Preparation method of myofibrillar protein / luteolin complex

Abstract

The present invention relates to the field of luteolin application technology, and in particular to a method for preparing a myofibrillar protein / luteolin complex. The preparation method comprises: dissolving myofibrillar protein in phosphate buffer to prepare a myofibrillar protein solution; uniformly dispersing luteolin in pure water by pre-homogenization to obtain a luteolin suspension; adding the luteolin suspension to the myofibrillar protein solution, mixing thoroughly, and then pre-homogenizing to obtain a complex solution; and incubating the complex solution after high-pressure homogenization to obtain a myofibrillar protein / luteolin complex. This method can effectively improve the loading rate of myofibrillar protein on luteolin while eliminating the dependence of luteolin on chemical cosolvents, and is easy to operate and has low cost. The myofibrillar protein / luteolin complex prepared by this method can be used as a functional meat product or as a food additive.

Description

Technical Field

[0001] The present invention relates to the technical field of luteolin application, and in particular to a method for preparing a fibrillin / luteolin complex. Background Art

[0002] Luteolin is a plant secondary metabolite and a natural flavonoid compound found in a variety of plants as a glycoside. Studies have shown that luteolin has a variety of pharmacological activities, including anti-inflammatory, anti-allergic, uric acid-lowering, anti-tumor, antibacterial, and antiviral. It is primarily used clinically for cough relief, expectorant, anti-inflammatory, uric acid-lowering, and cardiovascular disease treatment.

[0003] Luteolin is chemically very stable. Its molecule possesses a conjugated, planar structure, with two hydroxyl groups on each benzene ring, resulting in strong intermolecular hydrogen bonding. However, like most other flavonoids, luteolin is poorly water-soluble, even at high temperatures. Furthermore, luteolin alone can cause irritation to the mouth and stomach. Luteolin's hydrophobicity and irritation severely limit its application.

[0004] To expand the application of luteolin, related technologies include using polyethylene glycol or gelatin as carriers to load luteolin. However, on the one hand, polyethylene glycol, as a medium- and high-molecular-weight polymer, is not a food-grade raw material, which limits its application in the food industry. On the other hand, although gelatin is a food-grade raw material, gelatin extracted from pig skin has problems with the limited population and product cleanliness. If fish skin is used as a raw material, it will cause high costs. In addition, the process of using gelatin as a carrier to compound luteolin requires a large amount of auxiliary agents such as ethanol, which poses certain food safety risks. Summary of the Invention

[0005] In order to solve or partially solve the problems existing in the related art, the present invention provides a method for preparing a myofibrillar protein / luteolin complex.

[0006] The present invention provides a method for preparing a myofibrillar protein / luteolin complex, which comprises:

[0007] Step a), dissolving myofibrillar protein in phosphate buffer to prepare a myofibrillar protein solution;

[0008] Step b), uniformly dispersing luteolin in pure water by pre-homogenization treatment to obtain a luteolin suspension; there is no order restriction for step a) and step b);

[0009] Step c), adding the luteolin suspension to the myofibrillar protein solution, mixing thoroughly, and then performing a pre-homogenization treatment to obtain a complex solution;

[0010] Step d), the complex solution is subjected to high-pressure homogenization and then incubated to obtain a myofibrillar protein / luteolin complex.

[0011] Furthermore, the pressure of the high-pressure homogenization treatment is 5000-25000 psi and the temperature is 4-10°C.

[0012] Furthermore, the incubation temperature is 4-10° C. and the incubation time is 30-60 min.

[0013] Furthermore, the concentration of the luteolin suspension is 60-200 μmol / g.

[0014] Furthermore, the pre-homogenization treatment in step b) and step c) is specifically as follows: using a high-speed homogenizer to perform pre-homogenization twice; setting the speed of the high-speed homogenizer to 6000-8000 rpm, and the treatment time of each pre-homogenization is 30-50 seconds.

[0015] Furthermore, the concentration of the myofibrillar protein solution is 8-12 mg / mL.

