolates;

[0028] Figure 6 Carbonyl content of composite emulsions prepared for natural and modified pea protein isolates;

[0029] Figure 7 POV values ​​of composite emulsions prepared for natural and modified pea protein isolates;

[0030] Figure 8 Conjugated diene values ​​of composite emulsions prepared for natural and modified pea protein isolates;

[0031] Figure 9 Fluorescence intensity of Schiff bases of composite emulsions prepared from natural and modified pea protein isolates;

[0032] Figure 10 Determination of cooking loss of emulsified meatballs prepared from vegetable oil pre-emulsion;

[0033] Figure 11 Changes in TBARS values ​​of emulsified meatballs prepared from vegetable oil pre-emulsion during storage;

[0034] Figure 12 Sensory evaluation of emulsified meatballs prepared with vegetable oil pre-emulsion. DETAILED DESCRIPTION

[0035] The present invention will be described in further detail below with reference to specific embodiments and the accompanying drawings. Unless otherwise specified, the experimental methods described in the present invention are all conventional methods; and the biological materials described can all be obtained from commercial channels.

[0036] Example 1

[0037] Preparation of pea protein isolate modified by quercetin grafted with arginine catalyzed by laccase

[0038] (1) Laccase catalysis: 100 mL of pea protein isolate (80% purity, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) solution was prepared with deionized water to a protein concentration of 30 mg / mL. 0.1%, 0.2%, and 0.3% arginine (Arg) were added. Magnetic stirring was performed for at least 3 h and the protein was kept in a refrigerator at 4°C overnight to ensure that the protein was fully hydrated. Separately, a 1 mg / mL quercetin solution was prepared with deionized water and mixed with the protein solution under continuous stirring to a final concentration ratio of pea protein isolate to quercetin of 30:1. Laccase (1.0 U / mL) was then added to the mixture and reacted at room temperature for 4 h.

[0039] (2) Termination of the reaction: When the reaction is complete, the mixture is heated in a water bath at 85°C for 2 min to inactivate the laccase, and then cooled in an ice-water bath.

[0040] (3) Dialysis: The mixed solution was placed in a dialysis bag and dialyzed with deionized water at 4°C for 48 h with a molecular weight cutoff of 12-14 kDa. The deionized water was replaced every 8 h to remove free quercetin to obtain a pea protein isolate-arginine-quercetin covalent complex. The dialyzed solution was freeze-dried for 72 h to obtain a pea protein isolate-arginine-quercetin complex powder (modified pea protein isolate).

[0041] Natural PPI was treated under the same conditions and served as a control. Arginine-modified and quercetin-grafted composites with different concentrations were obtained and labeled as PA1 (0.1% Arg-modified PPI), PA2 (0.2% Arg-modified PPI), PA3 (0.3% Arg-modified PPI), PA0-Q (quercetin-grafted natural PPI), PA1-Q (quercetin-grafted 0.1% Arg-modified PPI), PA2-Q (quercetin-grafted 0.2% Arg-modified PPI), and PA3-Q (quercetin-grafted 0.3% Arg-modified PPI).

[0042] Experimental methods

[0043] 1. Determination of phenolic acid grafting rate of modified pea protein isolate

[0044] A UV-visible spectrophotometer was used to measure the absorbance at 760 nm of various pyrogallol standard solutions (0-0.2 mg / mL) and plot a standard curve. The sample solution (1 mg / mL) was mixed with Folin-phenol reagent (0.25 mol / L). After 6 minutes of reaction, 1 mL of 15% (w / v) sodium carbonate solution was added and the mixture was allowed to stand at room temperature in the dark for 1 hour. The absorbance at 760 nm was recorded. The polyphenol grafting equivalent was expressed as mg pyrogallol equivalent / g dry matter. The polyphenol grafting rate (%) was calculated as follows:

[0045]

[0046] Wherein, Qdialysis refers to the concentration of polyphenols in the complex solution after dialysis, and Qinitial refers to the concentration of polyphenols before the reaction.

[0047] 2. Determination of antioxidant activity of pea protein isolate before and after modification

[0048] Prepare a 0.2 mM DPPH solution using anhydrous ethanol. Then, add 2 mL of the 1 mg / mL sample solution to 2 mL of the DPPH solution (A S The mixed solution was reacted in the dark at room temperature for 1 h. The absorbance was measured at a wavelength of 517 nm using a spectrophotometer, and the DPPH free radical scavenging rate was calculated using the following formula:

[0049]

[0050] Where A s is the absorbance value of a mixed solution consisting of 2 mL of solution and 2 mL of DPPH, A c is the absorbance value of DPPH solution.

[0051] ABTS free radical scavenging assay: ABTS stock solution (7.4 mM) and potassium persulfate solution (2.6 mM) were mixed (1:1, v / v), stirred in the dark for 12 h (overnight), and then appropriately diluted 40-50 times with PBS (0.2 mol / L, pH = 7.4) to make the solution in A 734 nm =0.7±0.02 to obtain ABTS working solution. Mix 0.2 mL of sample solution (1 mg / mL) with 1 mL of ABTS working solution. Let the mixture stand for 10 minutes and measure the absorbance at 734 nm. Use the following formula:

[0052]

[0053] Where A 对照 Indicates the original absorbance of ABTS working solution, A 样品 Indicates the sample absorbance.

