A ha-egcg graft copolymer and its use in nuts

Abstract

The application provides an HA-EGCG graft copolymer, a preparation method thereof comprises using hyaluronic acid and EGCG as reaction substrates, and adopting an ascorbic acid / hydrogen peroxide redox system to mediate a crosslinking reaction to obtain the HA-EGCG graft copolymer. The HA-EGCG graft copolymer has excellent biological activity, and has a better improvement on nut coating effect, and provides a new idea and a new way for developing biological activity of nut coating.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to an HA-EGCG graft copolymer and application thereof in nuts. BACKGROUND

[0002] Epigallocatechin gallate (EGCG) is the main active component of flavanol in polyphenols and the highest content catechin in green tea, and has multiple effects such as antioxidant, antibacterial and antitumor. Free radical grafting is to form a covalent bond by the reaction of the hydroxyl radical of polyphenol and the active group on the side chain of the grafting compound, and the covalent bond generation involves irreversible interaction, thereby forming a more stable combination. Compared with enzyme method and alkali method, the free radical grafting method does not involve organic solvents in the reaction process, and has high safety, and is widely used in the food industry. However, how to improve the grafting rate of HA-EGCG is one of the problems to be solved by researchers.

[0003] Hyaluronic acid (HA) is a non-branching glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through β-(1→4) and β-(1→3) glycosidic bonds, and widely exists in the intercellular matrix of animal tissues and the capsules of some bacteria. The relative molecular mass (Mr) is generally 1×10 5 ~1×10 7 D, and the molar ratio of the two monosaccharides in the molecule is 1:1. HA is one of the main components of the extracellular matrix, has good biocompatibility, biodegradability and non-immunogenicity, and is rich in reactive functional groups such as carboxyl and hydroxyl groups in the molecular structure for further chemical modification and modification. People have conducted a relatively full study on the structure, properties and physiological functions of HA, and the average Mr of the HA used at present is generally greater than 1×10 5 D. Hyaluronic acid has been widely used in drug carriers, medical and cosmetic fields due to its excellent biocompatibility, modifiability and biodegradability. SUMMARY

[0004] The purpose of the application is to provide a HA-EGCG graft copolymer with high grafting rate, and to study the application thereof in film-coated nuts.

[0005] To achieve the purpose of the application, the technical scheme adopted by the application is as follows:

[0006] A HA-EGCG graft copolymer has the structure shown in formula I or formula II:

[0007] ;

[0008] Wherein, n, m are non-0 natural numbers.

[0009] Further, the grafting rate of the HA-EGCG graft copolymer is 40-90%.

[0010] On the other hand, the present application provides a preparation method of the HA-EGCG graft copolymer, which adopts the following technical scheme:

[0011] A preparation method of a HA-EGCG graft copolymer, comprising using hyaluronic acid and EGCG as reaction substrates, and using ascorbic acid / hydrogen peroxide redox system to mediate cross-linking reaction to obtain the HA-EGCG graft copolymer.

[0012] Specifically, a preparation method of a HA-EGCG graft copolymer, comprising the following steps:

[0013] Dissolve the hyaluronic acid with deionized water, add ascorbic acid and EGCG, and mix uniformly, then stand at room temperature;

[0014] Add hydrogen peroxide solution to the reaction system, and react under nitrogen or helium atmosphere;

[0015] After the reaction is completed, dialysis is performed with a dialysis bag, the dialysis solution is replaced at intervals to remove unreacted EGCG, and the solution after dialysis is freeze-dried to obtain the graft copolymer.

[0016] As preferred, the hyaluronic acid is H-HA or L-HA or a mixture thereof, wherein H-HA is a macromolecule with Mr≥100000, L-HA is a small molecule with Mr≤3000, and the mixture has 0≤Mr≤100000.

[0017] As preferred, the standing time in step (1) is 10-20 min, the reaction time in step (2) is 24-36 h, and the dialysis solution is replaced every 6-8 h to remove unreacted EGCG, and the cycle is repeated for 3 times.

