A high-stability protein-monascus pigment complex and preparation method thereof

Abstract

The present invention discloses a high-stability protein-monascus pigment complex and a preparation method thereof. The preparation method includes: 1) adding a hydrolytic protease containing glutamyl endopeptidase to a 0.5-2wt% protein solution for hydrolysis, the amount of the hydrolytic protease added makes the concentration of the hydrolytic protease in the protein solution 0.05-0.3wt%, and the pH value of the reaction system is guaranteed to be 7.0±0.2 during the hydrolysis process; 2) when the degree of hydrolysis is ≤10%, the pH value of the system is reduced to inactivate the enzyme to terminate the reaction, and then the pH value is adjusted to 7.0±0.5; 3) adding monascus pigment to the system obtained in step 2) so that the color value of the monascus pigment in the system is 0.1-50U / mL, and a protein-monascus pigment complex is obtained by combining. The present invention significantly improves the stability of monascus pigment, and effectively solves the problem that the color value is unstable and easy to decompose and fade when monascus pigment is applied to food coloring.

Description

Technical Field

[0001] The invention belongs to the field of pigments, and more specifically, relates to a high-stability protein-monascus pigment complex and a preparation method thereof. Background Art

[0002] Food coloring is a kind of food additive that can change or improve the color of food. It is now widely used in food processing and production. According to the properties of the pigment, food coloring is often divided into natural food coloring and artificial synthetic food coloring. In recent years, due to the doubts about the safety of artificial synthetic pigments, natural food coloring has been widely favored by consumers. Natural food coloring is a colorant separated from animals, plants, microorganisms and their metabolites. In addition to the ability to color food, this type of food coloring also has certain physiological activities such as antibacterial, blood pressure lowering, and antioxidant. At the same time, due to its high safety, natural pigments have received more and more attention. However, the disadvantages of poor stability and high price make it impossible for natural pigments to completely replace synthetic pigments. With the continuous improvement of people's living standards and the development of science and technology, it has become an inevitable trend for natural food pigments to replace artificial pigments in large quantities. At present, red yeast rice pigments, purple potato anthocyanins, and red date skin red pigments are all hot spots in the research of natural pigments.

[0003] Monascus pigment is a polyketide secondary metabolite secreted by Monascus purpurogenous fungi, including three types: Monascus red pigment, Monascus yellow pigment, and Monascus orange pigment. It is recognized as a food pigment with high safety. Studies have shown that in addition to its coloring ability, Monascus pigment also has high biological activities such as cholesterol-lowering, antibacterial, anti-cancer, anti-inflammatory, anti-tumor, anti-mutagenic and antioxidant. In addition, the main raw material for Monascus pigment fermentation is grain crops, which are easy to obtain and have a higher yield than general natural pigments, so it has better application prospects and higher commercial value. However, Monascus pigment has poor stability, especially at higher temperatures (>60°C), the degradation rate and amplitude are accelerated, which makes it easy to cause great color loss when the food containing Monascus pigment is subjected to traditional high-temperature sterilization treatment; at the same time, Monascus pigment has poor storage stability. During the storage process, the color of the food with Monascus pigment as the colorant will gradually become lighter, which makes the shelf life of the product short; at the same time, oxidants, pH and metal ions such as Fe 2+ and Zn 2+ etc. will also significantly affect the stability of Monascus pigment, which has obviously restricted the promotion and application of Monascus pigment in the food industry.

[0004] Whey protein isolate is a protein extracted from milk. It is a small, compact globular protein, of which the content of β-lactoglobulin is 55% to 61%, the content of α-lactalbumin is 19% to 22%, and the content of bovine serum albumin is 6% to 8%. Whey protein isolate has high protein content, low fat content, low cholesterol, is easy to digest by the human body, and has high nutritional value. It is called the "king of protein" by the academic community. In addition, whey protein isolate has excellent functional properties (foaming, emulsifying and gelling properties), and can act as a foaming agent, gelling agent, emulsifier, etc. in food, thereby improving the texture characteristics of food.

[0005] Purple potato protein is a type of protein derived from purple potatoes, and its content in purple potatoes is 0.012-0.1g / g dry matter. The amino acid composition pattern of purple potato protein meets the WHO / FAO recommended standards, and the essential amino acid content is higher than most other plant proteins. It has a high biological value and its nutritional value is comparable to that of milk and meat.