[0054] 3. Fourier transform infrared spectroscopy

[0055] To accurately characterize the chemical structure of the complex, Fourier transform infrared (FTIR) spectra of the complex were measured using a Cary 610 / 670 infrared microscope. The specific operation process is as follows: First, the obtained complex solution was freeze-dried and then ground into a powder. Next, 5 mg of the complex powder was accurately weighed and placed in a mortar with 495 mg of dry potassium bromide. The mixture was thoroughly ground until uniform. The mixed powder was then pressed into uniform, transparent thin sheets using a tablet press. During the spectrometer scanning process, the following parameters were set: spectral range 500–4000, number of scans 32, and resolution set to 0.1. In addition, a blank group was set up, with potassium bromide as a blank control; unmodified PPI was also used as a control group to ensure the accuracy and reliability of the experimental results.

[0056] Experimental results

[0057] 1. Phenolic acid grafting rate of modified pea protein isolate

[0058] Pea protein isolate (PPI) was modified with three concentrations of arginine (0.1%, 0.2%, and 0.3%). The arginine-modified pea protein isolate was then covalently bound to quercetin (Q) to investigate the effect of arginine addition on the degree of covalent binding between pea protein isolate and quercetin. The total phenolic content of the modified pea protein isolate-quercetin complex (PPI-Q) was determined using the Folin-phenol method, and the binding rate of polyphenols to pea protein isolate was calculated as a percentage.

[0059] like Figure 2 As shown, changes in arginine concentration significantly affected the pH of the PPI solution (p < 0.05). The pH of the control (native pea protein isolate) was 6.73 ± 0.32. Increasing arginine concentration gradually increased the pH of the PPI solution, indicating that arginine has a concentration-dependent effect on the pH of the PPI solution. Table 1 shows that PA0-Q (quercetin-grafted native pea protein isolate) has a total phenolic content of 11.49 ± 0.34 mg pyrogallol equivalents / g dry matter and a polyphenol binding rate of approximately 32.83%. The covalent complexes formed between pea protein isolate and quercetin modified with 0.1% to 0.3% arginine significantly increased both the total phenolic content and the polyphenol binding rate (p < 0.05). The highest polyphenol binding rate was achieved at an arginine concentration of 0.2%. Although the polyphenol binding rate decreased slightly at a 0.3% Arg concentration, the total phenol content was still higher than the polyphenol binding rate of natural pea protein isolate and quercetin. This result shows that moderate arginine modification can significantly increase the covalent binding rate of quercetin and pea protein isolate.

[0060] During the modification process of pea protein isolate, increasing the concentration of arginine (Arg) increases the number of basic groups in the system, leading to an increase in the pH value. This change may affect the structure and properties of the protein, thereby affecting subsequent reactions such as phenolic acid grafting. Under alkaline conditions, polyphenols are easily oxidized to form quinone derivatives. These quinones are highly reactive and can react with nucleophilic groups such as free amino groups, lysine, cysteine, and tryptophan in proteins, forming stable covalently bound products. This may also contribute to the high polyphenol grafting efficiency of arginine-modified pea protein isolate. The introduction of arginine (Arg) enhances interactions between protein and phenolic acids, such as electrostatic interactions and hydrogen bonding, further promoting the grafting efficiency of polyphenols. However, when the Arg modification concentration reaches 0.3%, the polyphenol binding rate and total phenol content show a downward trend. This may be because excessive Arg concentrations excessively alter the protein structure, resulting in the masking of some active sites or protein aggregation, which is not conducive to the quercetin grafting reaction. Alternatively, excessive Arg may compete with quercetin for reactive sites, thereby inhibiting the quercetin grafting efficiency. Further investigations are underway to explore the potential chemical structure modification of PPIs caused by the covalent grafting of polyphenol oxidation products onto PPIs during laccase catalysis.

[0061] Table 1 Determination of total phenol content and polyphenol binding rate of natural and modified pea protein isolates

[0062]

[0063] 2. Analysis of the antioxidant activity of covalent complexes

[0064] The effects of different modification treatments on the antioxidant properties of pea protein isolate were investigated by measuring the ABTS free radical scavenging rate and DPPH free radical scavenging rate. Figure 3As shown, in terms of ABTS free radical scavenging ability, native pea protein isolate (i.e., the control group) had the lowest scavenging rate, at only 26.48%. However, the scavenging rate of PPI modified with 0.2% arginine (PA2 group) was significantly increased, reaching 51.23%, indicating that the addition of arginine can indeed enhance the ABTS free radical scavenging rate of PPI. Further observation revealed that the ABTS free radical scavenging rates of the pea protein isolate groups modified with quercetin grafted with arginine (PA0-Q to PA3-Q) were all higher than those of the arginine-modified groups. The PA2-Q group achieved the highest ABTS free radical scavenging rate of 82.58%, the highest among all groups. This result demonstrates a synergistic effect between quercetin grafting and arginine modification, effectively enhancing the ABTS free radical scavenging ability of PPI. In the DPPH radical scavenging rate assay, the control group had a lower scavenging rate. Compared with the control group, the DPPH radical scavenging rates of the covalent complexes formed by quercetin and pea protein isolate (PA0-Q to PA3-Q) increased by 24.45%, 24.75%, 24.88%, and 24.60%, respectively. This fully demonstrates that quercetin grafting significantly improves the DPPH radical scavenging performance of PPI.

