Application of mannoprotein as protective agent of anthocyanin of oriental cherry

Abstract

The invention discloses an application of mannoprotein as a protective agent of anthocyanin of oriental cherry and a color protection method of anthocyanin of oriental cherry. According to the preparation method, a non-covalent bond binding mechanism can be formed by the mannoprotein and the anthocyanin of the oriental cherry, and the structural stability of the anthocyanin of the oriental cherry is maintained by the mannoprotein, so that the light stability, the heat stability and the antioxidant stability of the anthocyanin of the oriental cherry are improved. According to the method, the problem that the anthocyanin of the oriental cherry is easy to oxidize and fade is solved, and the application field of the anthocyanin of the oriental cherry in research and development of food and beverage products is widened.

Description

Technical Field

[0001] The invention relates to a technology for protecting anthocyanins of Sakura guanshanensis by utilizing mannoprotein based on the anthocyanin-mannoprotein covalent binding principle. Background Art

[0002] Cherry blossoms (Cerasua serrulata) are plants of the genus Cerasus in the family Rosaceae, and are cultivated all over the world. Cherry blossoms have high ornamental value. They have beautiful plant shapes, bright colors, and strong adaptability. They are highly ornamental and are widely used in greening parks, schools, streets, courtyards, etc.

[0003] Guanshan cherry (Prunus serrulata) is a representative variety of Yaezakura, widely planted in Zhejiang, Sichuan and Shandong, China. The flowering period is late March or early April, with flowers and leaves blooming at the same time, showing white to pink. In addition to being ornamental, cherry blossoms can also be served on the table as a delicacy. They can be processed into cherry blossom tea, cherry blossom wine, and cherry blossom cakes for people to eat. Cherry blossoms can also be used as medicine. Cherry leaves and cherry blossoms are traditional Chinese medicinal materials with the effects of relieving cough, relieving asthma, moistening the intestines, and sobering up.

[0004] The pigment of Guanshan cherry is mainly composed of anthocyanin. Anthocyanin is a polyphenol and a water-soluble pigment commonly found in mulberries, grapes, berries and other fruits and vegetables. Anthocyanin is formed by anthocyanidins in a free state in nature and glycosyl ligands. It is a secondary metabolite of higher plants and the main coloring substance of plant petals and fruits. Anthocyanin is the glycoside form of anthocyanidins, which differs in the number of methoxyl groups and / or hydroxyl groups and the type and number of sugar parts attached to the aglycone structure. The most common sugar groups (glucose, galactose, arabinose, rutinose, rhamnose and xylose) are combined with anthocyanins in the form of monosaccharides, disaccharides or trisaccharides. The basic structure of anthocyanin is 3,5,7-trihydroxy-2-phenylbenzopyran, which has a typical C6-C3-C6 carbon skeleton structure and can be combined with one or more sugar molecules through glycosidic bonds. Due to the differences in the number and position of hydroxylation, methylation and acylation of anthocyanins, as well as the nature, position and number of sugars present in their structures, there are many types of anthocyanins. It is reported that more than 550 anthocyanins have been identified so far, among which cyanidin, pelargonidin, delphinidin, peonydin, morning glorydin and malvidin are the six most common ones in the edible parts of plants.

[0005] Due to the unique properties of anthocyanins, they are increasingly used as a bioactive compound and functional food. However, the instability of Guanshan cherry anthocyanins has seriously affected the application of cherry blossoms in food, beverages, daily chemicals and other products. Generally, cherry blossom anthocyanins fade significantly within 6 hours under sunlight conditions and within 1 hour under traditional sterilization heat treatment conditions, which limits the application of Guanshan cherry anthocyanins in food processing.

[0006] In order to solve the problem of low stability of anthocyanins, some studies have used the interaction between proteins and anthocyanins to improve the physical and chemical stability of anthocyanins. However, the anthocyanins of Guanshan cherry blossoms contain unique ingredients, and their protective agents need to be further developed. Summary of the invention

[0007] The technical purpose of the present invention is to construct a Prunus guanshanensis anthocyanin-mannoprotein complex to improve the stability of Prunus guanshanensis anthocyanin under light and heat treatment.

[0008] The technical solution adopted by the present invention is:

[0009] Application of mannoprotein as a protective agent for anthocyanins of Prunus guanshanensis.

[0010] Furthermore, the application method is: adding mannoprotein as a protective agent to the raw material containing the relevant mountain cherry anthocyanins.