[0016] Furthermore, the myofibrillar protein in step a) is prepared according to the following method:

[0017] The raw meat is removed from the surface muscular membrane and fat tissue, cut into pieces and minced to obtain minced meat;

[0018] The minced meat was mixed with a standard salt solution, homogenized, filtered, and centrifuged to obtain a precipitate. The precipitate was mixed with a KCl solution, homogenized, filtered, and centrifuged to obtain myofibrillar protein.

[0019] Furthermore, in the step c), the mixing ratio of luteolin to myofibrillar protein in the complex solution is (10-120) μmol:1 g.

[0020] The present invention also provides a myofibrillar protein / luteolin complex, which comprises myofibrillar protein and luteolin embedded in the myofibrillar protein.

[0021] Furthermore, the embedding rate of luteolin in myofibrillar protein is greater than 95%.

[0022] The preparation method of the myofibrillar protein / luteolin complex provided by the present invention can have the following beneficial effects:

[0023] 1) This method uses myofibrillar protein to encapsulate and compound luteolin. Myofibrillar protein is a food-grade raw material with sufficient sources and is suitable for an unlimited population, thereby expanding the product's field of use and applicable population. This method can not only achieve stable preservation of luteolin, but also eliminate the oral and gastric irritation caused by high-concentration luteolin alone. Finally, due to the antioxidant properties of luteolin, it can also effectively delay the oxidative deterioration of lipids and proteins in myofibrillar protein, improve food safety, and extend shelf life. The myofibrillar protein / luteolin complex prepared by this method can be used as a functional meat product or as a food additive (such as an antioxidant).

[0024] 2) This method uses high-pressure homogenization technology to effectively increase the loading rate of myofibrillar protein on luteolin while eliminating luteolin's dependence on chemical cosolvents, reducing waste and improving product safety.

[0025] 3) This method is easy to operate and has low cost. It can provide a theoretical basis for the deep processing and utilization of myofibrillar proteins and provide new ideas for the development of functional meat products.

[0026] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0028] Figure 1 This is a graph showing the embedding efficiency test results of Experiment 1 of the present invention;

[0029] Figure 2 This is a graph showing the antioxidant activity of the compound in Experiment 2 of the present invention;

[0030] Figure 3 This is a graph showing the digestion characteristics of the complex in Experiment 3 of the present invention. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] The terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the invention. The singular forms "a," "the," and "the" used in this invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0033] It should be understood that although the terms "first", "second", "third", etc. may be used to describe various information in the present invention, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "plurality" means two or more, unless otherwise clearly and specifically defined.

[0034] In order to expand the application scope of luteolin, especially to the food-grade field, the inventors of this application first considered using food-grade materials to embed luteolin, which can achieve stable preservation of luteolin and reduce irritation to the mouth and stomach. Furthermore, combined with the characteristics of luteolin itself, the inventors of this application found that myofibrillar protein can be an embedding material for luteolin. Specifically: on the one hand, myofibrillar protein (MP) is a highly nutritious, low-allergenic amphiphilic meat protein, and therefore can be combined with luteolin; on the other hand, myofibrillar protein can be extracted from meat proteins such as chicken, and the content of myofibrillar protein in meat proteins is high, so the source of myofibrillar protein is sufficient and there is no restriction on the application population. Therefore, the applicant considered using myofibrillar protein to embed and compound luteolin to form a myofibrillar protein-luteolin complex. On the one hand, it can achieve the stable preservation of luteolin, and on the other hand, it can eliminate the oral and gastric irritation caused by the presence of high-concentration luteolin alone. Finally, due to the antioxidant properties of luteolin, it can also effectively delay the oxidative deterioration of lipids and proteins in myofibrillar protein, improve food safety performance, and extend shelf life. The prepared complex can be used as a functional meat product or as a food additive (such as an antioxidant).

[0035] Furthermore, the inventors of this application have found through research that as the amount of active substance added increases, the encapsulation efficiency of MP for small molecules decreases significantly, and the final encapsulation rate is generally 50-70%. On the other hand, compared with the good solubility of hydrophilic small molecules in different systems, luteolin, as a hydrophobic active substance, often requires the use of cosolvents such as DMSO and ethanol. Although methods such as distillation can be used to reduce the ethanol content in subsequent operations, or by constructing core-shell colloidal particles to encapsulate hydrophobic substances without relying on cosolvents, there are still problems such as containing unhealthy ingredients or low efficiency. Therefore, how to achieve a relatively simple combination of the two without introducing other ingredients and improve their encapsulation efficiency has become the focus of this application.