[0065] 3. Fourier transform infrared spectroscopy analysis of the complex

[0066] FTIR spectra of natural and modified pea protein isolates Figure 4 Infrared wavelength (1800-600 cm -1 ) range, the chemical structure changes between natural pea protein isolate and modified pea protein isolate can be observed. In the amide I band region (1600-1700 cm -1 , which represents the stretching vibration of COO-). The absorption peaks of both Arg-modified PPI and PPI grafted with quercetin have undergone red shifts. This phenomenon indicates that the secondary structure of the protein has changed after the natural PPI is modified with arginine and covalently grafted with quercetin. In the amide II band region (1500-1600 cm -1 , this region represents NH bending and CN stretching). The absorption peak of Arg-modified PPI showed a blue shift. In addition, in the covalent complex of quercetin and pea protein isolate, whether it is quercetin-grafted natural PPI (PA0-Q) or quercetin-grafted PPI modified with different concentrations of arginine (PA1-Q, PA2-Q, PA3-Q), the absorption peak of the amide II band showed a blue shift. The red shift of the amide I band and the blue shift of the amide II band are due to the interaction between quercetin and the C=O and NH groups of PPI, which leads to structural changes in the protein. Located at 1170.89 cm -1The absorption bands near 900 cm-1-800 cm-1 belong to the weak stretching of carboxyl and CO. Finally, PA0-Q, PA1-Q, PA2-Q and PA3-Q have the following absorption bands: -1 The absorption peak at is , which is a manifestation of the para-substituted aromatic ring of the polyphenol oxidation product during the catalytic reaction. This further confirms that quercetin and pea protein isolate have achieved covalent grafting. After the pea protein isolate and quercetin are covalently bound, the polyphenols interact with the relevant groups of the pea protein isolate, causing the protein structure to change. At the same time, the modified pea protein isolate shows a new absorption peak in the infrared spectrum. The above results confirm that the modified pea protein isolate has successfully covalently bound to quercetin and that the chemical structure has changed significantly.

[0067] Example 2

[0068] Preparation of composite emulsions of natural and modified pea protein isolates and myofibrillar protein

[0069] (1) Extraction of myofibrillar protein: Take fresh pork tenderloin, remove fat and connective tissue, and cut into small pieces to obtain chopped pork tenderloin; mix the chopped pork tenderloin with phosphate buffer, homogenize, filter, and centrifuge to obtain a precipitate; add 0.1 mol / L NaCl solution to the obtained precipitate, homogenize again, filter, and centrifuge to obtain a precipitate; finally, adjust the pH to 6-6.2, and centrifuge to obtain a precipitate, which is the desired myofibrillar protein.

[0070] (2) Preparation of pea protein isolate-myofibrillar protein composite emulsion: Myofibrillar protein was diluted with PIPES buffer to prepare a 2 wt% myofibrillar protein dilution and a 1 wt% modified pea protein isolate dilution respectively; then the mixture was stirred in a ratio of 1:1 to prepare a composite emulsion of 1% myofibrillar protein + 0.5% pea protein isolate; the composite solution of 1% myofibrillar protein + 0.5% pea protein isolate was mixed with 20% soybean oil; the myofibrillar protein-modified pea protein isolate composite emulsion containing 20% ​​soybean oil was homogenized at 12000 r / min for 1 min to obtain a composite emulsion of 1% myofibrillar protein + 0.5% pea protein isolate. The prepared composite emulsion should be stored in a refrigerator at 4°C or on crushed ice.

[0071] The experimental groups were myofibrillar protein-natural pea protein isolate composite emulsion (Control), myofibrillar protein-0.1% arginine-modified pea protein isolate composite emulsion (MP+PA1), myofibrillar protein-0.2% arginine-modified pea protein isolate composite emulsion (MP+PA2), myofibrillar protein-0.3% arginine-modified pea protein isolate composite emulsion (MP+PA3), myofibrillar protein-quercetin composite emulsion (MP+PA4), and myofibrillar protein-natural pea protein isolate composite emulsion (Control), myofibrillar protein-0.1% arginine-modified pea protein isolate composite emulsion (MP+PA1), myofibrillar protein-0.2% arginine-modified pea protein isolate composite emulsion (MP+PA2), myofibrillar protein-0.3% arginine-modified pea protein isolate composite emulsion (MP+PA3), and myofibrillar protein-quercetin composite emulsion (MP+PA4). Grafted natural pea protein isolate composite emulsion (MP+PA0-Q), myofibrillar protein-quercetin grafted 0.1% arginine-modified pea protein isolate composite emulsion (MP+PA1-Q), myofibrillar protein-quercetin grafted 0.2% arginine-modified pea protein isolate composite emulsion (MP+PA2-Q), myofibrillar protein-quercetin grafted 0.3% arginine-modified pea protein isolate composite emulsion (MP+PA3-Q).

[0072] Experimental methods

[0073] 1. Determination of emulsification properties of composite emulsion

[0074] First, extract 20 μL of sample from the bottom of the freshly prepared emulsion and transfer it to a centrifuge tube containing 5 mL of 0.1% (w / v) SDS solution for dilution. Next, vortex the sample for 2 minutes, then add 200 μL of the mixture to a microplate and remove any bubbles. Measure the absorbance at 500 nm using a microplate reader, while using a buffer solution containing SDS as a control. Emulsification activity is calculated using the following formula:

[0075]

[0076] where A0 is the absorbance at 500 nm, V is the dilution factor (251), Φ is the volume fraction of oil in the emulsion, and C is the protein concentration in the emulsion (mg / mL).

[0077] Emulsion stability: After the emulsion has been allowed to stand on crushed ice for 10 minutes, follow the above steps to aspirate the sample from the bottom and add it to SDS. The absorbance at 500 nm is read to determine the emulsion stability (ESI). The calculation formula is as follows:

[0078]

[0079] Where A t is the absorbance value read after standing for 10 min, and A0 is the initial absorbance value mentioned above.

