Gamma-glutamyl peptide and application thereof in improving stability of anthocyanin

Abstract

The invention discloses gamma-glutamyl peptide and application thereof in improving the stability of anthocyanin. The gamma-glutamyl peptide comprises the following sequence: gamma-(Glu) x-Cys-(Glu) y-Cys-(Glu) z, x, y and z represent the number of glutamic acid residues, x is 1-3, y is 0-2, and z is 0-3. The gamma-glutamyl peptide is further applied to improve the stability of anthocyanin. The compound can be combined with anthocyanin in an acid solution to form a compound, the degradation rate of the anthocyanin can be effectively reduced through formation of the compound, the stability of the anthocyanin is improved, guarantee is provided for industrial application of the anthocyanin in various fields, and the compound has good application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to an oligopeptide and its application, in particular to a gamma-glutamyl peptide and its application in improving stability of anthocyanins. BACKGROUND

[0002] Anthocyanins are compounds combined by anthocyanidins and sugar groups (such as glucose, rhamnose, etc.) through glycosidic bonds, i.e. the form of "anthocyanidin + sugar", and the stability of anthocyanins is higher than that of anthocyanidins due to the protection of sugar groups. The difference of sugar groups in anthocyanins extracted from different plants is large, so the stability of anthocyanins from different sources also differs greatly. As a kind of natural pigment, anthocyanins have a variety of unique physiological activities, such as preventing heart disease, reducing blood sugar, anti-aging, anti-cancer and anti-inflammatory effects. However, anthocyanins from some plants are sensitive to environmental stimuli, have poor chemical stability and low bioavailability in human body, which limits their application in food processing.

[0003] Gamma-glutamyl peptides are a kind of special peptide compounds formed by dehydration condensation of the gamma-carboxyl group of glutamic acid and the amino group of other amino acids or peptides. The gamma-glutamyl bond (gamma-peptide bond) in its structure is different from the common alpha-peptide bond, which endows it with unique physicochemical properties and biological activities, such as antioxidant, anti-inflammatory and flavor-enhancing effects. However, there is no report on the application of gamma-glutamyl peptides to improve the stability of anthocyanins. SUMMARY

[0004] The purpose of the present application is to provide a gamma-glutamyl peptide capable of improving the stability of blueberry anthocyanins. Another purpose of the present application is to provide the application of the gamma-glutamyl peptide in improving the stability of anthocyanins, and to solve the problem of how to improve the stability of anthocyanins.

[0005] The gamma-glutamyl peptide comprises the following sequence:

[0006] Gamma-(Glu) x Cys-(Glu) y Cys-(Glu) z wherein x, y and z each represent the number of glutamic acid residues, x is 1-3, y is 0-2, and z is 0-3.

[0007] In the gamma-glutamyl peptide, glutamic acid forms a peptide bond with adjacent amino acids through the gamma-carboxyl group and the alpha-amino group.

[0008] Preferably, x+y+z = 3-7.

[0009] Preferably, x is 1-2, y is 1-2, and z is 1-2.

[0010] Another aspect of the present application discloses the use of the above-mentioned gamma-glutamyl peptide in improving the stability of anthocyanins.

[0011] The present application finds that inserting cysteine at a specific position and in a specific amount in the glutamic acid sequence can not only ensure the efficient binding of the gamma-glutamyl peptide to the glycosyl of anthocyanins, but also make the cysteine residues attached to the surface of anthocyanins, efficiently play the antioxidant role, avoid the rapid degradation of anthocyanins and glycosyl, and effectively improve the stability of anthocyanins in solution environment or solid powder.

[0012] The method for improving the stability of anthocyanins by using the above-mentioned gamma-glutamyl peptide comprises the following steps:

[0013] The gamma-glutamyl peptide and the anthocyanins are dissolved in water, and the pH is adjusted to be acidic to obtain a mixed solution.

[0014] Preferably, the mass ratio of the gamma-glutamyl peptide to the anthocyanins is 1-100:1.

[0015] Preferably, the method for adjusting the pH is to add citric acid to adjust the pH to 3-5.

[0016] Preferably, the final concentration of the gamma-glutamyl peptide in the mixed solution is 1-20 g / L, and the final concentration of the anthocyanins is 0.5-10 g / L.

[0017] The above-mentioned method further comprises the following steps:

[0018] The mixed solution is freeze-dried to obtain a gamma-glutamyl peptide-anthocyanin complex solid powder.