[0036] High-pressure homogenization (HPH) is a mature food processing technology widely used in beverage refinement, biomacromolecule modification, and non-thermal sterilization. The inventors of this application considered introducing HPH to address the aforementioned key issues. The MP undergoes oscillatory shearing and turbulent mixing within the cavity, achieving changes in protein structure, such as unfolding and refolding, and protein particle dispersion and reaggregation. This could theoretically significantly impact protein encapsulation.

[0037] Based on the above inventive concept, the present invention provides a method for preparing a myofibrillar protein / luteolin complex, which comprises:

[0038] Step a), dissolving myofibrillar protein in phosphate buffer to prepare a myofibrillar protein solution;

[0039] Step b), uniformly dispersing luteolin in pure water by pre-homogenization treatment to obtain a luteolin suspension; there is no order restriction for step a) and step b);

[0040] Step c), adding the luteolin suspension to the myofibrillar protein solution, mixing thoroughly, and then performing a pre-homogenization treatment to obtain a complex solution;

[0041] Step d), the complex solution is subjected to high-pressure homogenization and then incubated to obtain a myofibrillar protein / luteolin complex.

[0042] The method provided in this embodiment uses luteolin and myofibrillar protein as raw materials to prepare a luteolin pure water suspension and a myofibrillar protein solution, respectively. The two are then mixed and treated with high-pressure homogenization to achieve a high encapsulation rate of luteolin by myofibrillar protein without relying on a solvent.

[0043] The above-mentioned step a) is a step of preparing a myofibrillar protein solution. The protein concentration of the myofibrillar protein solution is preferably 8 to 12 mg / mL. Too low a protein concentration makes it difficult to obtain an ideal amount of luteolin grafting, while too high a protein concentration easily cross-links to form a gel, affecting subsequent operations. In this embodiment, the protein concentration of the myofibrillar protein solution is most preferably 10 mg / mL. The phosphate buffer used preferably includes: NaCl, K2HPO4 and KH2PO4, and the pH value is 7.0. More specifically, the phosphate buffer includes: 0.6 mol / L NaCl, 50 mmol / L K2HPO4 and 50 mmol / L KH2PO4.

[0044] The above-mentioned myofibrillar protein is preferably prepared according to the following method:

[0045] Step a1), removing the surface muscular membrane and fat tissue from the raw meat, cutting it into pieces and then mincing it to obtain minced meat;

[0046] Step a2) The minced meat is mixed with a standard salt solution, homogenized, filtered, and centrifuged to obtain a precipitate, and the precipitate is mixed with a low-concentration salt solution, homogenized, filtered, and centrifuged to obtain myofibrillar protein.

[0047] The above processes are preferably carried out at 4-10°C, more preferably at 4°C. The standard salt solution preferably comprises: 0.1 mol / L KCl, 20 mmol / L K2HPO4, 20 mmol / L KH2PO4, 2 mmol / L MgCl2, and 1 mmol / L EGTA, and the pH value of the standard salt solution is 7.0. The concentration of the KCl solution is preferably 0.1 mol / L. The volume ratio of the standard salt solution to the minced meat is preferably 2-8:1, more preferably 3-5:1. The low-concentration salt solution can specifically be a low-concentration KCl solution, and its concentration is preferably 0.1 mol / L. The volume ratio of the low-concentration salt solution added to the precipitate is 2-8:1, more preferably 3-5:1.

[0048] The above step a2) may specifically be:

[0049] The minced meat was added to a standard saline solution with a volume ratio of 1:4 between the minced meat and the standard saline solution, and homogenized at 6900 rpm for 30 seconds, twice, and then filtered through a double layer of gauze and centrifuged at 2000×g for 10 minutes. The resulting precipitate was then added to a standard saline solution with a volume four times that of the minced meat, homogenized, filtered, and centrifuged under the same conditions. The resulting precipitate was again eluted with a standard saline solution with a volume four times that of the precipitate, homogenized, and centrifuged. The precipitate was mixed with a 0.1 mol / L KCl solution with a volume four times that of the minced meat, homogenized again, filtered through four layers of gauze, and centrifuged at 2500×g for 10 minutes, and the process was repeated once. The resulting precipitate is myofibrillar protein.