[0080] 2. Changes in the chemical structure of composite emulsion protein

[0081] 2.1 Determination of free sulfhydryl content in myofibrillar protein

[0082] Thiol content was determined using the Ellman method. The composite emulsion was diluted 20-fold with phosphate buffer. 1 mL of the dilution was mixed with 8 mL of Tris-Gly buffer (0.086 mol / L Tris, 0.09 mol / L Gly, 0.00443 mol / L EDTA) and centrifuged at 8000 × g for 15 minutes. After centrifugation, 4.5 mL of the supernatant was mixed with 50 μL of Ellman's reagent (4 mg / mL DTNB) and allowed to stand for 15 minutes. After the reaction, the supernatant was centrifuged at 12,000 × g for 10 minutes, and the absorbance of the supernatant was measured at λ = 412 nm. The thiol content was calculated according to the following formula:

[0083]

[0084] Where A 412 is the absorbance of the sample emulsion at λ = 412 nm, D is the dilution factor, and C is the sample protein concentration (mg / mL).

[0085] 2.2 Determination of myofibrillar protein carbonyl content

[0086] To 0.1 mL of the protein solution (1 mg / mL), add 0.5 mL of 10 mM DPPH (dissolved in 2 mol / L HCl) and mix. After the mixture reacts for 1 hour, add 1 mL of 20% TCA to the reaction sample and mix. Collect the precipitate by centrifugation (12,000 × g, 4°C, 15 min) and wash three times with 1 mL of ethanol / ethyl acetate (1:1, v / v) (11,000 × g, 10°C, 3 min, and then collect the precipitate). Dissolve the resulting precipitate in 6 M guanidine hydrochloride. Incubate in a 37°C water bath for 15 min. Record the absorbance at 370 nm. The formula is as follows:

[0087]

[0088] Where A is the absorbance of the sample emulsion at λ=370 nm, C is the sample protein concentration (1 mg / mL), 10 6 is the unit conversion factor, is the molar absorption coefficient (22400 mol -1 cm -1 ).

[0089] 3. Oxidation process of composite emulsion oil

[0090] 3.1 Determination of POV value of myofibrillar protein composite emulsion oil

[0091] Take 1 mL of the sample emulsion and add 5 mL of a 3:1 isooctane-isopropanol (volume ratio) mixture. Mix thoroughly and centrifuge at 3000 × g for 2 min. Aspirate 2 mL of the supernatant and add 20 μL of potassium thiocyanate. Then, adjust the volume to 5 mL with a methanol-n-butanol mixture and vortex for 10 s. Incubate in the dark for 25 min and measure the absorbance at 510 nm, which is recorded as A. 脂肪空白 Take 2 mL of distilled water instead of the sample and measure the absorbance according to the above steps, which is recorded as A 试剂空白 Take another 2 mL of supernatant and add 20 μL of potassium thiocyanate and ferrous chloride solution into a test tube, then dilute to 5 mL with methanol-n-butanol mixed solvent. Measure the absorbance under the same conditions and record it as A 样品 To quantify the peroxide content in the emulsion, the formula is as follows:

[0092]

[0093] Where A = A sample minus (A fat blank plus A reagent blank), K is Fe 2+ The slope of the standard curve is (1.5328), n is the volume fraction of the supernatant liquid, and m is the mass of oil in the sample (g).

[0094] 3.2 Determination of conjugated diene hydroperoxides in oils and fats

[0095] An emulsion sample (20 μL) was added to 10 mL of a mixture of isooctane and isopropanol (volume ratio 2:1) and vortexed for 1 min. The absorbance was measured at 234 nm. For protein-based emulsions, the sample was filtered through a Macherey-Nagel filter (25 mm, 0.2 μm pore size) before measurement to remove protein and reduce spectral interference in this wavelength region. The conjugated diene content in the oxidized emulsion was calculated by monitoring the absorbance at 234 nm:

[0096]

[0097] Where A represents the absorbance of the sample at 234 nm, V represents the volume of isooctane / isopropanol (mL), and ε represents the molar absorption coefficient (27000 M). -1 cm -1 , v represents the volume of the emulsion (mL)

[0098] 4. Determination of Schiff bases of oil and protein co-oxidation products

[0099] The fluorescence intensity of the Schiff base was measured using a fluorescence spectrophotometer. The emulsion was diluted 50-fold with phosphate buffer (10 mM, pH 7.0). The fluorescence intensity of each diluted emulsion was measured at 455 nm using a microplate reader. The excitation wavelength was set to 372 nm, and the fluorescence intensity was recorded within the scanning range of 420-600 nm.

[0100] Experimental results

[0101] 1. Emulsification properties of composite emulsion

[0102] Table 2 Emulsifying activity and emulsifying stability of composite emulsions prepared from natural and modified pea protein isolates

[0103]

[0104] As can be seen from Table 2, the natural pea protein isolate-myofibrillar protein composite emulsion (Control group) performed poorly in terms of emulsification activity and stability, with an emulsification activity of 20.62±0.30 m 2 / g, and the emulsification stability was 78.54±0.04%. This is because the hydrophilicity of natural pea protein isolate is low, making it difficult to form a stable adsorption layer at the oil-water interface, resulting in poor emulsification performance. The emulsification activity and stability of the pea protein isolate composite emulsion modified with arginine (MP+PA1, MP+PA2, MP+PA3 groups) were significantly improved (p<0.05). The emulsification activity was 31.84±0.5 m 2 / g, 32.90±1.10 m 2 / g and 32.03±0.60 m 2 / g, and the emulsification stability was 88.04±0.02%, 92.12±0.04% and 89.29±0.01%, respectively. The addition of arginine increased the hydrophilicity of pea protein isolate, thereby promoting its adsorption at the interface, reducing the aggregation of oil droplets, and improving the stability of the composite emulsion. The guanidine group (-NH-C(NH)-NH2) of arginine can increase the positive charge density on the protein surface and inhibit droplet aggregation through electrostatic repulsion. Basic amino acids improve the stability of the emulsion by enhancing the interfacial adsorption capacity of protein. The emulsification performance of the composite emulsion groups containing pea protein isolate-quercetin covalent complex (MP+PA1-Q, MP+PA2-Q, MP+PA3-Q groups) was further improved. The emulsification activity was 33.62±0.46 m 2 / g, 34.64±0.73 m 2 / g and 33.24±0.41 m 2 / g, and the emulsion stability was 95.65±0.04%, 98.18±0.01%, and 94.59±0.01%, respectively. This may be because the covalent modification of PPI with quercetin promotes the formation of a dense network structure around the lipid droplets, providing a strong physical barrier for the oil droplets in the emulsion and effectively slowing down the aggregation of oil droplets. The results indicate that the introduction of quercetin has a significant synergistic effect on the emulsification properties of PPI. In contrast, although PPI modified with only arginine (e.g., MP+PA2) also significantly improved the emulsification properties of the composite emulsion, the effect was not as significant as that of the quercetin-grafted group.