[0011] The anthocyanins of the cherry blossoms can be obtained by extracting and purifying the cherry blossoms, and the anthocyanins of the cherry blossoms can be obtained by using techniques known in the art. Further, an acidified ethanol solution can be generally used as an extraction solvent to extract the cherry blossoms to obtain a crude extract. The crude extract can be purified by using a macroporous resin to obtain the anthocyanins of the cherry blossoms.

[0012] The raw materials containing the anthocyanins of Prunus guanosinica can be solid or liquid food raw materials, liquid commodity raw materials such as beverages, wine, etc., into which the anthocyanins of Prunus guanosinica are added.

[0013] The present invention also provides a color protection method for anthocyanins from Prunus guanshanensis, which comprises adding mannoprotein as a protective agent to a raw material containing anthocyanins from Prunus guanshanensis.

[0014] In the method, the mass ratio of the anthocyanins of Prunus guanshanensis to mannoprotein is 100:1-5, preferably 100:2.

[0015] Furthermore, the method is preferably as follows: mannoprotein is added as a protective agent to the raw material containing the anthocyanins of the relevant wild cherry, and the pH value of the system is adjusted to 3-5, preferably the pH value of the system is adjusted to 3. In an acidic environment, mannoprotein has a better color protection effect on the anthocyanins of the wild cherry.

[0016] Furthermore, in the method, the added concentration of the anthocyanins of Prunus guanshanensis in the raw material is 0.1-100 mg / mL, and a suitable concentration can be added according to the requirements of the food formula.

[0017] The present invention also provides a method for improving the thermal stability and light stability of the anthocyanin of the cherry blossoms, the method comprising: adding mannoprotein as a stabilizer to a raw material containing anthocyanin of the cherry blossoms, and adjusting the pH value of the system to 3 to 5. Mannoprotein can improve the thermal stability and light stability of the anthocyanin of the cherry blossoms, maintain the structural stability of the anthocyanin of the cherry blossoms, so that the anthocyanin of the cherry blossoms is not easy to change color during the processing and storage of food, thereby improving its usability as a food additive.

[0018] The present invention also provides a protective agent for anthocyanins of Prunus guanshanensis, wherein the protective agent is mannoprotein.

[0019] The mannoprotein and the anthocyanin of Prunus guanshanensis are bound by non-covalent bonds, and the binding forces are mainly hydrogen bonds and hydrophobic forces.

[0020] Furthermore, the binding sites of mannoprotein are Ser43, Ser44, Asp321, Glu215, Lys217, Val225, Val226, Ile323, Ala54, Glu281, Asn283, Asp290, Tyr316, Gln318, Glu51, Val53 and Asp224, thereby improving the light stability and thermal stability of the anthocyanins of the Guanshan cherry tree.

[0021] The anthocyanins of Guanshan cherry are a mixture of various glycosides, the main components of which include petunidin-3,5-O-diglucoside (PD) and cyanidin-3-O-(6”-malonyl glucoside).

[0022] The beneficial effects of the present invention are:

[0023] The present invention constructs a complex of anthocyanin-mannoprotein of cherry blossoms, and improves the stability of anthocyanin of cherry blossoms under conditions of light and heat treatment. Under conditions of light-proof and room temperature, the retention rate of anthocyanin of cherry blossoms by mannoprotein is increased by 53.1% within 15 days of storage. Under heat treatment at 85°C, the retention rate of anthocyanin of cherry blossoms by mannoprotein can be increased by up to 22.8%. Under ultraviolet light irradiation, the retention rate of anthocyanin of cherry blossoms by mannoprotein is increased by 35%. The present invention also determines the optimal pH conditions for the construction of the complex. Under the condition of pH 3.0, the retention rate of anthocyanin of cherry blossoms by mannoprotein is the best, and the retention rate reaches 87.27%. The present invention utilizes the binding mechanism that mannoprotein and anthocyanin of cherry blossoms can form non-covalent bonds with each other, and the solution containing anthocyanin of cherry blossoms is mixed with the mannoprotein solution to maintain the structural stability of anthocyanin of cherry blossoms, thereby improving the light, heat and antioxidant stability of anthocyanin. The invention solves the problem that anthocyanins of Prunus guanshanensis are easily oxidized and faded, and broadens the application field of anthocyanins of Prunus guanshanensis in the research and development of food and beverage products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The LC-MS results of anthocyanins from Sakura guanshanensis, where Figure A is petunidin-3,5-O-diglucoside (PD); Figure B is cyanidin-3-O-(6”-malonyl glucoside) (CD).

[0025] Figure 2 This is the anthocyanin retention rate result of the Guanshan cherry anthocyanin monomer solution and the Guanshan cherry anthocyanin-mannoprotein mixture stored in the dark at room temperature for 15 days.