[0018] As preferred, the volume-to-mass ratio of the deionized water to the hyaluronic acid is 1:5-10 ml / g, the mass ratio of the hyaluronic acid to the ascorbic acid is 1:0.01-0.05, and the mass molar ratio of the ascorbic acid to the hydrogen peroxide is 1:0.5-1 g / mol.

[0019] As preferred, the dialysis has a molecular weight cut-off of 25-500 Da.

[0020] As preferred, the weight ratio of the EGCG to the HA is selected from 1:10, 1:5, 2:5, 4:5, 1:1 or 8:5.

[0021] On the other hand, the present application also includes the application of the HA-EGCG graft copolymer in nuts.

[0022] The nuts include but are not limited to peanuts, melon seeds, almonds, torreya grandis, hazelnuts, pine nuts, cashews, pistachios, walnuts, pecans, etc.

[0023] The HA-EGCG graft copolymer is directly coated on the nuts, and the preservation value of the nuts is improved.

[0024] The graft copolymer provided by the application has excellent biological activity, for example, good antioxidant activity and thermal stability, which greatly expands the application space of the graft copolymer in food, daily chemicals and medicine.

[0025] The graft copolymer provided by the application has excellent biological activity, for example, good antioxidant activity and thermal stability, which greatly expands the application space of the graft copolymer in food, daily chemicals and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 HA-EGCG graft copolymer antioxidant activity analysis chart;

[0027] Figure 2 HA-EGCG graft copolymer 1 H chart;

[0028] Figure 3 Microstructure chart of HA-EGCG graft copolymer;

[0029] Figure 4 Fourier transform infrared spectrum analysis chart of HA-EGCG graft copolymer;

[0030] Figure 5 Thermal stability analysis chart of HA-EGCG graft copolymer;

[0031] Figure 6 HA-EGCG graft copolymer coated torreya grandis storage acid value content change chart;

[0032] Figure 7 HA-EGCG graft copolymer coated torreya grandis storage peroxide value change chart;

[0033] Figure 8 HA-EGCG graft copolymer coated torreya grandis storage malondialdehyde content chart;

[0034] Figure 9 HA-EGCG graft copolymer coated torreya grandis storage appearance chart. DETAILED DESCRIPTION

[0035] The technical content, purposes and effects of the application are described in detail below in combination with the embodiments and the drawings.

[0036] The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0037] Example 1

[0038] The steps of the preparation method of the HA-EGCG graft copolymer are as follows:

[0039] (1) 100 mL of deionized water was used to dissolve hyaluronic acid H-HA 0.5 g in a beaker, and after the hyaluronic acid was fully dissolved and swelled, 0.005 g of ascorbic acid and 0.05 g of EGCG were added to the beaker, and after mixing evenly, it was placed at room temperature for 10 min.

[0040] (2) 1 mL of 5M H2O2 was added to the reaction system, and the reaction was started under a nitrogen atmosphere for 24-36 h;

[0041] (3) After the reaction was completed, the unreacted EGCG was removed by dialysis, and the dialysis solution was changed every 6-8 h, and the cycle was repeated 3 times. The HA-EGCG graft copolymer solution obtained after dialysis was freeze-dried to obtain the graft copolymer. The molecular weight cut-off of the dialysis was 25-500 Da, and 200 Da was selected here.

[0042] According to the method of Example 1, Examples 2-6 were obtained by changing the amount of hyaluronic acid and EGCG, as shown in Table 1:

[0043] Table 1.

[0044] Example 2 Example 3 Example 4 Example 5 Example 6 EGCG g 0.1 0.2 0.4 0.5 0.8 Hyaluronic acid g 0.5 0.5 0.5 0.5 0.5

[0045] The grafting rate of the HA-EGCG graft copolymer in Examples 1-6 was calculated. The grafting rate calculation: 1 mL of HA-EGCG graft copolymer solution was mixed with 1 mL of Folin phenol reagent (diluted 10 times), and incubated at 30°C in the dark for 5 minutes. Then, 2 mL of 7.5% Na2CO3 was added to the mixture, and incubated in the dark for 1 h. The absorbance of the solution at 765 nm wavelength was measured, and its value was correlated with the standard curve of EGCG, wherein the grafting rate of HA-EGCG was expressed as a percentage of mg HA equivalent / g (mg HHA / g), and the results are shown in Table 2.