[0105] 2. Changes in the protein chemical structure of the composite emulsion

[0106] 2.1. Myofibrillar protein free thiol content

[0107] The sulfhydryl group (-SH) in cysteine ​​residues is a highly reactive group in proteins and easily reacts with reactive oxygen species (ROS, such as superoxide anion, hydrogen peroxide, hydroxyl radical, etc.). Therefore, the oxidation state of cysteine ​​was evaluated by free sulfhydryl determination. Figure 5 In the initial state (day 0), it was observed that the free thiol content of myofibrillar protein composite emulsions prepared with arginine-modified and quercetin-grafted PPIs increased. This is because arginine modification and quercetin grafting provide antioxidant protection for protein thiol groups. During protein oxidation, thiol groups serve as active sites susceptible to free radical attack and are typically oxidized to disulfide bonds, sulfinic acids, or sulfonic acids, resulting in a decrease in their content. In this study, the increased thiol content of the composite emulsions prepared with modified PPIs was attributed to the antioxidant activity of quercetin, which inhibited the oxidative chain reaction by scavenging reactive oxygen species (ROS) or competing with thiol groups for free radicals, thereby reducing the oxidative decomposition of thiol groups. Furthermore, arginine itself has free radical scavenging ability, and its guanidine group can neutralize hydroxyl radicals through electron transfer, thereby synergistically enhancing the antioxidant stability of the emulsion. After 7 days of storage, the free thiol content of all treatments decreased, but the decrease in the free thiol content of the composite emulsions in the arginine-modified and grafted groups was less than that in the unmodified group. This further illustrates that the composite emulsion prepared by arginine-modified PPI covalently bound to quercetin can effectively slow down the oxidation process of thiol groups, thereby maintaining the high antioxidant capacity of the emulsion.

[0108] 2.2 Determination of carbonyl content in composite emulsion

[0109] During protein oxidation, the formation of carbonyl compounds (such as aldehydes and ketones) is one of the important signs. These carbonyl groups mainly come from the direct oxidation of amino acid side chains such as lysine, threonine, arginine, proline and hydroxyl radicals. As the degree of oxidation increases, the carbonyl content increases significantly, which has become an important indicator for measuring protein oxidative damage. Figure 6In the study, the natural pea protein isolate-myofibrillar protein composite emulsion (control group) exhibited a high carbonyl content at the initial storage stage (day 0), indicating that protein oxidation had already reached a significant level. After 7 days of storage, the carbonyl content further increased significantly, indicating that protein oxidation in the composite emulsion had deepened. In contrast, myofibrillar protein composite emulsions prepared with arginine-modified PPI and PPI covalently bound to quercetin exhibited lower carbonyl contents at the initial storage stage (day 0) than the composite emulsions supplemented with natural pea protein isolate. This result indicates that the addition of modified pea protein isolate significantly inhibited protein oxidation at the initial stage of composite emulsion preparation. Although the carbonyl content also increased after 7 days of storage, the increase was significantly less than that in the unmodified group, further confirming the effectiveness of the modified PPI in retarding protein oxidation in the emulsions. This inhibitory effect on protein oxidation can be attributed to the hydroxyl and superoxide radical scavenging ability of phenolic compounds. The relative reduction in carbonyl content verified the positive effects of arginine-modified and quercetin-grafted PPIs on inhibiting protein oxidation in emulsions.

[0110] 3. Oil oxidation process of composite emulsion

[0111] 3.1 Determination of POV value of composite emulsion oil

[0112] Peroxide value (POV) is a key indicator for evaluating the degree of lipid oxidation and can effectively reflect the amount of primary lipid oxidation products produced. The oxidation process of lipids / proteins is usually initiated by free radicals, and certain metal ions (such as Fe 2+ 、Cu 2+ , etc.) accelerates the formation of free radicals and catalyzes the oxidation of lipids and proteins. Therefore, in theory, substances that can scavenge free radicals and / or chelate metal ions are effective in inhibiting lipid and protein oxidation.

[0113] In the study, the changes in the peroxide value (POV) of the composite emulsion after 0 and 7 days of storage were analyzed. Figure 7As shown, the myofibrillar protein composite emulsion prepared with native PPI (Control group) had a high POV value at the initial stage (day 0), indicating a high degree of oxidation. After 7 days of storage, the POV value further increased, indicating that oxidation of the unmodified composite emulsion increased during storage. In contrast, the composite emulsions prepared with arginine-modified PPI and PPI covalently bound to quercetin had lower POV values ​​at the initial stage, indicating that these treatments significantly inhibited lipid oxidation at the initial stage. After 7 days, the POV values ​​of the composite emulsions prepared with arginine-modified PPI and PPI covalently bound to quercetin increased, but the increase was significantly less than that of the composite emulsion prepared with native pea protein isolate. This is attributed to the natural antioxidant activity of quercetin and the strong ferrous ion chelating activity of Arg, as well as the DPPH and hydroxyl radical scavenging activities.