[0006] However, at present, the prior art has not yet added the above-mentioned protein to the monascus pigment to improve the stability of the monascus pigment. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems and provide a high-stability protein-monascus pigment complex and a preparation method thereof.

[0008] The inventors have found that the stability of natural pigments including red pigment can be significantly improved through the auxiliary coloring effect of auxiliary pigments. In addition to flavonoids, polyphenols, alkaloids, etc., proteins composed of amino acids are also an important type of auxiliary pigments. As protein is the main component of food, it is of great significance to study the stability of the complex composed of protein and red pigment.

[0009] In order to achieve the above object, the first aspect of the present invention provides a method for preparing a high-stability protein-monascus pigment complex, the preparation method comprising:

[0010] 1) adding a hydrolytic protease containing glutamyl endopeptidase to a 0.5-2 wt % protein solution for hydrolysis, wherein the amount of the hydrolytic protease added is such that the concentration of the hydrolytic protease in the protein solution is 0.05-0.3 wt %, and the pH value of the reaction system is ensured to be 7.0±0.2 during the hydrolysis process;

[0011] 2) Test the degree of hydrolysis of the protein solution. When the degree of hydrolysis is ≤10%, reduce the pH value of the system to inactivate the enzyme and terminate the reaction, and then adjust the pH value to 7.0±0.5;

[0012] 3) Adding monascus pigment to the system obtained in step 2) to make the color value of monascus pigment in the system be 0.1-50 U / mL, and obtaining a protein-monascus pigment complex through binding.

[0013] As a preferred embodiment of the present invention, the protein solution is a plant protein solution and / or an animal protein solution. The plant protein solution is more preferably selected from at least one of a purple potato protein solution, a soy protein solution and a gluten solution; the animal protein solution is more preferably selected from at least one of whey protein isolate, egg white protein and keratin.

[0014] As a preferred embodiment of the present invention, the monascus pigment is selected from at least one of monascus red pigment, monascus yellow pigment and monascus orange pigment.

[0015] As a preferred embodiment of the present invention, in step 2), the reaction is terminated when the degree of hydrolysis is 0.4-1.5%. The inventors have found that when the degree of hydrolysis of protein is 0.4-1.5%, the ability of protein to bind to Monascus pigment can be significantly or mostly increased, thereby increasing the binding amount.

[0016] As a preferred embodiment of the present invention, the content of glutamyl endopeptidase in the hydrolyzing protease is above 10 wt %.

[0017] As a preferred embodiment of the present invention, in step 3), the color value of the monascus pigment in the system is set to 0.1-10 U / mL.

[0018] As a preferred embodiment of the present invention, in step 3), the monascus pigment is added in the form of a monascus pigment solution, and the concentration of the monascus pigment solution is 0.01 to 0.5 U / μL.

[0019] As a preferred embodiment of the present invention, in step 1), before hydrolysis, the method further includes: keeping the protein solution in which the protein is completely dissolved at 40±2°C for 5 to 30 minutes, and then adjusting the pH of the system to 7.0±0.5. According to the present invention, under normal circumstances, the protein solution in which the protein is completely dissolved is obtained by mixing the protein with distilled water, stirring at room temperature for 1 to 5 hours, and then storing at 2 to 8°C.

[0020] In step 1), since the pH value of the system is constantly decreasing during the reaction, in order to ensure the reaction, the pH value of the system must be maintained at 7.0±0.2. As a preferred embodiment of the present invention, in step 1), the pH value of the system is maintained at 7.0±0.2 by continuously adding 0.1 to 0.5 mol / L of an alkaline solution to the system. The alkaline solution is an alkaline solution conventionally used by those skilled in the art, including but not limited to a NaOH solution.

[0021] As a preferred embodiment of the present invention, in step 2), the enzyme is inactivated by adjusting the pH value of the system to 3.4-3.9 and maintaining the temperature at 50-56° C. for 40-60 min.

[0022] As a preferred embodiment of the present invention, in step 2), the calculation formula of the hydrolysis degree is:

[0023] DH=(B×N b ×100%)×(1 / α) / (M p ×h tot ) Formula Ⅰ

[0024] In Formula I:

[0025] DH: degree of hydrolysis;

[0026] B: amount of alkali solution added, mL;

[0027] N b : alkali solution concentration, mol / L;

[0028] 1 / α: correction factor for protease hydrolysis including glutamyl endopeptidase;

[0029] M p : Protein content in the reaction system, g;

[0030] h tot : The total number of peptide bonds of protein in the reaction system, mmol / g protein.