[0050] The above-mentioned step b) is a step of uniformly dispersing luteolin in pure water. The concentration of the obtained luteolin suspension is preferably 60-200 μmol / g, more preferably 100-160 μmol / g, and most preferably 120 μmol / g. In this step, a high-speed homogenizer is preferably used to perform two pre-homogenization treatments; the speed of the high-speed homogenizer is set to 6000-8000 rpm, and the treatment time for each pre-homogenization is 30-50 s. More preferably, the speed of the high-speed homogenizer is set to 6900 rpm, and the treatment time for each pre-homogenization is 40 s. This step can specifically be: dispersing luteolin in pure water to form a suspension, setting the speed of the high-speed homogenizer to 6900 rpm, pre-homogenizing for 40 s, and repeating twice to obtain a short-term stable luteolin dispersion, that is, the above-mentioned luteolin suspension.

[0051] Following steps a) and b) above, a myofibrillar protein solution and a luteolin suspension can be obtained, which can then be uniformly mixed according to step c) to obtain a complex solution. The homogenization process in this step can be the same as that in step b). The mixing ratio of luteolin to myofibrillar protein in the complex solution is preferably (10-120) μmol:1g, that is, the added amount of luteolin is preferably (10-120) μmol / g protein.

[0052] Step d) is a step of performing high-pressure homogenization. During the high-pressure homogenization process, the myofibrillar protein undergoes oscillatory shearing and turbulent blending in the cavity to achieve changes such as protein structure unfolding-refolding, protein particle dispersion-reaggregation, etc., thereby improving the embedding effect of luteolin without the need for a cosolvent. In this step, the pressure of the high-pressure homogenization treatment is preferably 5000-25000 psi, and the temperature is 4-10°C. It is further preferably 12000-20000 psi, and most preferably 15000 psi. The temperature is most preferably 4°C. After the high-pressure homogenization treatment, incubation is required, and the incubation temperature is preferably 4-10°C, more preferably 4°C, and the incubation time is preferably 30-60 min, more preferably 35-50 min, and most preferably 40 min. After incubation, a myofibrillar protein-encapsulated luteolin complex, i.e., the aforementioned myofibrillar protein / luteolin complex, is obtained. This complex does not contain cosolvents such as DMSO and ethanol, and has a high luteolin encapsulation efficiency, reaching over 95%. Preferably, the method further includes a phenol removal treatment to remove unreacted luteolin from the mixture, thereby purifying the myofibrillar protein / luteolin complex.

[0053] From the above, it can be seen that the method for preparing the myofibrillar protein / luteolin complex provided by the embodiment of the present invention has the following advantages:

[0054] 1) This method uses myofibrillar protein to encapsulate and compound luteolin. Myofibrillar protein is a food-grade raw material with sufficient sources and is suitable for an unlimited population, thereby expanding the product's field of use and applicable population. This method can not only achieve stable preservation of luteolin, but also eliminate the oral and gastric irritation caused by high-concentration luteolin alone. Finally, due to the antioxidant properties of luteolin, it can also effectively delay the oxidative deterioration of lipids and proteins in myofibrillar protein, improve food safety, and extend shelf life. The myofibrillar protein / luteolin complex prepared by this method can be used as a functional meat product or as a food additive (such as an antioxidant).

[0055] 2) This method uses high-pressure homogenization technology to effectively increase the loading rate of myofibrillar protein on luteolin while eliminating luteolin's dependence on chemical cosolvents, reducing waste and improving product safety.

[0056] 3) This method is easy to operate and has low cost. It can provide a theoretical basis for the deep processing and utilization of myofibrillar proteins and provide new ideas for the development of functional meat products.

[0057] Another embodiment of the present invention provides a myofibrillar protein / luteolin complex comprising myofibrillar protein and luteolin encapsulated by the myofibrillar protein. The myofibrillar protein can have an encapsulation efficiency of greater than 95% for luteolin. This complex can be prepared using the above-described method. The specific embodiments and beneficial effects are the same as those of the above-described embodiment and are not further described here.