[0114] 3.2 Determination of conjugated diene hydroperoxides in oils and fats

[0115] During oil oxidation, the double bonds of unsaturated fatty acids are easily attacked by oxygen, forming unstable free radicals. These free radicals can then trigger double bond rearrangements, generating hydroperoxides with conjugated diene structures. Therefore, changes in the conjugated diene content of an emulsion can serve as an important indicator for assessing the extent of primary oxidation in oils and fats.

[0116] from Figure 8 As can be seen in the results, the conjugated diene content of the emulsions on days 0 and 7 showed similar trends. The native pea protein isolate-myofibrillar protein composite emulsion had the highest conjugated diene content and exhibited severe lipid oxidation. In contrast, the myofibrillar protein composite emulsion prepared using arginine-modified pea protein isolate showed a slightly lower conjugated diene content, but this reduction was not significant. This suggests that arginine-modified pea protein isolate has a limited inhibitory effect on lipid oxidation in the composite emulsion. However, when a 0.2% arginine-modified pea protein isolate-quercetin complex was added to the myofibrillar protein composite emulsion, the antioxidant activity of the composite emulsion was significantly enhanced, exhibiting the best antioxidant performance. This result can be attributed to the highest total phenolic content and polyphenol binding rate of this complex. As a potent antioxidant, quercetin can effectively slow lipid oxidation in the myofibrillar protein composite emulsion by leveraging its antioxidant properties.

[0117] 4. Fluorescence intensity of composite emulsion Schiff base

[0118] The free amino groups of proteins (such as lysine, arginine and other side chain residues) can react with lipid oxidation products such as aldehydes to generate compounds containing Schiff base structures. Since Schiff bases have unique fluorescence properties, the degree of fat-protein co-oxidation in composite emulsions can be analyzed by fluorescence spectroscopy, which is used as an important indicator for detecting the mutual oxidation of lipids and proteins. Figure 9 As can be seen, the Schiff base fluorescence intensity of the composite emulsion prepared with natural pea protein isolate (Control group) was low at the beginning of storage. However, with extended storage (up to day 7), its fluorescence intensity increased significantly. This indicates that the reaction between protein and lipid oxidation products gradually intensified during storage, leading to an increase in the degree of oxidation. In contrast, the fluorescence intensity of the composite emulsions prepared with arginine-modified PPI (MP+PA1, MP+PA2, and MP+PA3) was lower than that of the Control group at the beginning of storage, and the increase in fluorescence intensity on day 7 was also relatively small. This indicates that arginine-modified PPI can effectively delay the co-oxidation process of the composite emulsions. In addition, the degree of co-oxidation of the emulsions containing quercetin was reduced, especially in the MP+PA2-Q group. This is due to the antioxidant properties of quercetin, which effectively inhibited the reaction between protein and lipid oxidation products, thereby significantly delaying the co-oxidation process.

[0119] Example 3

[0120] Preparation of emulsified pork meatballs with modified pea protein isolate-myofibrillar protein composite emulsion

[0121] Table 3 Basic formula of emulsified minced meat pork balls

[0122]

[0123] (1) Mince the meat: Mince the longissimus dorsi muscle of the pig. Add an appropriate amount of salt and 1 / 3 of ice water. Stir at medium speed for 1 minute. Then add the minced pork back fat, the compound emulsion, and another 1 / 3 of ice water. Continue stirring for 1 minute. Finally, add the remaining 1 / 3 of ice water and stir for 2 minutes. Keep the minced meat temperature below 12°C throughout the process.

[0124] (2) Cooking emulsified pork balls: Knead the minced meat into round balls of a certain mass. Place the emulsified balls in a retort bag and cook them in a constant temperature water bath at 85°C for 30 min until the core temperature of the emulsified balls reaches 72°C. After cooking, cool the emulsified balls to room temperature and then store them in a refrigerator at 4°C.

[0125] The experimental groups included emulsified meatballs prepared with animal oil (Control), and emulsified meatballs prepared with myofibrillar protein-natural pea protein isolate / modified pea composite emulsion instead of 50% animal oil, namely MP+PA0, MP+PA1, MP+PA, MP+PA3, MP+PA0-Q, MP+PA1-Q, MP+PA2-Q, and MP+PA3-Q.

[0126] Meatballs with modified pea protein isolate-myofibrillar protein composite emulsion can reduce fat intake and have better antioxidant properties.

[0127] Performance testing experimental methods

[0128] 1. Determination of cooking loss of emulsified pork balls

[0129] The emulsified pellets were marked and weighed before cooking. After cooking, they were cooled to room temperature, wiped dry, and weighed again. The cooking loss of the emulsified pellets was calculated according to the following formula.

[0130]

[0131] 2. Texture Analysis of Emulsified Pork Meatballs

[0132] The cooked emulsified pork meatballs were removed from the refrigerator and allowed to stand at room temperature for 2 hours before being cut into cylinders (3 cm diameter, 2 cm height). A texture analyzer was used to measure the hardness, springiness, adhesiveness, chewiness, and elasticity of the samples. The initial force was 0.5 N, the compression ratio was 60%, and the test speed was 1 mm / s. Each experimental group was tested three times, and the average value was calculated.