[0031] As a preferred embodiment of the present invention, in step 3), the binding time is 2 to 8 hours, and the binding temperature is 40±5°C.

[0032] The second aspect of the present invention provides a highly stable protein-monascus pigment complex prepared by the above-mentioned preparation method.

[0033] Advantages and positive effects of the present invention:

[0034] The present invention significantly improves the stability of monascus pigment, and effectively solves the problem that the color value of monascus pigment is unstable and easy to decompose and fade when used for food coloring. During storage at 4°C in the dark, the stability of monascus pigment in the protein-monascus pigment complex of the present invention is significantly higher than the stability of the monascus pigment aqueous solution. At the same time, the present invention utilizes the ability of glutamyl endopeptidase to act on acidic amino acids (Glu / Asp), and performs hydrophobic enzymatic modification on the protein to enhance its surface hydrophobicity, thereby improving the ability to bind to monascus pigment.

[0035] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The stability curves of free Monascus red pigment, whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and the whey protein isolate-Monascus red pigment complex of Example 1 in a 60° C. water bath for 5 hours are shown;

[0037] Figure 2 The stability curves of free Monascus red pigment, whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and the whey protein isolate-Monascus red pigment complex of Example 1 after 5 hours in a 70° C. water bath;

[0038] Figure 3 The stability curves of free Monascus red pigment, whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and the whey protein isolate-Monascus red pigment complex of Example 1 after 80° C. water bath for 5 hours are shown;

[0039] Figure 4 The stability curves of free Monascus red pigment, a whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and a whey protein isolate-Monascus red pigment complex of Example 1 in a 90° C. water bath for 5 hours are shown;

[0040] Figure 5 The stability curves of free Monascus red pigment, a whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and the whey protein isolate-Monascus red pigment complex of Example 1 under storage conditions of 4° C. in the dark are shown;

[0041] Figure 6 The different concentrations of H 2 O 2 Stability curve diagram of the effects on free Monascus red pigment, whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and the whey protein isolate-Monascus red pigment complex of Example 1. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0043] Embodiment 1:

[0044] (1) preparing a 0.1 wt% whey protein isolate solution with distilled water, stirring at room temperature for 2 h, and then placing in a 4° C. refrigerator overnight to completely dissolve the whey protein isolate;

[0045] (2) placing the whey protein isolate solution in a 40°C water bath for 10 min and adjusting the pH to 7.0 with a NaOH solution;

[0046] (3) adding Alcalase 2.4L enzyme solution to the above whey protein isolate solution so that the concentration of Alcalase 2.4L enzyme in the whey protein isolate solution is 0.1wt%, and reacting at 40°C;

[0047] (4) 0.15 mol / L NaOH solution was continuously added dropwise to the reaction solution to maintain the pH value of the enzyme reaction system at 7.0. After the reaction was completed, the degree of hydrolysis (DH) of the whey protein isolate solution was calculated according to the following pH-stat method: DH = (B × N b ×100%)×(1 / α) / (M p ×h tot )

[0048] Note: B is the amount of alkali solution added (mL), N b is the concentration of alkali solution (mol / L), 1 / α is the correction factor of Alcalase enzyme, M p is the protein content in the reaction system (g), h tot is the total number of peptide bonds of the protein in the reaction system (mmol / g (protein));

[0049] (5) When the degree of hydrolysis is 0.5%, the pH value of the hydrolyzed whey protein isolate solution is adjusted to 3.5 with hydrochloric acid, and the solution is placed in a water bath at 53° C. for 40 min to inactivate the enzyme and terminate the reaction, and then the pH value is adjusted back to 7.0;

[0050] (6) Prepare 0.05 U / μL of Monascus red pigment;

[0051] (7) Add the monascus red pigment prepared in step (6) to the whey protein isolate solution so that the color value of the monascus red pigment in the system is 2 U / mL, and combine in a constant temperature water bath at 40° C. for 4 hours to obtain a whey protein isolate-monascus red pigment complex.

