Preparation method and application of polypeptide-perilla polyphenol compound
By performing multi-stage hydrolysis of food proteins and coupling them with perilla polyphenols to form polypeptide-perilla polyphenol complexes, the allergenicity problem of food proteins is solved and their food processing characteristics are improved.
Patent Information
- Application Number
- CN202510939333.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2025-11-21
AI Technical Summary
Allergenic proteins in food proteins can easily trigger allergic reactions, and current technologies are insufficient to effectively reduce their allergenicity and improve food processing characteristics.
Food proteins were subjected to continuous multi-stage hydrolysis using trypsin, papain, and bromelain to obtain polypeptides, which were then coupled with perilla polyphenols under alkaline conditions to form polypeptide-perilla polyphenol complexes.
It significantly reduced the allergenicity of the peptide-perilla polyphenol complex and improved its food processing properties, such as foaming properties, foam stability and emulsifying activity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of food processing, and particularly relates to a preparation method and application of a polypeptide-perilla polyphenol complex. BACKGROUND
[0002] Some food proteins contain allergenic proteins, which may cause severe allergic reactions such as anaphylactic shock after consumption. For example, the allergenic proteins contained in peanut protein mainly include Ara h 1, Ara h 2 and Ara h 3, etc. These proteins are prone to cause overreaction of the immune system due to their special structure and amino acid composition, thereby leading to the occurrence of allergic symptoms.
[0003] Perilla frutescens is a traditional medicinal plant with both food and medicinal uses, and is rich in various bioactive components. It has multiple pharmacological effects such as antioxidant, anti-inflammatory and immunomodulatory effects. Polyphenols are important active ingredients in Perilla frutescens, which have various biological activities such as antioxidant, anti-inflammatory and antibacterial activities, and can effectively scavenge free radicals to protect cells from oxidative damage. In addition, Perilla frutescens polyphenols have been studied for improving cardiovascular health, enhancing immunity and promoting metabolism, etc. Due to its good safety and multiple health benefits, Perilla frutescens polyphenols have attracted widespread attention in food, health products and drug research and development.
[0004] Therefore, it is of great significance to apply Perilla frutescens polyphenols to reduce the allergenicity of food proteins, so as to improve the safety, functionality and applicability of food proteins. SUMMARY
[0005] In view of this, the present application provides a preparation method and application of a polypeptide-Perilla frutescens polyphenol complex, which can reduce the allergenicity of edible proteins and improve the food processing characteristics of edible proteins.
[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides a preparation method of a polypeptide-Perilla frutescens polyphenol complex, comprising the following steps: S1, a food protein is sequentially hydrolyzed by trypsin, papain and bromelain to obtain a polypeptide; wherein the food protein contains allergenic proteins; S2, Perilla frutescens polyphenols are obtained; S3, the polypeptide and the Perilla frutescens polyphenols are coupled under alkaline conditions, and dried to obtain a polypeptide-Perilla frutescens polyphenol complex.
[0007] Preferably, in step S1, the food protein includes at least one of peanut protein, soybean protein isolate, whey protein and egg white protein; and / or, In step S3, the mass ratio of the polypeptide to the perilla polyphenol is (20-25):(1-5).
[0008] Preferably, step S1 comprises: Step S11, mixing food protein with water to obtain a food protein aqueous solution; Step S12, adding trypsin to the food protein aqueous solution for enzymolysis, inactivating the enzyme to obtain a first hydrolysate; Step S13, adding papain to the first hydrolysate for enzymolysis, inactivating the enzyme to obtain a second hydrolysate; Step S14, adding bromelain to the second hydrolysate for enzymolysis, inactivating the enzyme to obtain a third hydrolysate; Step S15, drying the third hydrolysate to obtain a polypeptide.
[0009] Preferably, in step S11, the mass fraction of food protein in the food protein aqueous solution is 2.5-4%; and / or, In step S12, the mass ratio of the food protein to the trypsin is 100:(1-2); and / or, In step S12, the pH value of enzymolysis is 7.5-8.0, the temperature of enzymolysis is 37°C, and the time of enzymolysis is 1-2 h; and / or, In step S13, the mass ratio of the first hydrolysate to the papain is 100:(0.5-1); and / or, In step S13, the pH value of enzymolysis is 6.0-7.0, the temperature of enzymolysis is 50-60°C, and the time of enzymolysis is 2-3 h; and / or, In step S14, the mass ratio of the second hydrolysate to the bromelain is 100:(0.5-1); and / or, In step S14, the pH value of enzymolysis is 6.0-7.0, the temperature of enzymolysis is 50-60°C, and the time of enzymolysis is 1-2 h.
[0010] Preferably, step S2 further comprises: S111, ultrasonic treatment of the food protein aqueous solution; wherein the power of ultrasonic treatment is 100-200 W, and the time of ultrasonic treatment is 10-15 min.