[0133] 3. Analysis of oxidative stability of emulsified pills

[0134] The vacuum-packed emulsified pellets were stored at 4°C for 15 days, and the TBARS values ​​of the emulsified pellets were measured on days 0, 5, 10, and 15. Lipid peroxidation was determined using TBARS (thiobarbituric acid reactants). 5 g of each emulsified pellet was weighed, 50 mL of 7.5% (mass-to-volume) trichloroacetic acid (TCA) solution was added, and the mixture was shaken on a thermostatic shaker at 50°C for 30 minutes. The sample solution was filtered through a double layer of quantitative slow filter paper, and then 5 mL of thiobarbituric acid (TBA) aqueous solution was added. The mixture was boiled in a water bath for 30 minutes. The sample was cooled to room temperature, and the absorbance of the supernatant was measured at 532 nm. The TBARS value of the sample was calculated as follows:

[0135]

[0136] Where: ABS represents the absorbance value of the sample at 532 nm; W represents the sample weight (g).

[0137] 4. Sensory Difference Analysis of Emulsified Pills

[0138] Table 4 Sensory evaluation table

[0139]

[0140] The emulsified meatballs were subjected to a sensory evaluation using a nine-point scale. A sensory evaluation panel of 10 food science students was selected. After undergoing sensory training, they were able to independently and accurately evaluate and score the meatballs' color, aroma, mouthfeel, texture, and other sensory characteristics using smell, taste, touch, and vision. Evaluators independently scored using a sensory scoring sheet to minimize any influence from interactions. They rinsed their mouths with water between evaluations of different sample groups. The sensory scoring sheet is shown below.

[0141] Experimental results

[0142] 1. Effect of composite emulsion on cooking loss of emulsified pork meatballs

[0143] The cooking loss rate is an important indicator for measuring the water retention of minced meat products, which can reflect the degree of water loss during the heating and cooking process. The higher the cooking loss rate, the weaker the water retention capacity of the minced meat product during the cooking process, and the worse the water retention. Figure 10 As shown, compared with the control (pure animal oil group), the application of arginine-modified PPI to meat emulsion significantly reduced the cooking loss of emulsified meatballs. For example, the MP+PA2 group showed a 57.87% reduction in cooking loss. Furthermore, meatballs containing quercetin showed a further reduction in cooking loss. Meatballs in which a portion of the fat is replaced with a pre-emulsified liquid generally experience a reduction in cooking loss. This is because during cooking, solid pork back fat becomes liquid below the gelation temperature of meat protein (>40°C). Without proper containment, the liquid fat will flow out of the product. The emulsified liquid forms a dense adsorption layer that encapsulates the water-oil mixture of the meat emulsion, thereby preventing water and oil loss during processing. The reduced cooking loss of muscle meat after the addition of quercetin is associated with the enhanced antioxidant properties of the meat emulsion. Protein oxidation disrupts protein structure, increasing the porosity of the gel network and reducing the protein's ability to bind water, leading to increased cooking loss in emulsified pork meatballs.

[0144] 2. Effect of composite emulsion on the texture of emulsified meatballs

[0145] Table 5 Determination of texture characteristics of emulsified meatballs prepared from vegetable oil pre-emulsion

[0146]

[0147] Texture is a key indicator for objectively evaluating the overall quality of food, as well as the quality and acceptability of meat products. As shown in Table 5, the hardness of the animal oil control group was 11.43 ± 1.10 N, elasticity was 2.09 ± 0.17, cohesion was 0.23 ± 0.01, and chewiness was 12.26 ± 0.52. The product was generally soft, with poor elasticity and low chewiness. For meatballs prepared with arginine-modified pea protein isolate (MP+PA0 to MP+PA3), the hardness increased from 11.70 ± 0.87 N (MP+PA0) to 14.23 ± 1.33 N (MP+PA3), and the chewiness increased from 13.14 ± 1.72 to 22.02 ± 4.67. This indicates that the composite emulsion prepared with arginine-modified pea protein isolate significantly enhanced the firmness, hardness, and chewiness of the meatballs, while also improving the toughness and internal structural integrity of the meatballs. The hardness of the quercetin-grafted groups (MP+PA0-Q to MP+PA3-Q) further increased significantly, particularly the MP+PA2-Q group, which achieved a hardness of 27.97±2.06 N and a chewiness of 28.28±5.07. This indicates that the quercetin-grafted PPI-based myofibrillar protein composite emulsion further enhanced the firmness and chewiness of the emulsified meatballs. Compared with the animal oil-based emulsions, the elasticity and cohesion increased by 54.5% and 82.61%, respectively, demonstrating that the quercetin-grafted PPI-based myofibrillar protein composite emulsion has a certain effect on improving the toughness and elasticity of the emulsified meatballs. The emulsifying properties of the composite emulsion containing quercetin are improved, and the vegetable oil in the pre-emulsion is wrapped by protein to form uniform and dense small droplets. The pre-emulsion with good emulsification properties forms a dense and orderly network structure with the meatballs prepared with minced pork, which can tightly wrap the oil-water mixture, so that the water and oil in the emulsified meatballs can be better retained.

[0148] 3. Effect of composite emulsion on oxidative stability of emulsified meatballs