[0019] Preferably, the source of the anthocyanins is at least one of blueberry, purple sweet potato, black fruit medlar, black rice, purple cabbage, blackberry, black currant, mulberry, cherry, black corn, and purple perilla.

[0020] In some embodiments, the method for extracting anthocyanins from blueberries comprises:

[0021] After the blueberry freeze-dried powder is mixed with the DES system, the extraction liquid is obtained by oscillation extraction at 40-45°C, the solid impurities in the extraction liquid are removed to obtain supernatant, the supernatant is purified by using a macroporous resin, and the blueberry anthocyanins are obtained by freeze-drying after removing the elution solvent. The DES system can select a system containing 50% (V / V) of water, and the molar ratio of choline chloride to citric acid is 1:1.

[0022] Advantages: Compared with the prior art, the present application has the following obvious advantages:

[0023] The anthocyanin can be combined with the complex in an acidic solution, the complex can effectively reduce the degradation rate of the anthocyanin, improve the stability of the anthocyanin, provide protection for industrial application of the anthocyanin in various fields, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 UV-visible spectrum detection results of the combination of blueberry anthocyanin extract and No. 1 peptide;

[0025] Figure 2 Infrared FTIR detection results of the combination of blueberry anthocyanin extract and No. 1 peptide;

[0026] Figure 3 High performance liquid chromatography detection results of the combination of blueberry anthocyanin extract and No. 1 peptide. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be further described below in combination with the drawings.

[0028] Example 1: Screening of gamma glutamyl peptides capable of improving the stability of anthocyanin, the method is as follows:

[0029] (1) Chemically synthesize the gamma glutamyl peptides with the sequences shown in Table 1:

[0030] Table 1 Different gamma glutamyl peptide sequences

[0031]

[0032] The glutamic acid in the gamma glutamyl peptides in Table 1 forms a peptide bond with the adjacent amino acid through the gamma carboxyl and alpha amino group.

[0033] (2) Extraction and purification of anthocyanin, the method is as follows:

[0034] 5g of blueberry freeze-dried powder was weighed into a 200mL conical flask, and a DES system with a water content of 50%(volume fraction) was prepared using molar ratio of 1:1 of choline chloride and citric acid. The blueberry freeze-dried powder was mixed with the DES system according to the solid-liquid ratio of 1:6(g / mL), and extracted at 43℃ for 135min with oscillation. The solid impurities in the extraction liquid were removed by suction filtration, and the filtrate was centrifuged in a centrifuge at 8000r / min at 4℃ for 15min. The supernatant was taken and stored at 4℃ in the dark.

[0035] The collected supernatant was purified with a chromatographic column filled with AB-8 macroporous resin, and the loading amount was 1 / 2. After standing for 4 hours, the solvent and other impurities were eluted with distilled water.

[0036] The target sample solution was eluted with 75% ethanol, the eluent was evaporated at 40°C to remove ethanol, and the obtained concentrated solution was freeze-dried into powder to obtain blueberry anthocyanins, which were stored at -20°C for standby use.

[0037] (3) Preparation of γ-glutamyl peptide-anthocyanin complex, the steps are as follows:

[0038] The γ-glutamyl peptides in Table 1 were respectively dissolved in distilled water, citric acid was added to adjust the pH value of the solution to 4.0, and the solution was stirred with a magnetic stirrer until it was completely dissolved to obtain a γ-glutamyl peptide aqueous solution. Then the anthocyanins extracted in step (2) were added to the γ-glutamyl peptide aqueous solution, and the solution was again stirred with a magnetic stirrer until it was completely dissolved to obtain an acidic mixed solution.

[0039] The final concentration of polypeptides 1-12 in each acidic mixed solution was 0.015 mol / L, the final concentration of peptides 13-19 was 0.045 mol / L, and the final concentration of anthocyanins was 3 g / L. The blank control group was an anthocyanin solution without the addition of γ-glutamyl peptides.

[0040] After heat treatment (95°C, 30 min) of different acidic mixed solutions, cooling to room temperature, the degradation rate of anthocyanins was measured after the heat-treated acidic mixed solution samples were stored at 37°C under light for 7 days. The method for determining the stability of anthocyanins by high performance liquid chromatography is as follows:

[0041] Take 200 μL of the acidic mixed solution stored for 7 days, add 200 μL of 5% formic acid methanol solution and react for 15 min, filter through a 0.45 μm filter membrane, remove the precipitate, and analyze by high performance liquid chromatography.