[0011] Preferably, step S2 comprises: S21, crushing perilla leaves, adding 70% ethanol aqueous solution, constant temperature water bath at 45-50°C, and extracting for 1-3 h to obtain an extraction liquid; wherein the mass ratio of the perilla leaves to the 70% ethanol aqueous solution is 1:(10-20). S22. The extract is filtered, and the filtrate is concentrated by rotary evaporation at 65~70℃ and dried to obtain perilla polyphenols.
[0012] Preferably, step S3 includes: S31. Mix the polypeptide with water to obtain a polypeptide solution; S32. Adjust the pH value of the polypeptide solution, add the perilla polyphenol, and carry out a coupling reaction to obtain a reaction solution; S33. Dialyze the reaction solution and dry it to obtain the polypeptide-perilla polyphenol complex.
[0013] Preferably, in step S31, the mass percentage of the polypeptide in the polypeptide solution is 4-5%; and / or, In step S32, the pH value is 8.5~9.5; and / or, In step S32, the coupling reaction temperature is 20~25℃, and the coupling reaction time is 6~8 h; and / or, In step S33, dialysis is performed using a dialysis bag with a pore size of 3.5 kDa, the dialysis solution is distilled water, the dialysis time is 12 h, and the dialysis solution is replaced every 4 h.
[0014] Secondly, the present invention provides a milkshake comprising the polypeptide-perilla polyphenol complex prepared by the aforementioned preparation method.
[0015] Preferably, the method for preparing the milkshake includes the following steps: Milk, sucrose, honey, and concentrated orange juice are stirred at 500-600 r / min for 5-6 min. Then, the polypeptide-perilla polyphenol complex is added, and the pH is adjusted to 4.8-6.5 with sodium citrate solution. The mixture is homogenized at 4000-6000 r / min until foam appears, and then sterilized at 120℃ for 3-5 s to obtain the milkshake.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention involves sequentially hydrolyzing food proteins containing allergenic proteins using trypsin, papain, and bromelain to obtain polypeptides. These polypeptides are then coupled with perilla polyphenols. By covalently modifying the polypeptides with perilla polyphenols, the allergenicity of the edible protein can be reduced, and the food processing characteristics of the edible protein can be improved, thereby obtaining a polypeptide-perilla polyphenol complex with lower allergenicity and better food processing characteristics. Attached Figure Description
[0017] Figure 1 The perilla polyphenol binding equivalents in the polypeptide-perilla polyphenol complex, peanut protein, and soy protein isolate provided in Examples 1-5 and Comparative Examples 1-3 of this invention; Figure 2 The binding capacity of the polypeptide-perilla polyphenol complex, peanut protein and soy protein isolate provided in Examples 1-5 and Comparative Examples 1-3 of this invention to IgG; Figure 3 The binding capacity of the polypeptide-perilla polyphenol complex, peanut protein and soy protein isolate to IgE provided in Examples 1-5 and Comparative Examples 1-3 of this invention; Figure 4 The foaming properties of the polypeptide-perilla polyphenol complex, peanut protein and soy protein isolate provided in Examples 1-5 and Comparative Examples 1-3 of the present invention; Figure 5 The foam stability of the polypeptide-perilla polyphenol complex, peanut protein and soy protein isolate provided in Examples 1-5 and Comparative Examples 1-3 of this invention; Figure 6 The emulsifying activity of the polypeptide-perilla polyphenol complex, peanut protein and soy protein isolate provided in Examples 1-5 and Comparative Examples 1-3 of this invention; Figure 7 This study examines the emulsification stability of the polypeptide-perilla polyphenol complex, peanut protein, and soy protein isolate provided in Examples 1-5 and Comparative Examples 1-3 of this invention. Detailed Implementation
[0018] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.
[0019] In a first aspect, the present invention provides a method for preparing a polypeptide-perilla polyphenol complex, comprising the following steps: S1. Food protein is hydrolyzed sequentially by trypsin, papain and bromelain to obtain polypeptides; wherein, the food protein contains allergenic proteins; S2. Obtain perilla polyphenols; S3. Under alkaline conditions, the polypeptide and the perilla polyphenol are coupled together and dried to obtain a polypeptide-perilla polyphenol complex.
[0020] This invention involves sequentially hydrolyzing food proteins containing allergenic proteins using trypsin, papain, and bromelain to obtain polypeptides. These polypeptides are then coupled with perilla polyphenols. By covalently modifying the polypeptides with perilla polyphenols, the allergenicity of the edible protein can be reduced, and the food processing characteristics of the edible protein can be improved. This results in a polypeptide-perilla polyphenol complex with lower allergenicity and better food processing characteristics.