[0046] Table 2. Grafting rate of Examples 1-6

[0047] Group Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Grafting rate 40.4% 52.6% 61.8% 63.6% 65.7% 62.3%

[0048] Example 7

[0049] The steps of the preparation method of the HA-EGCG graft copolymer are as follows:

[0050] (1) Take 100 mL of deionized water to dissolve hyaluronic acid L-HA 1 g in a beaker, after the hyaluronic acid is fully dissolved and swelled, add 0.05 g of ascorbic acid and 0.1 g of EGCG to the beaker, mix uniformly, and stand at room temperature for 10 min.

[0051] (2) Add 0.5 mL of 5M H2O2 to the reaction system, start the reaction under nitrogen atmosphere for 24-36h;

[0052] (3) After the reaction is completed, remove the unreacted EGCG by dialysis bag, change the dialysate every 6-8h, cycle 3 times, and the HA-EGCG graft copolymer solution obtained after dialysis is completed is freeze-dried to obtain the graft copolymer, the molecular weight cut-off of the dialysis is 25-500Da, and 25Da is selected here.

[0053] According to the method of Example 7, the amount of hyaluronic acid and EGCG is changed to obtain Examples 8-12, see Table 3:

[0054] Table 3.

[0055] Example 8 Example 9 Example 10 Example 11 Example 12 EGCG g 0.2 0.4 0.8 1 1.6 Hyaluronic acid g 1 1 1 1 1

[0056] The grafting rate of HA-EGCG graft copolymer in Examples 7-12 is calculated. The grafting rate is calculated as in Example 1, and the results are shown in Table 4.

[0057] Table 4. Grafting rate of Examples 7-12

[0058] Group Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Grafting rate 45.3% 58.5% 78.2% 86.6% 90.0% 80.7%

[0059] Example 13

[0060] The steps of the preparation method of HA-EGCG graft copolymer are as follows:

[0061] (1) Take 100 mL of deionized water to dissolve a mixture of 0.8 g of hyaluronic acid H-HA and L-HA in a beaker, after the hyaluronic acid is fully dissolved and swelled, add 0.024 g of ascorbic acid and 0.08 g of EGCG to the beaker, mix uniformly, and stand at room temperature for 10 min.

[0062] (2) Add 0.8 mL of 5M H2O2 to the reaction system, start the reaction under nitrogen atmosphere for 24-36h;

[0063] (3) After the reaction is completed, remove the unreacted EGCG by dialysis bag, change the dialysate every 6-8h, cycle 3 times, and the HA-EGCG graft copolymer solution obtained after dialysis is completed is freeze-dried to obtain the graft copolymer, the molecular weight cut-off of the dialysis is 25-500Da, and 25Da is selected here.

[0064] The procedure of Example 13 was followed, varying the amounts of hyaluronic acid and EGCG to give Examples 14-18, see Table 5:

[0065] Table 5.

[0066] Example 14 Example 15 Example 16 Example 7 Example 18 EGCG g 0.16 0.32 0.64 0.8 1.28 Hyaluronic acid g 0.8 0.8 0.8 0.8 0.8

[0067] The grafting ratio of the HA-EGCG graft copolymer in Examples 13-18 was calculated. The grafting ratio was calculated as in Example 1, and the results are shown in Table 6.

[0068] Table 6. Grafting ratio of Examples 13-18

[0069] Group Example 13 Example 14 Example 15 Example 16 Example 17 Example 18 Grafting rate 42.7% 55.3% 62.2% 65.8% 69.3% 60.7%

[0070] Stability index analysis of HA-EGCG graft copolymer

[0071] The HA-EGCG graft copolymer solution with the highest grafting ratio from the Examples was taken for stability analysis.