[0058] The technical solution of the present invention will be further described below in conjunction with specific embodiments:

[0059] Example

[0060] 1. Preparation of myofibrillar protein

[0061] The surface fascia and fat tissue of the meat are removed, and the meat is cut into pieces and minced to obtain minced meat;

[0062] The processed minced meat was added with four times the volume of standard saline solution and homogenized at 6900 rpm for 30 s, homogenized twice, and then filtered through double gauze and centrifuged at 2000×g for 10 min; the standard saline solution was a saline solution with a pH value of 7.0 composed of 0.1 mol / L KCl, 20 mmol / L K2HPO4, 20 mmol / L KH2PO4, 2 mmol / L MgCl2 and 1 mmol / L EGTA.

[0063] The obtained precipitate is then added to a standard saline solution having a volume four times that of the aforementioned minced meat, and homogenized, filtered, and centrifuged under the same conditions; the obtained precipitate is again eluted with a standard saline solution having a volume four times that of the precipitate, homogenized, and centrifuged; the precipitate is mixed with a 0.1 mol / L KCl solution having a volume four times that of the aforementioned minced meat, and the mixture is homogenized again, filtered through four layers of gauze, and centrifuged at 2500×g for 10 minutes. This is repeated once, and the obtained precipitate is the myofibrillar protein. The entire process is carried out at 4°C.

[0064] 2. Preparation of myofibrillar protein solution

[0065] The obtained myofibrillar protein was dissolved in a phosphate buffer solution comprising 0.6 mol / L NaCl, 50 mmol / L K2HPO4 and 50 mmol / L KH2PO4. The protein concentration of the myofibrillar protein solution was 10 mg / mL.

[0066] 3. Preparation of luteolin pure water suspension

[0067] Luteolin was dispersed in pure water to form a suspension, and the suspension was placed in a high-speed homogenizer for pre-homogenization. The speed of the high-speed homogenizer was set to 6900 rpm, and the pre-homogenization was performed for 40 seconds. This was repeated twice to obtain a short-term stable luteolin suspension. The concentration of the luteolin suspension was 120 μmol / g.

[0068] 4. Mix the luteolin suspension in pure water with myofibrillar protein. The luteolin contents in the complex solutions were 10 μmol / g protein, 30 μmol / g protein, and 120 μmol / g protein, respectively. The mixed complex solutions were pre-homogenized as in step 3. The complex solutions were then subjected to high-pressure homogenization at 4°C and 15,000 psi, followed by incubation at 4°C for 40 min.

[0069] 5. Phenol removal: Remove unreacted luteolin from the mixture to obtain purified myofibrillar protein / luteolin complex. The prepared myofibrillar protein / luteolin complex is denoted as MP-HPH-Lut 10 、MP-HPH-Lut 30 and MP-HPH-Lut 120 .

[0070] Comparative Example 1

[0071] The difference between the embodiment and the method is that it only includes step 1 and does not include steps 2 to 5.

[0072] Comparative Example 2

[0073] Steps 1 and 2 are the same as those in the embodiment.

[0074] 3. Preparation of luteolin ethanol solution

[0075] Luteolin was dissolved in ethanol to prepare a stock solution, and the concentration of the luteolin solution was 120 μmol / g.

[0076] 4. Add luteolin solution to myofibrillar protein solution. The luteolin contents in the complex solutions were 10 μmol / g protein, 30 μmol / g protein, and 120 μmol / g protein, respectively. After thorough mixing, the mixed complex solutions were incubated at 4°C for 1 hour, vortexed every 15 minutes, and the prepared myofibrillar protein / luteolin complexes were recorded as MP-Lut 10 、MP-Lut 30 and MP-Lut 120 .

[0077] 5. Phenol removal: Remove the unreacted luteolin in the mixture to obtain the purified myofibrillar protein / luteolin complex. The prepared myofibrillar protein / luteolin complex is recorded as MP-Lut 10 、MP-Lut 30 and MP-Lut 120 .