[0021] It should be noted that the order of steps S1 and S2 is not specifically limited. Step S1 can be performed before step S2, after step S2, or steps S1 and S2 can be performed simultaneously.
[0022] Further, in step S1, the food protein includes at least one of peanut protein, soy protein isolate, whey protein, and egg white protein.
[0023] Further, in step S3, the mass ratio of the polypeptide to the perilla polyphenol is (20~25):(1~5).
[0024] Further, step S1 includes: Step S11: Mix food protein with water to obtain a food protein aqueous solution; Step S12: Add trypsin to the food protein aqueous solution, hydrolyze, and inactivate the enzyme to obtain the first hydrolysate; Step S13: Add papain to the first hydrolysate, hydrolyze, and inactivate the enzyme to obtain the second hydrolysate; Step S14: Add bromelain to the second hydrolysate, hydrolyze, and inactivate the enzyme to obtain the third hydrolysate; Step S15: Dry the third hydrolysate to obtain the polypeptide.
[0025] It should be noted that the inactivation in steps S12, S13 and S14 can be performed at 85°C for 10-15 minutes; in step S15: the third hydrolysate can be filtered before drying; the drying can be spray drying, and the inlet air temperature of the spray drying can be 160°C and the outlet air temperature can be 90°C.
[0026] Furthermore, in step S11, the mass percentage of food protein in the food protein aqueous solution is 2.5-4%.
[0027] Further, in step S12, the mass ratio of the food protein to the trypsin is 100:(1~2).
[0028] Furthermore, in step S12, the pH value of the enzymatic hydrolysis is 7.5~8.0, the temperature of the enzymatic hydrolysis is 37℃, and the time of the enzymatic hydrolysis is 1~2 h.
[0029] Further, in step S13, the mass ratio of the first hydrolysate to the papain is 100:(0.5~1).
[0030] Furthermore, in step S13, the pH value of the enzymatic hydrolysis is 6.0~7.0, the temperature of the enzymatic hydrolysis is 50~60℃, and the time of the enzymatic hydrolysis is 2~3 h.
[0031] Further, in step S14, the mass ratio of the second hydrolysate to the bromelain is 100:(0.5~1).
[0032] Furthermore, in step S14, the pH value of the enzymatic hydrolysis is 6.0~7.0, the temperature of the enzymatic hydrolysis is 50~60℃, and the time of the enzymatic hydrolysis is 1~2 h.
[0033] Furthermore, the procedure before step S12 includes: S111. The food protein aqueous solution is ultrasonically treated; wherein the ultrasonic power is 100~200 W and the ultrasonic treatment time is 10~15 min. Ultrasonic treatment of the food protein aqueous solution before adding trypsin for enzymatic hydrolysis is beneficial to improving the enzymatic hydrolysis effect of the food protein.
[0034] Further, step S2 includes: S21. Pulverize the perilla leaves, add 70% ethanol aqueous solution, and extract in a constant temperature water bath at 45~50℃ for 1~3 h to obtain an extract; wherein the mass ratio of the perilla leaves to the 70% ethanol aqueous solution is 1:(10~20). S22. The extract is filtered, and the filtrate is concentrated by rotary evaporation at 65~70℃ and dried to obtain perilla polyphenols.
[0035] Further, step S3 includes: S31. Mix the polypeptide with water to obtain a polypeptide solution; S32. Adjust the pH value of the polypeptide solution, add the perilla polyphenol, and carry out a coupling reaction to obtain a reaction solution; S33. Dialyze the reaction solution and dry it to obtain the polypeptide-perilla polyphenol complex.
[0036] It should be noted that in step S33, the drying can be spray drying, and the inlet air temperature of spray drying can be 160°C and the outlet air temperature can be 90°C.
[0037] Furthermore, in step S31, the mass percentage of the polypeptide in the polypeptide solution is 4-5%.
[0038] Furthermore, in step S32, the pH value is 8.5~9.5.
[0039] Furthermore, in step S32, the temperature of the coupling reaction is 20~25℃, and the time of the coupling reaction is 6~8 h.
[0040] Furthermore, in step S33, dialysis is performed using a dialysis bag with a pore size of 3.5 kDa, the dialysis solution is distilled water, the dialysis time is 12 h, and the dialysis solution is replaced every 4 h.
[0041] Secondly, the present invention provides a milkshake comprising the polypeptide-perilla polyphenol complex prepared by the aforementioned preparation method.
[0042] Furthermore, the method for preparing the milkshake includes the following steps: Milk, sucrose, honey, and concentrated orange juice are stirred at 500-600 r / min for 5-6 min. Then, the polypeptide-perilla polyphenol complex is added, and the pH is adjusted to 4.8-6.5 with sodium citrate solution. The mixture is homogenized at 4000-6000 r / min until foam appears, and then sterilized at 120℃ for 3-5 s to obtain the milkshake.