[0072] Experimental index 1

[0073] Antioxidant activity analysis: 0.1 mL of HA-EGCG graft copolymer was mixed with 2.8 mL of Tris-HCl buffer (100 mM, pH 8.0) and incubated at 25°C for 10 min. Then 3 mM of pyropyrol solution was added, preheated in a 25°C water bath, mixed and the absorbance at 325 nm was measured by spectrophotometry. The results are shown in Figure 1 .0 2•− The clearance capacity was calculated as

[0074] 0 2•− The clearance rate % = (1 - sample absorbance value / blank absorbance value) x 100%

[0075] Experimental index 2

[0076] NMR analysis: 50 mg of HA-EGCG graft copolymer lyophilized powder was weighed, dissolved in 0.5 mL of heavy water and freeze-dried. Subsequently, the freeze-dried powder was redissolved in 0.5 mL of heavy water and freeze-dried again, repeating the above process to fully exchange the active hydrogen. Then the sample was dissolved in 0.5 mL of heavy water and placed in a nuclear magnetic resonance spectrometer at 600 MHz at room temperature 25°C to determine the one-dimensional spectrum of the 1 HNMR spectrum, the results are shown in Figure 2 .

[0077] Experimental index 3

[0078] The 10 mg HA-EGCG graft copolymer freeze-dried powder was placed on a sample net rack coated with 200 mesh copper for 2 min, then dyed with 2% phosphotungstic acid for 3 min, and the excess dyeing solution was absorbed with filter paper. The dried sample was used for TEM observation. The surface morphology of hyaluronic acid and its conjugate was observed by scanning electron microscopy (SEM). The prepared sample was fixed on a 1*1 mm single-side polished silicon wafer and sputtered with gold. The sample was observed under a scanning electron microscope, and the results are shown in Figure 3 .

[0079] Experimental index 4

[0080] Fourier transform infrared spectroscopy analysis: 2 mg of HA-EGCG graft copolymer freeze-dried powder was mixed with dry KBr (1:100 ratio), then pressed into 1 mm compressed particles (5 ~ 8 tons per square centimeter) using a hydraulic press to obtain a transparent film. The prepared tablets were placed in a test tube for Fourier transform infrared spectroscopy analysis, and the results are shown in Figure 4 .

[0081] Experimental index 5

[0082] Thermal stability analysis: 2.5 mg of HA-EGCG graft copolymer freeze-dried powder was placed in a thermogravimetric furnace under the conditions of nitrogen flow of 20 mL / min and heating rate of 10℃ / min. The TGA curve of the sample in the range of 50-800℃ was obtained by thermogravimetric analysis, and the results are shown in Figure 5 .

[0083] Application of HA-EGCG graft copolymer

[0084] Implementation 1

[0085] Film coating process: Take mature processed seedless Chinese torreya and coat the surface with copolymer solution, then dry to complete the film coating.

[0086] Quality index detection of film-coated Chinese torreya

[0087] Application index 1: acid value content. Take 0.5 g of the prepared sample (accurate to 0.001 g) and place it in a 250 mL iodometric flask. Add 7.5 mL of chloroform-glacial acetic acid solution, gently shake the sample until it is completely dissolved. Accurately add 0.25 mL of potassium iodide saturated solution, tightly cap the bottle, and gently shake for 0.5 min. Place in the dark for 3 min. Add 25 mL of water, shake well, and immediately titrate with sodium thiosulfate standard titration solution; titrate the precipitated iodine until it turns light yellow, add 1 mL of starch indicator, continue titration and shake vigorously until the solution turns blue. The blank test was also conducted. The results are shown in Figure 6As shown, compared with the non-coated group, the acid value of the coated group was 0.26367 mg / g after three months, and the acid value of the non-coated group was 0.40392 mg / g.

[0088] Application index 2: peroxide value. Measurement is determined according to GB 5009.227-2023: 2g~3g of the prepared sample is weighed (accurate to 0.001g), placed in a 250mL iodine flask, 30mL of chloroform-glacial acetic acid solution is added, and the sample is shaken gently until it is completely dissolved. Accurately add 1.00mL of saturated potassium iodide solution, tightly plug the bottle cap, and shake gently for 0.5min, and place in the dark for 3min. Take out 100mL of water, shake well, and immediately titrate the precipitated iodine with sodium thiosulfate standard titration solution until it is light yellow, add 1mL of starch indicator, continue to titrate and shake vigorously until the solution turns blue, and the end point is reached. At the same time, a blank test is carried out. The volume of sodium thiosulfate standard titration solution consumed in the blank test is V. If Figure 7 As shown, compared with the non-coated group, the peroxide value of the coated group was 0.022842 g / 100g after three months, and the peroxide value of the non-coated group was 0.073602 g / 100g.