[0026] Figure 3 This is the result graph of anthocyanin retention rate under 85℃ heat treatment conditions for the anthocyanin monomer solution of Prunus guanshanensis and the anthocyanin-mannoprotein mixed solution of Prunus guanshanensis.

[0027] Figure 4 This is the result graph of anthocyanin retention rate of the Guanshan cherry anthocyanin monomer solution and the Guanshan cherry anthocyanin-mannoprotein mixed solution under ultraviolet light conditions.

[0028] Figure 5 The graph shows the anthocyanin retention rate results of the anthocyanin monomer solution of Prunus guanshanensis and the anthocyanin-mannoprotein mixture of Prunus guanshanensis under different pH conditions.

[0029] Figure 6 These are the fluorescence spectra of the interaction between mannoprotein (MP) and PD and CD, where Figure A is the MP-PD complex and Figure B is the MP-CD complex.

[0030] Figure 7 This is the Fourier infrared spectrum of the interaction between mannoprotein (MP) and CD and PD.

[0031] Figure 8 It is the Ramachandran diagram of mannoprotein homology.

[0032] Fig. 9 It is a 3D diagram of molecular docking of MP-PD and MP-CD complex, where Figure A is the MP-PD binding site; Figure B is the 3D schematic diagram of MP-PD docking; Figure C is the MP-CD binding site; and Figure D is the 3D schematic diagram of MP-CD docking.

[0033] Fig.10 The 3D diagram of molecular docking of PD-MP and CD-MP complexes, where Figure A is the PD-MP complex and Figure B is the CD-PD complex. DETAILED DESCRIPTION

[0034] In order to make the invention purpose, technical scheme and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, not for limiting the present invention. The experimental materials and reagents used, unless otherwise specified, are all consumables and reagents that can be obtained from commercial channels.

[0035] Example 1: Identification of anthocyanin composition of Prunus guanshanensis

[0036] 1. Sample pretreatment

[0037] Extraction of anthocyanins from cherry blossoms

[0038] Weigh 10 g of sieved Sakura guanyin powder and add 600 mL of acidified ethanol solution (adjust the pH of ethanol to 3 with hydrochloric acid, and the ethanol concentration is 80%). Stir evenly and extract by ultrasonic for 1 hour, centrifuge to obtain the extract. Rotate and evaporate the extract at 38°C to obtain the crude extract of anthocyanins for later use.

[0039] Purification of anthocyanins from Prunus guanshanensis

[0040] Add X-AB macroporous resin to the crude anthocyanin extract in a conical flask at a ratio of 1:100 (g / mL). Oscillate at 100 rpm for 12 hours at 25°C until adsorption equilibrium is reached. Centrifuge at low temperature (4°C, 10000 rpm / min, 10 min) and filter the supernatant through a 0.22 μm organic filter for later use. Freeze-dry at a final concentration of 20%-30%.

[0041] 2. Liquid chromatography parameters

[0042] Chromatographic column: Waters HSS T3 (100×2.1mm, 1.8μm); mobile phase: phase A is ultrapure water solution (containing 0.1% formic acid), phase B is acetonitrile solution (containing 0.1% formic acid); flow rate 0.3mL / min; column temperature 40℃; injection volume 2μL; elution gradient: 0min A phase / B phase (95:5, v / v), 2min A phase / B phase (95:5, v / v), 15min A phase / B phase (70:30, v / v), 15.1min A phase / B phase (5:95, v / v), 20min A phase / B phase (5:95, v / v), 20.1min A phase / B phase (95:5, v / v), 26min A phase / B phase (95:5, v / v).

[0043] 3. Mass spectrometry parameters

[0044] The Q Exactive HFX high-resolution mass spectrometer system from Thermo Corporation of the United States was used to collect primary and secondary spectra. It was equipped with an electrospray ionization (ESI) source, sheath gas 40arb, auxiliary gas 10arb, ion spray voltage +3000V, temperature 350℃, and ion transfer tube temperature 320℃. The scanning mode was FSMS2, and the scanning mode was positive ion. The primary mass spectrometer scan range was (scan m / z range): 200-700Da.

[0045] 4. Identification results

[0046] Figure 1 As shown, the molecular formula of the compound was characterized by identifying the molecular ion and comparing it with the literature. In addition, the isotope peak abundance and fragmentation pattern of the molecular ion ([M]+) were investigated to determine the structure and characteristics of typical anthocyanins. After purification, two anthocyanins with higher contents were identified. They are Petunidin 3,5-O-diglucoside (PD), with a content of 25.57%; and Cyanidin-3-O-(6-O-malonyl-beta-D-glucoside); CD), with a content of 12.48%..