[0078] Comparative Example 3

[0079] Steps 1 and 2 are the same as those in the embodiment.

[0080] 3. Preparation of luteolin ethanol solution

[0081] Luteolin was dissolved in ethanol to prepare a stock solution, and the concentration of the luteolin solution was 120 μmol / g.

[0082] 4. Add luteolin solution to the myofibrillar protein solution at the addition amounts of luteolin of 10 μmol / g protein, 30 μmol / g protein, and 120 μmol / g protein, respectively. Place the mixed complex in a high-speed homogenizer for pre-homogenization at a speed of 6900 rpm and pre-homogenize for 40 seconds. Then, perform high-pressure homogenization on the complex solution at an ambient temperature of 4°C and a pressure of 15,000 psi, and then incubate at 4°C for 40 minutes.

[0083] 5. Phenol removal: Remove the unreacted luteolin in the mixture to obtain purified myofibrillar protein successfully grafted with luteolin. The prepared myofibrillar protein / luteolin complexes are respectively recorded as MP-HPH (Ethanol) -Lut10 、MP-HPH (Ethanol) -Lut 30 and MP-HPH (Ethanol) -Lut 120 .

[0084] The products prepared in the above examples and comparative examples were subjected to the following tests:

[0085] Test Example 1 Embedding Rate Test

[0086] Test objects: myofibrillar protein / luteolin complex samples prepared in Example, Comparative Example 2 and Comparative Example 3.

[0087] Test method:

[0088] The complex samples prepared in Example, Comparative Example 2, and Comparative Example 3 were diluted to 2 mg / mL (based on protein) using the above-mentioned phosphate buffer. 1 mL of 20% trichloroacetic acid and 0.4% phosphotungstic acid was added to the diluted samples from Comparative Examples 2 and 3 to cause flocculation and precipitation. After standing at 4°C for 30 minutes, the precipitated protein was washed and centrifuged at 15,000 g for 15 minutes at 4°C to obtain a supernatant. 480 μL of the supernatant was collected and 265 μL of 1 mol / L NaOH was added to neutralize the trichloroacetic acid used to precipitate the protein. The final volume was adjusted to 500 μL for determination of free phenol content. The diluted samples from Example were dialyzed against 4°C phosphate buffer for 24 hours using an 8-10 kDa dialysis bag. 0.5 mL of the supernatant was collected and used to determine the free phenol content.

[0089] To 0.5 mL of each test solution, add 0.5 mL of 1 mol / L Folin phenol reagent. After incubation at room temperature for 30 minutes, measure the absorbance at 650 nm. A standard absorbance curve with a concentration range of 0–2 mg / mL free luteolin was established to estimate the amount of luteolin in the supernatant. The grafting efficiency of the myofibrillar protein / luteolin complex was calculated using the following equation:

[0090] Luteolin grafting rate = (total amount of luteolin added - luteolin content in the supernatant) / total amount of luteolin added × 100%

[0091] Test results:

[0092] like Figure 1As shown, the Lut embedding rate of the MP-Lut complex sample (Comparative Example 3) treated with high-pressure homogenization at different concentrations was significantly improved compared with the simple non-covalent grafted sample (Comparative Example 2) (P<0.001), and the embedding rate did not show a significant downward trend with increasing concentration, and was close to 100%. This shows that high-pressure homogenization treatment is effective in improving the MP protein embedding rate. Without the addition of ethanol, the embodiment can obtain an MP-Lut complex solution with good dispersibility and stability. Compared with Comparative Example 3, the embedding rate is close to 100% at a Lut concentration of 120 μmol / g, and there is no significant difference in the embedding rate between the embodiment and Comparative Example 3 (P>0.05). The above results show that high-pressure homogenization treatment effectively overcomes the dependence of protein on cosolvents when embedding insoluble active substances, and can empower new meat products in a purely green way.

[0093] Test Example 2 Antioxidant Activity of the Complex

[0094] Test objects: myofibrillar protein / luteolin complex samples prepared in Example, Comparative Example 1 and Comparative Example 3.