[0043] Furthermore, in the milkshake, the polypeptide-perilla polyphenol complex is a peanut peptide-perilla polyphenol complex. It should be noted that the peanut peptide-perilla polyphenol complex is prepared using peanut protein.
[0044] Furthermore, in the milkshake, the amount of peanut peptide-perilla polyphenol complex added is 8-15%, the amount of sucrose added is 3%, the amount of honey added is 5-8%, the amount of concentrated orange juice added is 2-5%, and the amount of sodium citrate added is 0.05-0.12%.
[0045] Example 1 A method for preparing a polypeptide-perilla polyphenol complex includes the following steps: S1. Prepare a 2.5% peanut protein aqueous solution and sonicate it at room temperature for 10 min with an ultrasonic power of 200 W. S2. The ultrasonically treated peanut protein aqueous solution was hydrolyzed with trypsin to inactivate the enzyme and obtain the first hydrolysate. The ratio of peanut protein to trypsin was 100:2, the pH value of the hydrolysis was 7.5, the hydrolysis temperature was 37℃, the hydrolysis time was 1 h, and the enzyme was inactivated by treatment at 85℃ for 10 min. S3. The first hydrolysate is hydrolyzed with papain to inactivate the enzyme and obtain the second hydrolysate. The ratio of the first hydrolysate to papain is 100:1, the pH value of the hydrolysis is 7.0, the hydrolysis temperature is 50℃, the hydrolysis time is 2 h, and the enzyme is inactivated by treatment at 85℃ for 10 min. S4. The second hydrolysate is hydrolyzed with bromelain to inactivate the enzyme and obtain the third hydrolysate. The ratio of the second hydrolysate to bromelain is 100:1, the pH value of the hydrolysis is 7.0, the hydrolysis temperature is 50℃, the hydrolysis time is 2 h, and the enzyme is inactivated by treatment at 85℃ for 10 min. S5. Filter the third hydrolysate and spray dry it to obtain polypeptide powder; wherein, the inlet air temperature of the spray dryer is 160℃ and the outlet air temperature is 90℃. S6. Pulverize the perilla leaves, add 70% ethanol at a material-to-liquid ratio of 1:20, and extract in a 50°C constant temperature water bath for 2 hours. Filter the extract, concentrate the filtrate by rotary evaporation at 70°C, and dry to obtain perilla polyphenols. S7. Prepare a 5% polypeptide solution from polypeptide powder, adjust the pH of the polypeptide solution to 9.0, add perilla polyphenols, and carry out a coupling reaction to obtain a reaction solution; wherein, the mass ratio of polypeptide to perilla polyphenols is 20:5, the temperature of the coupling reaction is 25℃, and the time of the coupling reaction is 6.5 h. S8. Dialyze the reaction solution and spray dry it to obtain the peanut peptide-perilla polyphenol complex; wherein the dialysis bag has a pore size of 3.5 kDa, the dialysis solution is distilled water, the dialysis time is 12 h, the dialysis solution is replaced every 4 h, the inlet air temperature of the spray dryer is 160℃, and the outlet air temperature of the spray dryer is 90℃.
[0046] Example 2 A method for preparing a polypeptide-perilla polyphenol complex, the steps are the same as in Example 1, except that: S1. Prepare a 4% peanut protein aqueous solution and sonicate it at room temperature for 15 min with an ultrasonic power of 100 W. S2. The ultrasonically treated peanut protein aqueous solution was hydrolyzed with trypsin to inactivate the enzyme and obtain the first hydrolysate. The ratio of peanut protein to trypsin was 100:1, the pH value of the hydrolysis was 8.0, the hydrolysis temperature was 37℃, the hydrolysis time was 2 h, and the enzyme was inactivated by treatment at 85℃ for 10 min. S3. The first hydrolysate is hydrolyzed with papain to inactivate it, and the second hydrolysate is obtained. The ratio of the first hydrolysate to papain is 100:0.5, the pH value of the hydrolysis is 6.0, the hydrolysis temperature is 60℃, the hydrolysis time is 3 h, and the enzyme is inactivated by treatment at 85℃ for 8 min. S4. The second hydrolysate is enzymatically hydrolyzed with bromelain to inactivate it, thus obtaining the third hydrolysate. The ratio of the second hydrolysate to bromelain is 100:0.5, the pH value of the enzymatic hydrolysis is 6.0, the temperature of the enzymatic hydrolysis is 60℃, the time of the enzymatic hydrolysis is 1 h, and the enzyme is inactivated by treatment at 85℃ for 8 min.
[0047] Example 3 A method for preparing a polypeptide-perilla polyphenol complex, the steps are the same as in Example 1, except that: S6. Pulverize the perilla leaves, add 70% ethanol at a material-to-liquid ratio of 1:10, and extract in a constant temperature water bath at 45℃ for 3 hours. Filter the extract, concentrate the filtrate by rotary evaporation at 65℃, and dry to obtain perilla polyphenols.