[0149] Fat oxidation can have an adverse effect on the product. The thiobarbituric acid reactant (TBARS) value is often used to measure the degree of fat oxidation. Its essence represents the content of malondialdehyde, a secondary product of fat oxidation, and is of great significance in evaluating the oxidative stability of the product. Figure 11As can be seen, the TBARS values ​​of the emulsified meatballs in the animal oil control group (Control) increased most significantly during storage. This suggests that meatballs prepared with animal fat are more susceptible to oxidation during storage, leading to the accumulation of malondialdehyde, a fat oxidation product. In contrast, the TBARS values ​​of the emulsified meatballs prepared with pea protein isolate-myofibrillar protein composite emulsions modified with arginine (Arg) (MP+PA0, MP+PA1, MP+PA2, and MP+PA3) increased at a significantly slower rate. Furthermore, the TBARS values ​​of the emulsified meatballs prepared with pea protein isolate-myofibrillar protein composite emulsions modified with quercetin (MP+PA0-Q, MP+PA1-Q, MP+PA2-Q, and MP+PA3-Q) during storage were significantly lower than those of the control group and other treatments without quercetin (P<0.05). Arginine itself has certain antioxidant properties. Its strong hydroxyl radical scavenging and iron ion chelating abilities can inhibit protein and fat oxidation, thereby improving the oxidative stability of meat products. During storage, the TBARS value of the emulsified meat emulsion containing quercetin was significantly lower than that of the control (p<0.05), confirming that quercetin can inhibit fat oxidation and reduce malondialdehyde production. Quercetin, with its strong free radical scavenging ability, can scavenge free radicals generated by unsaturated fatty acids and prevent the formation of peroxides. In summary, the antioxidant activity of emulsified meatballs prepared with quercetin-modified pea protein isolate remained stable during storage, demonstrating its potential as a functional additive in meat emulsion products and potentially providing strong support for the improvement of meat emulsion products.

[0150] 4. Sensory Difference Analysis of Emulsified Pills Prepared from Composite Emulsions

[0151] In order to evaluate the feasibility of using modified pea protein isolate composite emulsion to replace animal fat in emulsion-type meat products, and to comprehensively evaluate the overall quality of emulsion balls, sensory evaluation was conducted to predict consumer acceptance of the modified emulsion balls. Figure 12 As shown, the overall sensory evaluation of emulsified meatballs prepared with animal fat was low. However, when the animal oil group was partially replaced with a vegetable oil pre-emulsion (50% replacement rate), the sensory evaluation was significantly improved. The composite emulsion prepared with quercetin-modified PPI, which partially replaced animal fat, generally scored higher in terms of texture. Its antioxidant activity protects the myofibrillar proteins in the product system, interacting with the proteins during processing to help form a more stable internal structure and achieve an optimal texture. Juiciness and cooking loss results were generally consistent, with the product receiving a higher taste score due to its juiciness and smooth texture when chewed. The emulsified pork meatballs containing quercetin exhibited improved firmness, elasticity, and chewiness, making them more palatable.

Claims

1. A method for preparing a pea protein isolate composite emulsion, characterized in that: The following steps are involved: The myofibrillar protein dilution and the modified pea protein isolate dilution are mixed and stirred, and vegetable oil is added and homogenized to obtain a pea protein isolate composite emulsion; The preparation method of the modified pea protein isolate comprises: adding arginine to a pea protein isolate solution, magnetically stirring, fully hydrating the solution, adding a quercetin solution, fully mixing the solution, adding laccase, reacting the solution at room temperature, heating to inactivate the enzyme after the reaction, cooling the solution, and dialyzing the solution to obtain a pea protein isolate-arginine-quercetin covalent complex; and freeze-drying the solution to obtain the modified pea protein isolate.

2. The method for preparing the pea protein isolate composite emulsion according to claim 1, wherein The concentration of the myofibrillar protein diluent is 1.5-2.5wt%, the concentration of the modified pea protein isolate diluent is 0.8-1.2wt%, and the mass ratio of the myofibrillar protein diluent to the modified pea protein isolate diluent is 1:1; the vegetable oil is soybean oil, and the volume percentage of the vegetable oil in the pea protein isolate composite emulsion is 15-25%.

3. The method for preparing the pea protein isolate composite emulsion according to claim 1, wherein The concentration of the pea protein isolate is 25-35 mg / mL, the mass concentration ratio of the pea protein isolate to quercetin is 25-35:1, and the concentration of the arginine is 0.1-0.3%.

4. The method for preparing the pea protein isolate composite emulsion according to claim 1, wherein The final concentration of the laccase in the mixed solution is 1.0 U / mL, and the reaction time is 4-5 h.

5. The method for preparing the pea protein isolate composite emulsion according to claim 1, wherein The enzyme inactivation method includes: placing the reaction solution in an 80-85°C water bath and heating it for 2-3 minutes to inactivate the laccase; the dialysis method includes: placing the enzyme-inactivated and cooled reaction solution into a dialysis bag, dialysis using deionized water at 4-6°C for 48-60 hours, with a molecular weight cutoff of 12-14 kDa, and replacing the deionized water every 6-8 hours.

6. The method for preparing the pea protein isolate composite emulsion according to claim 1, wherein The method for preparing myofibrillar protein comprises: removing fat and connective tissue from fresh pork tenderloin and then cutting it into small pieces to obtain chopped pork tenderloin; mixing the chopped pork tenderloin with phosphate buffer, homogenizing, filtering, and centrifuging to obtain a precipitate; adding NaCl solution to the precipitate, homogenizing again, filtering, and centrifuging to obtain a precipitate; and finally adjusting the pH to 6-6.2, centrifuging to obtain a precipitate to obtain myofibrillar protein.

7. A pea protein isolate composite emulsion, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the pea protein isolate composite emulsion according to claim 7 in preparing emulsified meatballs.

9. The use according to claim 8, characterized in that The preparation method of the emulsified meatballs comprises the following steps: mincing the longissimus dorsi muscle of a pig, adding salt and ice water, stirring thoroughly, adding minced pork back fat, the pea protein isolate composite emulsion according to claim 4, and ice water, continuing stirring, adding ice water again and stirring to obtain raw meat paste, kneading the raw meat paste into meatballs, and cooking and cooling to obtain emulsified meatballs.

10. Use of the pea protein isolate composite emulsion according to claim 7 in improving the antioxidant properties and mouthfeel of emulsified meatballs.

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