[0095] Test method:

[0096] The complex samples prepared in Example, Comparative Example 1 and Comparative Example 3 were diluted with the above-mentioned phosphate buffer to prepare a 0.5 mg / mL (protein) solution of the sample to be tested.

[0097] ABTS Radical Scavenging Activity: Dissolve 10 mg of ABTS powder in 2.6 mL of 2.45 mmol / L potassium persulfate solution. Incubate this 7 mmol / L ABTS stock solution in the dark at room temperature for 12-16 hours. Then dilute the ABTS stock solution with distilled water to an absorbance of 0.70 ± 0.02 at 734 nm. Mix 1 mL of the test sample solution with 3 mL of the diluted ABTS solution and incubate at room temperature for 1 hour. The ABTS concentration in the mixture is measured at 734 nm. ABTS scavenging activity is calculated and expressed as μmol Trolox equivalents (TE) per g of sample using a Trolox calibration curve.

[0098] Iron(III) Reducing Capacity: Mix 1 mL of the test sample solution with 2.5 mL of 0.2 mol / L sodium phosphate buffer (pH 6.6), then add 2.5 mL of 1% (w / v) potassium ferricyanide to initiate the reaction. Incubate the mixture at 50°C for 20 minutes. Add 2.5 mL of 10% (w / v) TCA to the mixture, and centrifuge at 3000 × g for 10 minutes. After centrifugation, mix 2.5 mL of the supernatant with 0.5 mL of distilled water and 0.1 mL of 0.1% (w / v) ferric chloride. After mixing thoroughly, measure the absorbance of the mixture at 700 nm and analyze the reducing capacity of all test protein samples.

[0099] Test results:

[0100] After the high-pressure homogenization treatment, the antioxidant capacity of the composite prepared in Example 1 was significantly increased, and the difference was significant compared with that in Comparative Example 1 (P<0.001). Figure 2 It can be seen that the results of the ABTS scavenging activity and iron reducing ability of the examples show the same trend of change. Comparative Example 1 shows a high level of IC50 (ABTS is 0.65 mg / mL, Fe 2+ The results showed that the antioxidant activity of Example 3 was slightly lower than that of Comparative Example 3, which may be due to the different diffusion patterns of Lut in protein. In the presence of ethanol, Lut is more easily dispersed in the system. However, this example has a relatively better sustained-release effect.

[0101] Test Example 3 Complex Digestion Characteristics Test

[0102] Test objects: Example, Comparative Example 1 and Comparative Example 3

[0103] Test method:

[0104] The composite samples prepared in Example 1, Comparative Example 1, and Comparative Example 3 were diluted to a sample solution of 10 mg / mL (in terms of protein) using the above-mentioned phosphate buffer. The sample solution was mixed with 9.5 ml of gastric digestion fluid, the pH of the digestion fluid was adjusted to 2.0 with 6 M hydrochloric acid, and the solution was incubated in a 37°C water bath. Appropriate amounts of pepsin (3000 U / mg) and trypsin (130 U / mg) were weighed and dissolved in gastric digestion fluid at pH 2.0 and pancreatic digestion fluid at pH 7.5, so that the concentrations of pepsin and trypsin were both 20 mg / mL. 0.67 ml of pepsin solution was added to the sample, the total volume was considered to be 20 mL, and the final enzyme activity of the system was 2000 U / mL. The sample was digested in a shaking incubator at 37°C and 200 rpm for 60 min. During the simulated in vitro digestion process, 1 mL of sample was removed from the digestion system every 15 minutes and quickly replaced with an equal volume of digestion fluid. The sample was placed in a 2 mL centrifuge tube and mixed with an equal volume of 30% TCA solution to terminate the reaction and precipitate the protein. The mixture was then placed on ice for 15 minutes and centrifuged at 10,000 g for 10 minutes. The supernatant was discarded, and 1 mL of 1 M NaOH was added to the centrifuge tube to resolubilize the protein. The protein concentration was determined using the biuret method. The in vitro digestibility was defined as follows:

[0105] Digestibility = (Ct-Cp) / Ct×100%

[0106] Ct and Cp refer to the total protein concentration and TCA-precipitated protein concentration, respectively.