[0042] Liquid phase conditions: flow rate 0.6 mL / min; injection volume 20 μL; elution gradient 0 min 10%, 30 min 60%, 33 min 100%, 35 min 100%, 37 min 10%, 40 min 10%. The formula for calculating the degradation rate of anthocyanins is:

[0043] Anthocyanin degradation rate = (initial solution peak area - solution peak area after 7 days of storage) / initial solution peak area.

[0044] The results are as follows:

[0045] Table 2 Effect of different γ-glutamyl peptides on the degradation rate of anthocyanins

[0046]

[0047]

[0048] From the results of Table 2, the anthocyanin degradation rate of the polypeptide group 1-3 was significantly lower than that of other polypeptide groups, indicating that polypeptide 1-3 can significantly improve the stability of anthocyanin, while polypeptides of other sequences do not have this effect.

[0049] The polypeptide 1 with the best anthocyanin stability improvement effect was used for subsequent complexation detection, and the method was as follows:

[0050] 1. UV absorption spectrum determination of complexation

[0051] Take 10 μL of anthocyanin aqueous solution (1.92 × 10 -4 g / mL) in the reaction plate, add different concentrations of 1 peptide PBS solution (1 × 10 -3 , 2 × 10 -3 , 3 × 10 -3 , 4 × 10 -3 , 5 × 10 -3 g / mL) 100 μL, take 10 μL of anthocyanin aqueous solution and 100 μL of phosphate buffer solution as control, and stand at 25℃ for 10 min. After reaction, scan the spectrum with the enzyme marker, and observe the change of the spectrum after combination. The results are shown in Figure 1 , Figure 1 Figure legends 1-5 represent 1 × 10 -3 , 2 × 10 -3 , 3 × 10 -3 , 4 × 10 -3 , 5 × 10 -3 g / mL, respectively.

[0052] Figure 1 Reflects the change of ultraviolet-visible spectrum of γ-glutamyl peptide and anthocyanin after interaction at 25℃. As can be seen from Figure 1 , γ-glutamyl peptide has a maximum absorption peak at 312 nm. The addition of anthocyanin makes the absorption peak appear near 354 nm, and with the increase of the concentration of anthocyanin (0, 50, 150, 250 mg / L and 350 mg / L), the solution becomes deeper red, the maximum absorption wavelength λ311 nm red shifts to λ320 nm, and λ354 nm red shifts to λ360 nm. The peak values of the two places are increased. This indicates that there is interaction between γ-glutamyl peptide and anthocyanin, and a complex may be formed.

[0053] 2. Infrared FTIR spectrum determination of complexation

[0054] The γ-glutamyl peptide-anthocyanin complex solution was prepared by mixing 0.2 g of the No. 1 peptide and 0.01 g of anthocyanin in deionized water. After freeze-drying for 48 h, the γ-glutamyl peptide-anthocyanin complex solid powder was obtained by grinding. The scanning range of the infrared spectrum was 4000-400 cm -1 . The results are shown in Table 1. Figure 1

[0055] The position, intensity, shape and other information of the absorption peaks in the infrared spectrum can be used to determine the characteristic groups contained in the molecule, thereby characterizing the composition and structure of the molecule. The amide I band (1600-1700 cm-1) of the protein is caused by the C=O stretching vibration, and the amide II band (1500-1600 cm –1 ) contains 40% C-N stretching vibration and 60% N-H bending vibration. The amide I band and the amide II band are often used for the analysis of the secondary structure of proteins. Although the structure of the peptide is simpler than that of the protein, the peptide also has a secondary structure in addition to the primary structure, and therefore the infrared spectrum can also be used to analyze the secondary structure of the peptide.

[0056] Figure 2 The characteristic peaks of the γ-glutamyl peptide are 1610, 1627, 1636 and 1655 cm -1 ; the characteristic peaks of the anthocyanin are 1627, 1657 and 1677 cm -1 ; and the characteristic peaks of the γ-glutamyl peptide-anthocyanin are 1630, 1637 and 1658 cm -1 . It is shown that the anthocyanin and the γ-glutamyl peptide interact with each other, and the two form a complex, which is consistent with the result of ultraviolet. Compared with the γ-glutamyl peptide alone, the absorption peak at 1610 cm -1 of the amide I band disappears after the addition of the anthocyanin, indicating that the content of the α-helix structure in the γ-glutamyl peptide-anthocyanin complex is reduced, which may be due to the combination of the hydroxyl group on the anthocyanin and the C=O group on the γ-glutamyl peptide through hydrogen bonding and hydrophobic interaction.