[0047] Example 2: Determination of the retention rate of anthocyanins in Guanshan cherry

[0048] 1. Determination of anthocyanin content

[0049] The anthocyanin content was determined by the pH differential method as follows: the anthocyanin solution was added to two test tubes separately, and pH 1.0 buffer (accurately weighed 1.86g KCl into a 2000mL beaker, added about 980mL of water to dissolve, first added 6mL of concentrated hydrochloric acid, then adjusted the pH to 1.0 with a 2mol / L dilute hydrochloric acid solution and then fixed the volume to 1L for storage) and 4mL of pH 4.5 (accurately weighed 54.43g sodium acetate into a 2000mL beaker, added about 960mL of water to dissolve, first added 19mL of concentrated hydrochloric acid, then adjusted the pH to 4.5 with a 2mol / L dilute hydrochloric acid solution and then fixed the volume to 1L for storage) buffer were added, and the solution was kept away from light for 10 minutes, and the absorbance at 520nm and 700nm was detected respectively, and the measurement was repeated 3 times. The absorbance value was calculated according to the following formula:

[0050] A=(A 520 -A 700 ) pH1.0 -(A 520 -A 700 ) pH4.5

[0051] Anthocyanin content (C):

[0052] C(mg / mL)=(A×DF×MW×V) / (ε×1×m×10)

[0053] Where: DF: dilution factor;

[0054] MW: relative molecular weight of anthocyanin;

[0055] V: system volume;

[0056] ε: average molar extinction coefficient, which is 26900 in the present invention;

[0057] m: sample mass.

[0058] 2. Calculation of anthocyanin retention rate

[0059] The calculation formula of anthocyanin retention rate is as follows:

[0060] Anthocyanin retention rate (%) = (C t / C0)×100%

[0061] Where: C t : The content of anthocyanins under target conditions (target time, target temperature, target light, etc.).

[0062] C0: Anthocyanin content under initial conditions (initial time, initial temperature, initial light, etc.).

[0063] Example 3: Effect of mannoprotein on the retention rate of anthocyanins in Cherry Blossoms of Guanshan during a 15-day storage period 1. Sample preparation

[0064] Citric acid buffers of pH 3.0 and pH 6.6 were prepared respectively, and the purified anthocyanin from Sakura guanyin was dissolved in the citric acid buffer of pH 3.0 to obtain an anthocyanin solution; mannoprotein was dissolved in the citric acid buffer of pH 6.6 to obtain a mannoprotein solution. The anthocyanin solution and the mannoprotein solution were then mixed to obtain a mixed solution sample with a final concentration of 5 mg / mL anthocyanin and 0.1 mg / mL mannoprotein, and a pH value of 4.5.

[0065] 2. Anthocyanin retention rate detection during 15-day storage period

[0066] Pipette 10 mL of 5 mg / mL anthocyanin solution as a reference, and pipette 10 mL of the above mixed solution sample. After repeated shaking, store the sample at room temperature in a dark place for 15 days. Starting from the third day, samples are taken every 2 days to determine the anthocyanin content.

[0067] 3. Analysis results

[0068] Figure 2 As shown:

[0069] After 3 days of storage, the retention rate of cherry blossom anthocyanins in the anthocyanin solution was 41.92%; the retention rate of anthocyanins in the mixed solution was 46.47%. The retention rate increased by 10.85%.

[0070] After 5 days, the retention rate of cherry anthocyanins in the anthocyanin solution was 24.54%; the retention rate of anthocyanins in the mixed solution was 27.24%. The retention rate increased by 11%.

[0071] After 7 days, the retention rate of cherry anthocyanins in the anthocyanin solution was 20.41%; the retention rate of anthocyanins in the mixed solution was 26.14%. The retention rate increased by 11.37%.

[0072] After 9 days of storage, the retention rate of cherry anthocyanins in the anthocyanin solution was 17.38%; the retention rate of anthocyanins in the mixed solution was 23.24%. The retention rate increased by 33.71%.

[0073] After 11 days, the retention rate of cherry anthocyanins in the anthocyanin solution was 16.41%; the retention rate of anthocyanins in the mixed solution was 22.04%. The retention rate increased by 34.30%.

[0074] After 13 days, the retention rate of cherry anthocyanins in the anthocyanin solution was 14.58%; the retention rate of anthocyanins in the mixed solution was 21.44%. The retention rate increased by 47%.