[0048] Example 4 A method for preparing a polypeptide-perilla polyphenol complex, the steps are the same as in Example 1, except that: S7. Prepare a 4% polypeptide solution from polypeptide powder, adjust the pH of the polypeptide solution to 9.5, add perilla polyphenols, and carry out a coupling reaction to obtain a reaction solution; wherein, the mass ratio of polypeptide to perilla polyphenols is 25:1, the temperature of the coupling reaction is 25℃, and the time of the coupling reaction is 8 h.
[0049] Example 5 A method for preparing a polypeptide-perilla polyphenol complex is the same as that in Example 1, except that in step S1, peanut protein is not used, but soy protein isolate is used.
[0050] Example 6 A method for preparing a milkshake includes the following steps: Milk, sucrose, honey and concentrated orange juice were stirred at low speed (600 r / min, 5 min) until homogenized. Then, the peanut peptide-perilla polyphenol complex prepared in Example 1 was added, and sodium citrate solution was added to adjust the acidity to pH 6.0. The mixture was homogenized at medium-high speed (5000 r / min) for 8 min and then sterilized at 120℃ for 5 s to obtain a milkshake containing peanut peptide-perilla polyphenol complex. In the milkshake, the added amount of peanut peptide-perilla polyphenol complex is 15%, the added amount of sucrose is 3%, the added amount of honey is 5%, the added amount of concentrated orange juice is 5%, and the added amount of sodium citrate is 0.1%.
[0051] Comparative Example 1 A method for preparing a polypeptide-perilla polyphenol complex is the same as that in Example 1, except that step S2 is not performed, i.e., the peanut protein is not hydrolyzed by trypsin.
[0052] Comparative Example 2 A method for preparing a polypeptide-perilla polyphenol complex is the same as that in Example 1, except that step S3 is not performed, i.e. the first hydrolysate is not hydrolyzed by papain.
[0053] Comparative Example 3 A method for preparing a polypeptide-perilla polyphenol complex is the same as that in Example 1, except that step S4 is not performed, i.e. the second hydrolysate is not hydrolyzed by bromelain.
[0054] Performance Tests and Results (1) Determination of the binding equivalent of perilla polyphenols in the polypeptide-perilla polyphenol complex The binding equivalent of perilla polyphenols in the peanut peptide-perilla polyphenol complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 3, and the soybean peptide-perilla polyphenol complex prepared in Example 5 were determined respectively; the results are shown in the figure. Figure 1 .
[0055] Depend on Figure 1 It can be seen that the binding equivalent of perilla polyphenols in the uncoupled peanut protein and uncoupled soy protein isolate (control) was 0. The binding equivalents of perilla polyphenols in the samples of Examples 1-5 and Comparative Examples 1-3 were 93.5 mg / kg, 87.5 mg / kg, 78.8 mg / kg, 87.8 mg / kg, 85.05 mg / kg, 65.2 mg / kg, 70 mg / kg, and 58.7 mg / kg of protein peptide, respectively. The binding equivalent of perilla polyphenols in the peanut peptide-perilla polyphenol complex prepared in Examples 1-4 was significantly higher than that in Comparative Examples 1-3. The binding equivalent of perilla polyphenols in the soy peptide-perilla polyphenol complex prepared in Example 5 was 85.05 mg / kg of protein peptide, indicating that perilla polyphenols effectively bind to soy peptides. This demonstrates that the polypeptide-perilla polyphenol complex prepared in this invention effectively binds to the polypeptide, and that sequential hydrolysis with trypsin, papain, and bromelain further enhances the coupling efficiency between the polypeptide and perilla polyphenols.
[0056] (2) Determine the binding capacity of the polypeptide-perilla polyphenol complex to IgG. The binding affinity of peanut peptide-perilla polyphenol complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 3, and soybean peptide-perilla polyphenol complex prepared in Example 5 to IgG was analyzed using a competitive inhibition enzyme-linked immunosorbent assay (ciELISA). The specific procedures were as follows: Protein solution (1 mg / mL) was coated onto 96-well microplates at 4°C for 12 h. After extensive washing with PBST buffer, blocking solution (PBST containing 2% fish gelatin) was used for 2 h at 37°C. Then, 50 μL of a pre-incubation mixture containing the sample (100 μg / mL) and a serum pool from allergic patients (diluted 1:30) was added to the plate and incubated at 37°C for 1 h. 100 μL of enzyme-labeled goat anti-human IgG (1:5000) was added, and the plate was incubated at 37°C for 1 h. Finally, 100 μL of TMB buffer was added, and the reaction was terminated with H2SO4. The absorbance was measured at 450 nm using a microplate reader. Results are shown in the figure. Figure 2 .