[0057] 3. Study on the combination of the anthocyanin extract and the γ-glutamyl peptide by high performance liquid chromatography

[0058] 100 μL of the anthocyanin aqueous solution with a concentration of 3 x 10 -3 g / mL was mixed with 100 μL of the No. 1 peptide aqueous solution with a concentration of 0.01 g / mL. After 15 min of reaction, 200 μL of 5% formic acid methanol solution was added for 15 min of reaction. After filtration through a 0.45 μm filter membrane and removal of the precipitate, liquid phase analysis was performed (100 μL of the anthocyanin solution was mixed with 100 μL of the phosphate buffer, and 200 μL of 5% formic acid methanol solution was added as a control group).

[0059] ​Liquid phase conditions: flow rate, 0.6 mL / min; injection volume, 20 μL; elution gradient, 10% at 0 min, 60% at 30 min, 100% at 33 min, 100% at 35 min, 10% at 37 min, and 10% at 40 min.

[0060] The results are as follows Figure 3 As shown by Figure 3 It can be seen that the peak area of ​​anthocyanins after binding to γ-glutamyl peptide has changed. Since anthocyanins and γ-glutamyl peptide bind to each other, the anthocyanins bound to γ-glutamyl peptide are filtered out after alcohol precipitation, resulting in a decrease in the anthocyanin peak area. Therefore, the change in peak area is used to analyze whether anthocyanins and γ-glutamyl peptide have bound to each other. A decrease in peak area indicates that a binding effect has occurred. Figure 3 The anthocyanin concentration decreased from 4379934 to 2128265 after the addition of γ-glutamyl peptide, indicating that anthocyanin interacted with γ-glutamyl peptide.

[0061] From Table 2 and Figures 1-3 The results show that peptide No. 1 combines with the sugar group of anthocyanin through H bonds to form a complex, which effectively inhibits the degradation of anthocyanin and can greatly improve the stability of anthocyanin. The peptide group No. 4-9 shows that changes in the location and number of cysteine ​​residues will directly inhibit the binding of polypeptides to anthocyanins, thereby affecting the stability improvement effect of the polypeptide. The peptide group No. 10-12 shows that the polypeptide relies on cysteine ​​residues to exert its degradation inhibition effect. When cysteine ​​is replaced by tryptophan or proline, the inhibitory effect disappears immediately. Peptides No. 13-19 are all dipeptides, tripeptides or tetrapeptides. They do not have a binding effect on sugar groups or a degradation inhibition effect, and therefore cannot improve the stability of anthocyanins.

[0062] Example 2: Peptide No. 1 in Example 1 was used to improve the stability of anthocyanins, as follows:

[0063] γ-glutamyl peptide No. 1 was dissolved in distilled water, citric acid was added to adjust the pH of the solution to 3.0, and the solution was stirred using a magnetic stirrer until fully dissolved to obtain a γ-glutamyl peptide aqueous solution. Anthocyanins extracted in step (2) of Example 1 were then added to the γ-glutamyl peptide aqueous solution, and the solution was stirred again using a magnetic stirrer until fully dissolved to obtain an acidic mixed solution. The final concentration of γ-glutamyl peptide No. 1 in the acidic mixed solution was 0.05 mol / L, and the final concentration of anthocyanins was 10 g / L.

[0064] Example 3: Peptide No. 1 in Example 1 was used to improve the stability of anthocyanins, as follows:

[0065] The No. 1 γ-glutamyl peptide was dissolved in distilled water, citric acid was added to adjust the pH value of the solution to 5.0, and the solution was stirred to complete dissolution using a magnetic stirrer to obtain a γ-glutamyl peptide aqueous solution. Then the anthocyanins extracted in step (2) of Example 1 were added to the γ-glutamyl peptide aqueous solution, and the solution was stirred to complete dissolution again using a magnetic stirrer to obtain an acidic mixed solution. The final concentration of the No. 1 γ-glutamyl peptide in the acidic mixed solution was 0.005 mol / L, and the final concentration of the anthocyanins was 1 g / L.