[0052] Embodiment 2:

[0053] (1) preparing a 2 wt % purple sweet potato protein solution with distilled water, stirring at room temperature for 4 h, and then placing in a 4° C. refrigerator overnight to completely dissolve the purple sweet potato protein;

[0054] (2) placing the purple potato protein solution in a 42°C water bath for 30 min and adjusting the pH to 7.5 with NaOH solution;

[0055] (3) adding Alcalase 2.4 L enzyme solution to the purple sweet potato protein solution to make the concentration of Alcalase 2.4 L enzyme in the purple sweet potato protein solution be 0.3 wt %, and reacting at 42° C.;

[0056] (4) 0.2 mol / L NaOH solution was continuously added to the above reaction solution to keep the pH value of the enzyme reaction system at 7.2. After the reaction was completed, the degree of hydrolysis (DH) of the purple sweet potato protein solution was calculated according to the following pH-stat method formula: DH = (B × N b ×100%)×(1 / α) / (M p ×h tot )

[0057] Note: B is the amount of alkali solution added (mL), N b is the concentration of alkali solution (mol / L), 1 / α is the correction factor of Alcalase enzyme, M p is the protein content in the reaction system (g), h tot is the total number of peptide bonds of the protein in the reaction system (mmol / g (protein));

[0058] (5) When the degree of hydrolysis is 1%, the pH value of the hydrolyzed purple sweet potato protein solution is adjusted to 3.9 with hydrochloric acid, and the solution is placed in a water bath at 53° C. for 60 min to inactivate the enzyme and terminate the reaction, and then the pH value is adjusted back to 7.5;

[0059] (6) Prepare 0.05U / μL Monascus yellow pigment solution;

[0060] (7) Add the monascus yellow pigment prepared in step (6) to the purple sweet potato protein solution so that the color value of the monascus yellow pigment in the system is 2 U / mL, mix thoroughly, and combine in a constant temperature water bath at 45° C. for 8 hours to obtain a purple sweet potato protein-monascus yellow pigment complex.

[0061] Test example:

[0062] In the test example, the amount of free Monascus red pigment, the relative amount of Monascus red pigment in the whey protein isolate-Monascus red pigment complex with a hydrolysis degree of 0 and the whey protein isolate-Monascus red pigment complex of Example 1 are the same.

[0063] Test Example 1:

[0064] The color value preservation rate of the whey protein isolate-monascus red pigment complex prepared in Example 1 at different temperatures and different storage times was tested. The results are as follows: Figure 1 to Figure 4 shown.

[0065] Depend on Figure 1 to Figure 4It can be seen that under heating conditions, the red pigment in free red pigment and whey protein isolate-red pigment complex is unstable. With the increase of temperature and the growth of treatment time, the color value preservation rate (A / A0, A0 is the absorbance of the solution before temperature treatment, A is the absorbance of the solution after temperature treatment) of the red pigment in free red pigment and whey protein isolate-red pigment complex is reduced to varying degrees, and the higher the temperature and the longer the time, the more the color value preservation rate of red pigment is reduced. However, at the same temperature and the same heating time, the color value preservation rate of red pigment in whey protein isolate-red pigment complex is significantly higher than the color value preservation rate of red pigment solution. This shows that red pigment will slow down the degradation rate under the auxiliary color effect of whey protein isolate, especially under high temperature conditions above 60°C, the auxiliary color effect of whey protein isolate plays a certain protective role in the temperature stability of red pigment.

[0066] Test Example 2:

[0067] The color value preservation rate of the whey protein isolate-monascus red pigment complex prepared in Example 1 was tested under the condition of 4°C in the dark and changed with time. The results are as follows: Figure 5 shown.

[0068] Depend on Figure 5 It can be seen that the color value preservation rate of free Monascus red pigment and Monascus red pigment in whey protein isolate-Monascus red pigment complex both decreases with the increase of storage time, but after the second day, the color value preservation rate of free Monascus red pigment gradually becomes significantly lower than the color value preservation rate of Monascus red pigment in whey protein isolate-Monascus red pigment complex, indicating that during this period, the stability of free Monascus red pigment is significantly lower than that of Monascus red pigment in whey protein isolate-Monascus red pigment complex. This shows that under the condition of 4°C storage in the dark, protein plays a certain protective role in the storage stability of Monascus red pigment.