[0075] After 15 days, the retention rate of cherry anthocyanins in the anthocyanin solution was 13.31%; the retention rate of anthocyanins in the mixed solution was 20.38%. The retention rate increased by 53.12%.

[0076] Example 4: Effect of mannoprotein on the retention rate of anthocyanins in Prunus guanshanensis under 85°C heat treatment 1. Sample preparation

[0077] Citric acid buffers of pH 3.0 and pH 6.6 were prepared respectively, and the purified anthocyanin from Sakura guanyin was dissolved in the citric acid buffer of pH 3.0 to obtain an anthocyanin solution; mannoprotein was dissolved in the citric acid buffer of pH 6.6 to obtain a mannoprotein solution. The anthocyanin solution and the mannoprotein solution were then mixed to obtain a mixed solution sample with a final concentration of 5 mg / mL anthocyanin and 0.1 mg / mL mannoprotein, and a pH value of 4.5.

[0078] 2. Heat treatment at 85℃

[0079] 10 mL of anthocyanin solution and complex mixture were respectively transferred into test tubes and placed in a constant temperature water bath set at 85°C. Samples were taken at 1 h, 2 h, and 3 h, and the anthocyanin content in the test tube solution was determined.

[0080] 3. Analysis results

[0081] Figure 3 As shown:

[0082] After heating at 85°C for 1 hour, the retention rate of cherry anthocyanins in the anthocyanin solution was 71.12% and the retention rate of anthocyanins in the cherry anthocyanin-protein mixture was 75.82%, which was an increase of 6.61%.

[0083] After heating at 85°C for 2 hours, the retention rate of cherry anthocyanins in the anthocyanin solution was 53.13% and the retention rate of anthocyanins in the cherry anthocyanin-protein mixture was 59.10%, which was an increase of 11.24%.

[0084] After heating at 85°C for 3 hours, the retention rate of cherry anthocyanins in the anthocyanin solution was 41.37% and the retention rate of anthocyanins in the cherry anthocyanin-protein mixture was 50.80%, which was an increase of 22.79%.

[0085] Example 5: Effect of mannoprotein on the stability of anthocyanins from Sakura guanshanensis under ultraviolet light 1. Sample preparation

[0086] Citric acid buffers of pH 3.0 and pH 6.6 were prepared respectively, and the purified anthocyanin from Sakura guanyin was dissolved in the citric acid buffer of pH 3.0 to obtain an anthocyanin solution; mannoprotein was dissolved in the citric acid buffer of pH 6.6 to obtain a mannoprotein solution. The anthocyanin solution and the mannoprotein solution were then mixed to obtain a mixed solution sample with a final concentration of 5 mg / mL anthocyanin and 0.1 mg / mL mannoprotein, and a pH value of 4.5.

[0087] 2. Ultraviolet irradiation treatment

[0088] 10 mL of anthocyanin solution and complex mixture were respectively transferred into test tubes, and placed under ultraviolet light for 2 h, 4 h, and 6 h, and samples were taken to determine the anthocyanin content in the test tubes.

[0089] 3. Analysis results

[0090] Figure 4 As shown:

[0091] UV irradiation for 2 hours: the retention rate of cherry anthocyanins in anthocyanin solution: 93.83%; the retention rate of anthocyanins in cherry anthocyanin-protein mixture: 95.63%. The retention rate increased by 1.8%.

[0092] UV irradiation for 4 hours: the retention rate of cherry anthocyanins in anthocyanin solution: 80.61%; the retention rate of anthocyanins in cherry anthocyanin-protein mixture: 85.80%. The retention rate increased by 6.44%.

[0093] UV irradiation for 6 hours: the retention rate of cherry anthocyanins in anthocyanin solution was 55.84%; the retention rate of anthocyanins in cherry anthocyanin-protein mixture was 75.10%. The retention rate increased by 34.49%.

[0094] Example 6: Determination of the optimal pH conditions for the color protection technology of mannoprotein for anthocyanins in Guanshan cherry 1. Sample preparation

[0095] Citric acid buffers of pH 3.0 and pH 6.6 were prepared respectively, and the purified anthocyanin from Sakura guanyin was dissolved in the citric acid buffer of pH 3.0 to obtain an anthocyanin solution; mannoprotein was dissolved in the citric acid buffer of pH 6.6 to obtain a mannoprotein solution. The anthocyanin solution and the mannoprotein solution were then mixed to obtain a mixed solution sample with a final concentration of 5 mg / mL anthocyanin and 0.1 mg / mL mannoprotein, and the pH was 4.5 at this time.