[0089] Application index 3: malondialdehyde content. Measurement is determined according to GB 5009.181-201. 5g of the sample is weighed and placed in a 100ml conical flask with a plug, 50mL of trichloroacetic acid mixture is accurately added, shaken well, sealed with a plug, placed on a constant temperature oscillator at 50℃ for 30min, taken out, cooled to room temperature, filtered with double-layer quantitative slow-speed filter paper, the initial filtrate is discarded, and the filtrate is reserved. Accurately transfer 5mL of the filtrate and the standard series solution into 25mL colorimetric tubes with plugs, respectively, and take another 5mL of trichloroacetic acid mixture as a sample blank, respectively. Add 5mL of thiobarbituric acid (TBA) aqueous solution, mix well, and place in a 90℃ water bath for 30min. Cool to room temperature. Adjust the zero point with the sample blank, measure the absorbance values of the sample solution and the standard series solution at 532nm, and draw the standard curve with the mass concentration of the standard series solution as the abscissa and the absorbance value as the ordinate. If Figure 8 As shown, compared with the non-coated group, the malondialdehyde content of the coated group was 762.40mg / kg after three months, and the malondialdehyde content of the non-coated group was 1136.87mg / kg.

[0090] The coated torreya nuts have the advantage of quality preservation during storage. The surface of the processed torreya nuts is easily oxidized and the texture becomes soft due to exposure to air. The surface of the coated torreya nuts can effectively maintain the quality due to the isolation of the HA-EGCG graft copolymer, and the strong antioxidant property of EGCG can maintain the normal color of the torreya nuts, as shown in Figure 9As shown, the quality of the coated torreya was maintained after 90 days of storage compared to the untreated torreya.

[0091] Similarly, other nuts such as peanuts, melon seeds, almonds, torreya, hazelnuts, pine nuts, cashews, pistachios, walnuts, pecans can also achieve the effect of torreya by coating with the copolymer solution of the present application.

[0092] These results show that the biological activity of torreya after coating is well maintained, which has great potential and application value for the preservation of nuts such as torreya.

Claims

1. A HA-EGCG graft copolymer, characterized in that, The HA-EGCG graft copolymer has a structure shown in Formula I or Formula II: ； Wherein, n, m are non-zero natural numbers.

2. A process for the preparation of HA-EGCG graft copolymer as claimed in claim 1, wherein, The method comprises: using hyaluronic acid and EGCG as reaction substrates, and using ascorbic acid / hydrogen peroxide redox system to mediate cross-linking reaction to obtain HA-EGCG graft copolymer, and comprises the following steps: (1) dissolving hyaluronic acid with deionized water, adding ascorbic acid and EGCG, and uniformly mixing, and then standing at room temperature; (2) adding hydrogen peroxide solution into the reaction system, and reacting under nitrogen or helium atmosphere; (3) after the reaction is completed, the reaction solution is dialyzed with a dialysis bag, the dialysis solution is replaced at intervals to remove unreacted EGCG, and the solution after dialysis is freeze-dried; The hyaluronic acid is H-HA or L-HA, wherein H-HA is a macromolecule with Mr≥100,000, and L-HA is a small molecule with Mr≤3000; The weight ratio of EGCG to HA is selected from 1:10, 1:5, 2:5, 4:5, 1:1 or 8:

5.

3. The method of claim 2, wherein the HA-EGCG graft copolymer is prepared by the steps of: The grafting rate of the HA-EGCG graft copolymer is 40-90%.

4. The method of claim 2, wherein the HA-EGCG graft copolymer is prepared by the steps of: The dialysis has a molecular weight cut-off of 25-500 Da.

5. Application of the HA-EGCG graft copolymer of claim 1 to nuts.

6. Use according to claim 5, characterized in that, The nuts are selected from peanuts, melon seeds, almonds, Japanese pine nuts, hazelnuts, pine nuts, cashews, pistachios, walnuts or pecans.

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