[0107] Data analysis and graphics:

[0108] The above experimental procedures were repeated three times, and statistical analysis, including one-way analysis of variance (ANOVA, P < 0.05) and multivariate comparative analysis (P < 0.05), was performed using IBM SPSS Statistics 27. Origin 2021 was used for data analysis and plotting.

[0109] Test results:

[0110] Changes in protein digestibility under HPH treatment Figure 3As shown, compared with comparative example 1, after a short heat treatment, the presence of Lut significantly improved (P < 0.05) the thermal tolerance of MP. The protein digestibility of the MP-Lut complex in comparative example 3 and the example at a concentration of 120 μmol / g reached more than 70%, which was significantly higher than that in comparative example 1 (P < 0.05). The improvement in thermal stability can be attributed to the hydrophobic interaction between MP and Lut, which reduces the thermal aggregation between proteins. In addition, the non-covalent binding sites of Lut and MP are mainly aromatic amino acids such as tyrosine and lysine, while digestive enzymes such as pepsin usually act on aromatic amino acid residues or peptide bonds adjacent to acidic amino acid residues, and trypsin usually acts on peptide bonds next to arginine or lysine. There may be a "polyphenol protection" effect during the heat treatment of meat protein. The above results show that regardless of whether there is a cosolvent, high-pressure homogenization treatment can alleviate the reduction in in vitro protein digestibility of the MP-Lut complex caused by heat treatment, so that the MP protein digestibility is maintained at a higher level.

[0111] The above experiments demonstrate that the myofibrillar protein / luteolin complex prepared using the methods of the present invention undergoes a high-speed pre-homogenization process combined with high-pressure homogenization, causing the MP protein structure to undergo an unfolding-refolding and dispersion-aggregation process. This significantly improves the product's encapsulation efficiency and antioxidant properties, while also eliminating the need for the addition of chemical reagents, achieving a completely green production process. The results demonstrate that HPH is an effective method for improving luteolin encapsulation efficiency, helping to expand the application range of myofibrillar protein and having practical significance for promoting the industrialized production of meat protein foods.

[0112] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a myofibrillar protein / luteolin complex, characterized in that: include: Step a), dissolving myofibrillar protein in phosphate buffer to prepare a myofibrillar protein solution; The concentration of the myofibrillar protein solution is 8-12 mg / mL; Step b), uniformly dispersing luteolin in pure water by pre-homogenization to obtain a luteolin suspension; the concentration of the luteolin suspension is 60 to 200 μmol / g; there is no order restriction for steps a) and b); Step c), adding the luteolin suspension to the myofibrillar protein solution, mixing thoroughly, and then performing a pre-homogenization treatment to obtain a complex solution; The mixing ratio of luteolin and myofibrillar protein in the complex solution is (10-120) μmol:1g; Step d), the complex solution is subjected to high-pressure homogenization and then incubated to obtain a myofibrillar protein / luteolin complex.

2. The preparation method according to claim 1, wherein The pressure of the high-pressure homogenization treatment is 5000-25000 psi and the temperature is 4-10°C.

3. The preparation method according to claim 1, wherein The incubation temperature is 4-10°C and the incubation time is 30-60 min.

4. The preparation method according to claim 1, wherein The pre-homogenization treatment in step b) and step c) is specifically as follows: performing pre-homogenization twice using a high-speed homogenizer; setting the speed of the high-speed homogenizer to 6000-8000 rpm, and the treatment time of each pre-homogenization is 30-50 s.

5. The preparation method according to claim 1, characterized in that The myofibrillar protein in step a) is prepared as follows: The raw meat is removed from the surface muscular membrane and fat tissue, cut into pieces and minced to obtain minced meat; The minced meat was mixed with a standard salt solution, homogenized, filtered, and centrifuged to obtain a precipitate. The precipitate was mixed with a KCl solution, homogenized, filtered, and centrifuged to obtain myofibrillar protein.

6. A myofibrillar protein / luteolin complex prepared by the method according to any one of claims 1 to 5, characterized in that: It includes myofibrillar protein and luteolin embedded in the myofibrillar protein.

7. The myofibrillar protein / luteolin complex according to claim 6, characterized in that The encapsulation rate of luteolin in myofibrillar protein was greater than 95%.

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