[0096] 2. Setting pH gradient conditions

[0097] 10 mL of anthocyanin solution and complex mixture were transferred into test tubes respectively, and the pH was adjusted to 3.0, 4.0, 5.0, and 6.0 respectively. The mixture was placed in a constant temperature water bath at 85°C, heated in the dark for 1 h, and stored in the dark at room temperature for 24 h. Then, samples were taken to determine the anthocyanin content in the test tubes.

[0098] 3. Analysis results

[0099] Figure 5 As shown:

[0100] pH = 3.0, the retention rate of cherry anthocyanin in anthocyanin solution: 72.46%; the retention rate of anthocyanin in cherry anthocyanin-protein mixture: 87.27%. The retention rate increased by 20.44%.

[0101] pH = 4.0, the retention rate of cherry anthocyanin in anthocyanin solution: 75.10%; the retention rate of anthocyanin in cherry anthocyanin-protein mixture: 84.87%. The retention rate increased by 13%.

[0102] pH = 5.0, the retention rate of cherry anthocyanin in anthocyanin solution: 74.13%; the retention rate of anthocyanin in cherry anthocyanin-protein mixture: 77.32%. The retention rate increased by 4.3%.

[0103] pH = 6.0, the retention rate of cherry anthocyanin in anthocyanin solution: 4.88%; the retention rate of anthocyanin in cherry anthocyanin-protein mixture: 5.02%. The retention rate increased by 2.86%.

[0104] The results determined that pH 3.0 was the optimal pH condition for mannoprotein to maintain the stability of anthocyanins from Sakura guanshanensis.

[0105] The reaction conditions were changed from heating in a constant temperature water bath at 85°C for 1 hour in the dark and storing at room temperature in the dark for 24 hours to storing at room temperature in the dark for 3 days. The retention rates of anthocyanin solution and anthocyanin-protein mixture under different pH conditions were tested. The results also showed that pH 3.0 was the optimal pH condition with the highest retention rate.

[0106] Example 7: Analysis of the binding mechanism between mannoprotein and Sakura anthocyanins based on fluorescence spectroscopy

[0107] The present invention analyzed and obtained that the anthocyanins of Prunus guanshanensis are mainly composed of petunidin-3,5-O-diglucoside (PD) and cyanidin-3-O-(6"-malonyl glucoside) (CD). Therefore, the present invention then selected PD and CD monomers as research objects to analyze the mechanism of binding of Prunus guanshanensis anthocyanins to mannoprotein. First, the relevant mechanism was analyzed using fluorescence spectroscopy.

[0108] 1. Sample preparation

[0109] In the experiment, 0.1 mol / L phosphate buffer (PBS, pH 7.4) was used to prepare a 10 μmol / L mannoprotein solution, which was stored at 4°C for use. PD and CD monomers were prepared into 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, and 50 μmol / L aqueous solutions with deionized water, respectively, and stored at 4°C for use.

[0110] 2. Fluorescence spectrum

[0111] The prepared 10 μmol / L mannoprotein solution was mixed with 0, 10, 20, 30, 40, 50 μmol / L PD and CD aqueous solutions at a volume ratio of 1:1, and mixed with a vortex oscillator to obtain a molar concentration ratio of 0:1, 1:1, 2:1, 3:1, 4:1, and 5:1 for PD, CD, and MP in the solution. The mixture was placed in a water bath at 24, 44, and 64 °C for 20 min, and the mixture was quickly transferred to a 1 cm quartz cuvette, and its fluorescence spectrum was scanned using a UV-200 fluorescence spectrophotometer. The instrument parameters were set as follows: 280 nm fixed excitation wavelength, 290-500 nm emission wavelength scanning range, 5 nm slit span, and 1000 nm / min scanning speed.

[0112] 3. Analysis results

[0113] 3.1MP causes fluorescence quenching

[0114] Figure 6 As shown in the fluorescence emission spectrum, it can be seen that the excitation wavelength of MP is 310nm. After adding gradient concentrations of MAE, the fluorescence intensity decreases in a dose-dependent manner, indicating that MP exhibits typical endogenous fluorescence quenching after addition.

[0115] 3.2 Fluorescence quenching mechanism

[0116] Fluorescence quenching follows the Stern-Volmer equation

[0117]

[0118] Where: F0: fluorescence intensity of MP when PD and CD are not added;

[0119] F: fluorescence intensity of MP after adding PD and CD;

[0120] K SV : Stern-Volmer quenching constant;

[0121] K q : Quenching rate constant in the quenching process of biomacromolecules;

[0122] c q : quencher concentration;

[0123] τ0: lifetime of the fluorescent molecule in the absence of quencher (10-8s).