[0057] Depend on Figure 2It can be seen that the binding capacity of uncoupled peanut protein and uncoupled soy protein isolate to IgG in the control group was 100%. The binding capacities of the samples in Examples 1 to 5 and Comparative Examples 1 to 3 to IgG were 32.2%, 44.5%, 50.2%, 43.7%, 36%, 67%, 73.3%, and 55.4%, respectively. The binding capacity of the peanut peptide-perilla polyphenol complex prepared in Examples 1 to 4 to IgG was significantly lower than that in Comparative Examples 1 to 3, and significantly lower than that of peanut protein. The binding capacity of the soy peptide-perilla polyphenol complex prepared in Example 5 to IgG was significantly lower than that of soy protein isolate. This indicates that the allergenicity of the peptide-perilla polyphenol complex prepared in this invention is significantly lower than that of food protein, and that hydrolysis with trypsin, papain, and bromelain can reduce the binding capacity of the peptide-perilla polyphenol complex to IgG.
[0058] (3) Determine the binding ability of the polypeptide-perilla polyphenol complex to IgE. The binding affinity of peanut peptide-perilla polyphenol complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 3, and soybean peptide-perilla polyphenol complex prepared in Example 5 to IgE was analyzed using a competitive inhibition enzyme-linked immunosorbent assay (ciELISA). The specific procedures were as follows: Protein solution (1 mg / mL) was coated onto 96-well microplates at 4°C for 12 h. After extensive washing with PBST buffer, the plates were blocked with blocking solution (PBST containing 2% fish gelatin) at 37°C for 2 h. Then, 50 μL of a pre-incubation mixture containing the sample (100 μg / mL) and a serum pool from allergic patients (diluted 1:30) was added to the plates and incubated at 37°C for 1 h. 100 μL of enzyme-labeled goat anti-human IgE (1:2000) was added, and the plates were incubated at 37°C for 1 h. Finally, the plates were reacted with 100 μL of TMB buffer, and the reaction was terminated with H2SO4. The absorbance was measured at 450 nm using a microplate reader. The results are shown in the figure below. Figure 3 .
[0059] Depend on Figure 3 It can be seen that the binding capacity of uncoupled peanut protein and uncoupled soy protein isolate to IgE was 100%. The binding capacities of the samples in Examples 1 to 5 and Comparative Examples 1 to 3 to IgE were 43.8%, 48.8%, 48.7%, 55.5%, 58%, 71.3%, 65%, and 70.3%, respectively. The binding capacity of the peanut peptide-perilla polyphenol complex prepared in Examples 1 to 4 to IgE was significantly lower than that of Comparative Examples 1 to 3, and significantly lower than that of peanut protein. The binding capacity of the soy peptide-perilla polyphenol complex prepared in Example 5 to IgE was significantly lower than that of soy protein isolate. This indicates that the allergenicity of the peptide-perilla polyphenol complex prepared in this invention is significantly lower than that of food protein, and that hydrolysis with trypsin, papain, and bromelain can reduce the binding capacity of the peptide-perilla polyphenol complex to IgE.
[0060] (4) Analysis of the foaming properties and foam stability of the polypeptide-perilla polyphenol complex Peanut protein, soy protein isolate, peanut peptide-perilla polyphenol complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 3, and soy peptide-perilla polyphenol complexes prepared in Example 5 were dissolved in phosphate buffer (pH 7.0) to a concentration of 10 g / L. 100 mL of the solution was homogenized at 12000 r / min for 1 min. The foaming properties and foam stability of the solution were calculated as follows: Foaming property = V0 (foam volume in mL when homogenization stops) / V L (Volume of solution before stirring, mL) × 100%, foam stability = V 30 (Foam volume in mL after stirring and standing for 30 min) / V0 (Foam volume in mL when stirring stops) × 100%. See the foaming results below. Figure 4 The results of foam stability are shown in Figure 5 .
[0061] Depend on Figure 4 It can be seen that the foaming property of peanut protein is 42%, and that of soy protein isolate is 63.17%. The foaming properties of the samples in Examples 1 to 5 and Comparative Examples 1 to 3 are 57.8%, 49.3%, 48.3%, 49.7%, 83.67%, 38.8%, 45.17%, and 45%, respectively. The foaming properties of the peanut peptide-perilla polyphenol complex prepared in Examples 1 to 4 are significantly higher than those in Comparative Examples 1 to 3 and significantly higher than those of peanut protein. The foaming property of the soy peptide-perilla polyphenol complex prepared in Example 5 is significantly higher than that of soy protein isolate. This indicates that the foaming property of the peptide-perilla polyphenol complex prepared in this invention is significantly better than that of food protein, and that sequential hydrolysis with trypsin, papain, and bromelain can improve the foaming property of the peptide-perilla polyphenol complex.