[0066] Example 4: The stability of anthocyanins was improved using the No. 1 peptide in Example 1, and the method was as follows:

[0067] The No. 1 γ-glutamyl peptide was dissolved in distilled water, citric acid was added to adjust the pH value of the solution to 4.0, and the solution was stirred to complete dissolution using a magnetic stirrer to obtain a γ-glutamyl peptide aqueous solution. Then the anthocyanins extracted in step (2) of Example 1 were added to the γ-glutamyl peptide aqueous solution, and the solution was stirred to complete dissolution again using a magnetic stirrer to obtain an acidic mixed solution. The final concentration of the No. 1 γ-glutamyl peptide in the acidic mixed solution was 0.02 mol / L, and the final concentration of the anthocyanins was 5 g / L.

[0068] Comparative Example 1: The rest were the same as Example 4, except that:

[0069] The blueberry anthocyanins extracted in step (2) of Example 1 were replaced with purple sweet potato anthocyanins, and the extraction method of the purple sweet potato anthocyanins was the same as that in step (2) of Example 1, except that the blueberry freeze-dried powder was replaced with purple sweet potato freeze-dried powder.

[0070] The degradation rate of anthocyanins was detected for Examples 2-4 and Comparative Example 1 using the degradation rate determination method in Example 1, and the results were as follows:

[0071] Table 3: Degradation inhibition effect of the No. 1 peptide on different anthocyanin extracts

[0072] Group 7d degradation rate (%) Example 2 45.6±6.3 Example 3 39.4±4.5 Example 4 26.3±3.8 Comparative Example 1 77.2±2.6 Purple sweet potato anthocyanin control group 95.8±2.1 Blueberry anthocyanin control group 97.8±1.4

[0073] As can be seen from the results of Table 3, the complexing ratio and concentration of anthocyanin extract and No. 1 peptide also have a greater impact on the action of polypeptide. Since the anthocyanin extract contains a variety of different anthocyanin monomers, and the types and proportions of anthocyanin monomers in anthocyanin extracts from different plant sources differ greatly, and the main difference between different types of anthocyanin monomers is the difference in sugar groups, the present application has confirmed that the combination of No. 1 peptide and anthocyanin mainly occurs on the sugar group, therefore, the difference in sugar groups in different types of anthocyanin monomers will directly affect the combination of No. 1 peptide and anthocyanin, and further affect its role in improving the stability of anthocyanin. As can be seen from the comparison of Example 4 and Comparative Example 1, although No. 1 peptide can greatly improve the stability of blueberry anthocyanin, its role in improving the stability of purple sweet potato anthocyanin is limited, because the types and proportions of anthocyanin monomers in blueberry anthocyanin and purple sweet potato anthocyanin differ greatly, and No. 1 peptide may only have good stability improvement effect on part of the characteristic monomers in blueberry anthocyanin.

Claims

1. A γ-glutamyl peptide, characterized in that The following sequences are included: γ-(Glu) x -Cys-(Glu) y -Cys-(Glu) z , where x, y, and z all represent the number of glutamic acid residues, with x ranging from 1 to 3, y ranging from 0 to 2, and z ranging from 0 to 3.

2. The γ-glutamyl peptide according to claim 1, characterized in that x+y+z=3~7.

3. The γ-glutamyl peptide according to claim 1, characterized in that x is 1-2, y is 1-2, and z is 1-2.

4. Use of the γ-glutamyl peptide according to any one of claims 1 to 3 in improving the stability of anthocyanins.

5. The use according to claim 4, characterized in that The steps include: The gamma-glutamyl peptide and anthocyanin are dissolved in water, and the pH is adjusted to acidic to obtain a mixed solution.

6. The use according to claim 5, characterized in that The mass ratio of the gamma-glutamyl peptide to anthocyanin is 1-100:

1.

7. The use according to claim 5, characterized in that The method for adjusting pH is to add citric acid to adjust pH to 3-5.

8. The use according to claim 5, characterized in that The final concentration of γ-glutamyl peptide in the mixed solution is 1-20 g / L, and the final concentration of anthocyanin is 0.5-10 g / L.

9. The use according to claim 5, characterized in that The following steps are also included: The mixed solution is freeze-dried to obtain a solid powder of the gamma-glutamyl peptide-anthocyanin complex.

10. The use according to claim 4, characterized in that The anthocyanin is sourced from at least one of blueberry, purple sweet potato, black wolfberry, black rice, purple cabbage, blackberry, black currant, mulberry, cherry, black corn, and perilla.

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