[0124] result:

[0125]

[0126] 3.3 Calculation of binding constants and binding sites

[0127] Binding sites and binding constants are generally calculated using the double logarithmic curve equation:

[0128]

[0129] Where: Ks: binding constant;

[0130] n: number of binding sites;

[0131] According to the formula, a curve is obtained by plotting log[(F0-F) / F] against log[cq].

[0132] 3.4 Thermodynamic analysis of interactions

[0133]

[0134] ΔG=ΔH-TΔS

[0135] Where: ΔG: free enthalpy change;

[0136] ΔH: enthalpy change;

[0137] ΔS: entropy change;

[0138] K S : The binding constant obtained by the formula;

[0139] T: temperature;

[0140] R: gas constant, equal to 8.314 J mol -1 K -1 .

[0141]

[0142] For MP-PD, ΔH = 9.61 kJ·mol -1 , greater than 0, ΔS = 111.53 J·mol - 1 K -1 , which is greater than 0, indicating that the binding forces between cherry anthocyanins and mannoprotein are mainly hydrogen bonds and hydrophobic forces.

[0143] Similarly, for the MP-CD group, according to the formula, ΔH = 24.90 KJ·mol-1, which is greater than 0, and ΔS = 183.41 J·mol -1 K -1 , which is greater than 0, indicating that the binding forces between cherry anthocyanins and mannoprotein are mainly hydrogen bonds and hydrophobic forces.

[0144] Example 8: Analysis of the binding mechanism between mannoprotein and Sakura anthocyanins based on Fourier transform infrared chromatography 1. Sample preparation

[0145] Place the prepared sample in a disposable culture dish and freeze-dry it for 48 hours before use. Mix the freeze-dried powder with the dried potassium bromide in a ratio of 1:100 and press into tablets. Instrument parameter setting: Scan spectrum range 4000-400cm -1 The scanning times were 16. Each group of samples was prepared and measured three times, and the data were smoothed and averaged.

[0146] 2. Analysis results

[0147] Figure 7 This is the Fourier infrared spectrum result.

[0148]

[0149] According to the results of Fourier infrared combined with peakfit software, the content of each structure was analyzed. Compared with MP alone, the secondary structure of MP-PD and MP-CD will change, as shown below: the α-helix and β-turn of MP-PD and MP-CD are significantly reduced, the β-fold is significantly increased, and the content of random coil is not significantly changed (p<0.05). After binding with PD and CD, the α-helix content of MP changed from 18.3 to 8.7 and 13.51%, indicating that the secondary structure of MP changed after binding with PD and CD; the binding of PD to the amino acid residues of the main chain of MP destroyed the hydrogen bond network structure of MP. In the presence of PD and CD, the content of α-helix structure decreased to 36.5±0.38%, while the content of β-fold and random coil increased to 55.71%, 51.88%, 16.33% and 20.27%, respectively.

[0150] Example 9: Analysis of the binding mechanism between mannoprotein and Sakura anthocyanins based on molecular docking simulation technology 1. Preparation and processing of mannoprotein and ligand models

[0151] First, the amino acid sequence of mannoprotein (UniProt identifier: Q03180) was obtained from the UniProt database (https: / / www.Un iProt.org). Since there is no three-dimensional model of mannoprotein in the protein database, the amino acid sequence Q03180 was used as a template and homology modeling was performed using the Swiss model. Secondly, the rationality of the constructed protein model was evaluated using the Ramachandran graph on SAVES v6.0. The structures of the two required ligands were searched in the PubChem database. Petunidin 3,5-diglucoside (CAS: 25846-73-5) and Cyanidin 3-O-(6-O-malonyl-beta-D-glucoside) (CAS: 171828-62-9).

[0152] The homology modeling results are as follows Figure 8 The Ramachandran diagram is used to describe the dihedral angles of amino acid residues in protein structures. Whether and Ψ are within a reasonable range can be used to characterize the rationality of homology modeling results. Figure 8 It shows that the sum of the total allowed area (88.3%), the relatively allowed area (11.0%) and the generally allowed area (0.3%) exceeds 99%, indicating that the MP structure obtained in the present invention is reasonable.

[0153] 2. Molecular docking of mannoprotein and anthocyanin

[0154] AutoDockTools was used to determine the parameters of the active pocket. Secondly, the 3D structure of the ligand needs to be completely contained in the Gridbox.

[0155] MP and PD molecular docking parameter settings: Gridbox center coordinates are set to center_x = 0.874, center_y = -0.409, center_z = -5.676. Therefore, the Gridbox size is set to size_x = 61.95, size_y = 47.2, size_z = 61.95. Other parameters are the default values.