[0062] Depend on Figure 5 It can be seen that the foam stability of peanut protein is 30.67%, and that of soy protein isolate is 35.33%. The foam stability of the samples in Examples 1 to 5 and Comparative Examples 1 to 3 are 45.50%, 28%, 37.83%, 30.67%, 57.50%, 31.33%, 31.00%, and 34.44%, respectively. The foam stability of the peanut peptide-perilla polyphenol complex prepared in Examples 1 to 4 is higher than that of Comparative Examples 1 to 3 and peanut protein. The foam stability of the soy peptide-perilla polyphenol complex prepared in Example 5 is significantly higher than that of soy protein isolate. This indicates that the foam stability of the peptide-perilla polyphenol complex prepared in this invention is significantly better than that of food protein, and that sequential hydrolysis with trypsin, papain, and bromelain can improve the foam stability of the peptide-perilla polyphenol complex.
[0063] (5) Analysis of emulsifying activity and emulsifying stability of polypeptide-perilla polyphenol complex Peanut protein, soy protein isolate, peanut peptide-perilla polyphenol complexes prepared in Examples 1 to 4 and Comparative Examples 1 to 3, and soy peptide-perilla polyphenol complexes prepared in Example 5 were dissolved in phosphate buffer (pH 7.0) to a concentration of 10 g / L. 4 mL of the solution was mixed with 1 mL of corn oil and homogenized at 12000 r / min for 2 min. 10 μL of the emulsion was thoroughly mixed with 1 mL of 0.1% (m / v) SDS solution, and the absorbance A0 of the sample was measured at 500 nm. The homogenized solution was allowed to stand for 10 min, and 10 μL of the emulsion was thoroughly mixed with 1 mL of 0.1% (m / v) SDS solution, and the absorbance A0 of the sample was measured at 500 nm. 10 The calculation methods for solution emulsifying activity and emulsifying stability are as follows: Emulsifying activity (m 2 / g) = 2 × 2.303 × A0 × 100 / (0.01 g / mL × 80%) × 10000, Emulsification stability (min) = A 10 / A0×100; Results of emulsification activity are shown in Figure 6 The results of emulsification stability are shown in Figure 7 .
[0064] Depend on Figure 6 It can be seen that the emulsifying activity of peanut protein is 18.5 m. 2 / g, the emulsifying activity of soy protein isolate is 21 m 2 / g, the emulsifying activity of the samples in Examples 1 to 5 and Comparative Examples 1 to 3 was 31.17 m. 2 / g、22.23 m 2 / g、28.33m 2 / g, 26.83 m 2 / g、33.4 m 2 / g, 24.51 m 2 / g、21 m 2 / g and 20 m 2 / g; The emulsifying activity of the peanut peptide-perilla polyphenol complexes prepared in Examples 1 to 4 was significantly higher than that of peanut protein. The emulsifying activity of Examples 1, 3, and 4 was higher than that of Comparative Examples 1 to 3. The emulsifying activity of the soybean peptide-perilla polyphenol complex prepared in Example 5 was significantly higher than that of soybean protein isolate. This indicates that the emulsifying activity of the peptide-perilla polyphenol complexes prepared in this invention is significantly better than that of food proteins, and that sequential hydrolysis with trypsin, papain, and bromelain can improve the emulsifying activity of the peptide-perilla polyphenol complexes.
[0065] Depend on Figure 7 It can be seen that the emulsification stability of peanut protein is 22.7 min, and that of soy protein isolate is 28.5 min. The emulsification stability of the samples in Examples 1 to 5 and Comparative Examples 1 to 3 are 40.8 min, 33.7 min, 44.3 min, 30 min, 42.5 min, 26.2 min, 25.7 min, and 23.7 min, respectively. The emulsification stability of the peanut peptide-perilla polyphenol complex prepared in Examples 1 to 4 is significantly higher than that of Comparative Examples 1 to 3 and peanut protein. The emulsification stability of the soy peptide-perilla polyphenol complex prepared in Example 5 is significantly higher than that of soy protein isolate. This indicates that the emulsification stability of the peptide-perilla polyphenol complex prepared in this invention is significantly better than that of food protein, and that sequential hydrolysis with trypsin, papain, and bromelain can improve the foam stability of the peptide-perilla polyphenol complex.
[0066] Conclusion: This invention utilizes trypsin, papain, and bromelain to sequentially hydrolyze food proteins in multiple stages, followed by covalent modification of the polypeptide with perilla polyphenols, to obtain a polypeptide-perilla polyphenol complex with low allergenicity and good food processing properties.
[0067] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.