[0069] Test Example 3:

[0070] The color value preservation rate of the whey protein isolate-monascus red pigment complex prepared in Example 1 was tested under the condition of 4°C in the dark and changed with time. The results are as follows: Figure 5 shown.

[0071] H 2 O 2 It is a strong oxidizing bleaching agent. It is generally believed that H 2 O 2 The principle of bleaching is that it can ionize hydrogen peroxide ions (HO 2 - ), and then react with the pigment to cause fading. 2 O 2It is stable, but adding a small amount of H 2 O 2 , due to H 2 O 2 The bleaching effect will still cause a small loss of pigment, resulting in a decrease in the color value preservation rate. Figure 6 It can be seen that after low concentration of H 2 O 2 After treatment with 0-1 mg / mL of H2O, the color value preservation rates of whey protein isolate-monascus red pigment complex (DH=0), whey protein isolate-monascus red pigment complex (DH=0.5%) and monascus red pigment solution were all above 95%, indicating that low concentrations of H2O 2 O 2 The effect on the stability of Monascus pigment is small. However, under the same conditions, the color value preservation rate of whey protein isolate-Monascus pigment complex (DH=0) and whey protein isolate-Monascus pigment complex (DH=0.5%) is greater than the color value preservation rate of Monascus pigment solution.

[0072] Table 1 shows the binding constants of whey protein isolate with different hydrolysis degrees and monascus red pigment.

[0073] Table 1

[0074]

[0075] As shown in Table 1, with the increase of the hydrolysis degree of whey protein isolate, the binding constant KA shows a trend of first increasing and then decreasing, and the binding capacity is strongest when the hydrolysis degree is about 0.50%.

[0076] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing a high-stability whey protein isolate-monascus pigment complex, characterized in that: The preparation method comprises: 1) adding a protease containing glutamyl endopeptidase to a 0.5-2wt% whey protein isolate solution for hydrolysis, wherein the amount of the protease added is such that the concentration of the protease in the whey protein isolate solution is 0.05-0.3wt%, and during the hydrolysis process, 0.1-0.5mol / L of an alkaline solution is continuously added dropwise to the system to ensure that the pH value of the reaction system is 7.0±0.2; the protease containing glutamyl endopeptidase is Alcalase 2.4L enzyme; 2) Testing the degree of hydrolysis of the whey protein isolate solution, when the degree of hydrolysis is 0.4-0.5%, lowering the pH value of the system to 3.4-3.9, and maintaining the temperature at 50-56° C. for 40-60 min to inactivate the enzyme and terminate the reaction, and then adjusting the pH value to 7.0±0.5; 3) Adding monascus pigment to the system obtained in step 2) to make the color value of monascus pigment in the system be 0.1-10 U / mL, and obtaining a protein-monascus pigment complex through binding.

2. The preparation method according to claim 1, wherein The monascus pigment is selected from at least one of monascus red pigment, monascus yellow pigment and monascus orange pigment.

3. The preparation method according to claim 1, wherein The content of glutamyl endopeptidase in the hydrolyzing protease is above 10wt%.

4. The preparation method according to claim 1, wherein In step 3), the monascus pigment is added in the form of a monascus pigment solution, and the color value of the monascus pigment solution is 0.01 to 0.5 U / μL.

5. The preparation method according to claim 1, wherein In step 1), before hydrolysis, the method further comprises: maintaining the whey protein isolate solution in which the protein is completely dissolved at 40±2° C. for 5 to 30 minutes, and then adjusting the pH of the system to 7.0±0.

5.

6. The preparation method according to claim 1, wherein In step 2), the calculation formula of the hydrolysis degree is: DH = (B × N b × 100%) × (1 / α) / (M p × h tot ) Equation Ⅰ In Formula I: DH: degree of hydrolysis; B: amount of alkali solution added, mL; N b : alkali solution concentration, mol / L; 1 / α: correction factor for protease hydrolysis including glutamyl endopeptidase; M p : Protein content in the reaction system, g; h tot : The total number of peptide bonds of the protein in the reaction system, mmol / g protein.

7. The preparation method according to claim 1, wherein In step 3), the binding time is 2 to 8 hours and the binding temperature is 40±5°C.

8. A highly stable whey protein isolate-monascus pigment complex prepared by the preparation method according to any one of claims 1 to 7.

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