[0156] Molecular docking parameter settings for MP and CD: Gridbox center coordinates are set to center_x = 0.874, center_y = -0.409, center_z = -5.676. Gridbox size is set to size_x = 65.66, size_y = 45.84, size_z = 63.18. Other parameters are the default values.

[0157] 3. Analysis results

[0158] The molecular docking mechanism of mannoprotein with two ligands was studied by AutoDock Vina software. The AutoDockVina program continuously adjusts the conformation of the ligand molecule within the Gridbox range, and then scores the different conformations of the ligand (including direction, position and energy, etc.). During the docking process, the two ligands produced 9 docking conformations respectively, and each conformation was sorted from high to low according to the affinity value. The conformation with the highest affinity value indicates that the ligand molecule and the receptor protein have achieved the best geometric match and energy match. Subsequently, PyMol software and ligPlus were used to analyze the binding mode and interaction between mannoprotein and the two ligands.

[0159] Petunidin-3,5-diglucoside(PD)

[0160] Affinity is an evaluation standard for the degree of binding between the target receptor and the small molecule ligand. The larger the absolute value of affinity, the stronger the ligand-receptor binding ability and the more stable the formed complex. The docking results are shown in the table below. According to the table, the affinity values ​​of the binding conformations of MP and PD docking are all negative, which means that the reactions are all spontaneous.

[0161] According to the docking results, when the docking affinity between MP and PD is -6.9 kcal / mol, hydrogen bond interaction occurs between the two, and the RMSD of PD is 5.681 when the affinity is -6.9 kcal / mol. Residues Ser43, Ser44, and Asp321 interact with PD through hydrogen bonds, and the interaction distances are and There are no pi-pi and pi-cation interactions. LigPlus analysis showed that residues Glu215, Lys217, Val225, Val226 and Ile323 have hydrophobic interactions with PD.

[0162] MP-PD binding mode

[0163]

[0164] Cyanidin-3-(6-malonyl glucoside)(CD)

[0165] The docking results are shown in the table below. According to the table, the affinity values ​​of the docking binding conformations of MP and CD are all negative, which means that the reactions are all spontaneous.

[0166] According to the docking results, when the docking affinity between MP and CD is -6.9 kcal / mol, hydrogen bond interaction occurs between the two, and the RMSD of CD is 5.845 when the affinity is -6.9 kcal / mol. Residue Ala54 interacts with CD through hydrogen bonds, and the interaction distance is Residue Glu281 forms a hydrogen bond interaction with CD with an interaction distance of 2.6 and Residue Asn283 interacts with CD through hydrogen bonds with a distance of 2.8 and Residue Asp290 interacts with CD through hydrogen bonds with a distance of Residue Tyr316 interacts with CD through hydrogen bonds with a distance of Residue Gln318 interacts with CD through hydrogen bonds with a distance of and There are no pi-pi and pi-cation interactions. LigPlus analysis showed that residues Glu51, Val53 and Asp224 have hydrophobic interactions with CD.

[0167] MP-CD binding mode

[0168]

Claims

1. Application of mannoprotein as a protective agent for anthocyanins of Prunus guanshanensis.

2. The use according to claim 1, characterized in that The application method is: adding mannoprotein as a protective agent to the raw material containing the anthocyanin of related mountain cherry.

3. A method for protecting the color of anthocyanins from Sakura guanshanensis, characterized in that The method comprises the following steps: adding mannoprotein as a protective agent to a raw material containing anthocyanins of related wild cherry.

4. The method according to claim 3, characterized in that The mass ratio of the anthocyanins from the cherry blossoms of Guanshanshan to mannoprotein is 100:1-5.

5. The method according to claim 4, characterized in that The mass ratio of the anthocyanins from the cherry blossoms of Guanshania guanshanica to mannoprotein is 100:

2.

6. The method according to claim 3, characterized in that The method comprises the following steps: adding mannoprotein as a protective agent to a raw material containing anthocyanins of related wild cherry, and adjusting the pH value of the system to 3-5.

7. The method according to claim 6, characterized in that The pH value of the system was adjusted to 3.

8. A method for improving the thermal stability and light stability of anthocyanins from Sakura guanshanensis, characterized in that The method comprises the following steps: adding mannoprotein as a stabilizer to a raw material containing anthocyanins of related wild cherry, and adjusting the pH value of the system to 3-5.

9. A protective agent for anthocyanins of Sakura guanshanensis, characterized in that The protective agent is mannoprotein.

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