[0068] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for preparing a polypeptide-perilla polyphenol complex, characterized in that, Includes the following steps: S1. Food protein is hydrolyzed sequentially by trypsin, papain and bromelain to obtain polypeptides; wherein, the food protein contains allergenic proteins; S2. Obtain perilla polyphenols; S3. Under alkaline conditions, the polypeptide and the perilla polyphenol are coupled together and dried to obtain a polypeptide-perilla polyphenol complex.
2. The method for preparing the polypeptide-perilla polyphenol complex according to claim 1, characterized in that, In step S1, the food protein includes at least one of peanut protein, soy protein isolate, whey protein, and egg white protein; and / or, In step S3, the mass ratio of the polypeptide to the perilla polyphenol is (20~25):(1~5).
3. The method for preparing the polypeptide-perilla polyphenol complex according to claim 1, characterized in that, Step S1 includes: Step S11: Mix food protein with water to obtain a food protein aqueous solution; Step S12: Add trypsin to the food protein aqueous solution, hydrolyze, and inactivate the enzyme to obtain the first hydrolysate; Step S13: Add papain to the first hydrolysate, hydrolyze, and inactivate the enzyme to obtain the second hydrolysate; Step S14: Add bromelain to the second hydrolysate, hydrolyze, and inactivate the enzyme to obtain the third hydrolysate; Step S15: Dry the third hydrolysate to obtain the polypeptide.
4. The method for preparing the polypeptide-perilla polyphenol complex according to claim 3, characterized in that, In step S11, the mass percentage of food protein in the food protein aqueous solution is 2.5% to 4%; and / or, In step S12, the mass ratio of the food protein to the trypsin is 100:(1~2); and / or, In step S12, the pH value for enzymatic hydrolysis is 7.5–8.0, the temperature for enzymatic hydrolysis is 37°C, and the time for enzymatic hydrolysis is 1–2 h; and / or, In step S13, the mass ratio of the first hydrolysate to the papain is 100:(0.5~1); and / or, In step S13, the pH value for enzymatic hydrolysis is 6.0–7.0, the temperature for enzymatic hydrolysis is 50–60°C, and the time for enzymatic hydrolysis is 2–3 h; and / or, In step S14, the mass ratio of the second hydrolysate to the bromelain is 100:(0.5~1); and / or, In step S14, the pH value of the enzymatic hydrolysis is 6.0~7.0, the temperature of the enzymatic hydrolysis is 50~60℃, and the time of the enzymatic hydrolysis is 1~2 h.
5. The method for preparing the polypeptide-perilla polyphenol complex according to claim 3, characterized in that, The steps preceding step S12 also include: S111. The food protein aqueous solution is ultrasonically treated; wherein the ultrasonic power is 100~200 W and the ultrasonic treatment time is 10~15 min.
6. The method for preparing the polypeptide-perilla polyphenol complex according to claim 1, characterized in that, Step S2 includes: S21. Pulverize the perilla leaves, add 70% ethanol aqueous solution, and extract in a constant temperature water bath at 45~50℃ for 1~3 h to obtain an extract; wherein the mass ratio of the perilla leaves to the 70% ethanol aqueous solution is 1:(10~20). S22. The extract is filtered, and the filtrate is concentrated by rotary evaporation at 65~70℃ and dried to obtain perilla polyphenols.
7. The method for preparing the polypeptide-perilla polyphenol complex according to claim 1, characterized in that, Step S3 includes: S31. Mix the polypeptide with water to obtain a polypeptide solution; S32. Adjust the pH value of the polypeptide solution, add the perilla polyphenol, and carry out a coupling reaction to obtain a reaction solution; S33. Dialyze the reaction solution and dry it to obtain the polypeptide-perilla polyphenol complex.
8. The method for preparing the polypeptide-perilla polyphenol complex according to claim 7, characterized in that, In step S31, the mass percentage of the polypeptide in the polypeptide solution is 4-5%; and / or, In step S32, the pH value is 8.5~9.5; and / or, In step S32, the coupling reaction temperature is 20~25℃, and the coupling reaction time is 6~8 h; and / or, In step S33, dialysis is performed using a dialysis bag with a pore size of 3.5 kDa, the dialysis solution is distilled water, the dialysis time is 12 hours, and the dialysis solution is replaced every 4 hours.
9. A milkshake, characterized in that, Including the polypeptide-perilla polyphenol complex prepared by the preparation method according to any one of claims 1 to 8.
10. The method for preparing a milkshake according to claim 9, characterized in that, Includes the following steps: Milk, sucrose, honey, and concentrated orange juice are stirred at 500-600 r / min for 5-6 min. Then, the polypeptide-perilla polyphenol complex is added, and sodium citrate solution is added to adjust the pH to 4.8-6.
5. The mixture is homogenized at 4000-6000 r / min until foam appears, and then sterilized at 120℃ for 3-5 s to obtain the milkshake.