Preparation method and application of modified tea polysaccharide-chlorogenic acid compound

Abstract

The invention provides a preparation method and application of a modified tea polysaccharide-chlorogenic acid compound, and belongs to the field of food. The preparation method comprises the following steps: preparing a mixed solution of tea polysaccharide and a nonionic surfactant, adjusting the pH value to 3-4, and freeze-drying to obtain modified tea polysaccharide; preparing a chlorogenic acid and modified tea polysaccharide mixed solution, adjusting the pH value to 6-8, fully stirring and compounding under a water bath condition, dialyzing, and freeze-drying to obtain the modified tea polysaccharide-chlorogenic acid compound. The tea polysaccharide is modified through the surfactant, a stable chlorogenic acid-modified tea polysaccharide composite system is constructed, and the chlorogenic acid-modified tea polysaccharide composite system is applied to fresh keeping of marine products such as fish meat.

Description

Technical Field

[0001] The present invention relates to the field of food, and in particular to a preparation method and application of a modified tea polysaccharide-chlorogenic acid complex. Background Art

[0002] Chlorogenic acid (CA) is a natural plant polyphenol that has attracted widespread attention due to its rich biological activity and strong antioxidant activity, but the active phenolic hydroxyl groups in its structure are easily inactivated by environmental influences. Therefore, liposomes or nano-encapsulation are often used to make it more stable and improve its utilization rate.

[0003] Tea polysaccharides (TPS) are a class of natural complex polysaccharides extracted from tea leaves. They have multiple activities such as antioxidant and hypoglycemic effects, and can combine with polyphenols to form a stable "sugar-phenol" complex. On the basis of retaining their respective characteristics, sugar-phenol complexes have significant synergistic effects, can improve the overall physical and chemical properties, and have good application potential in food preservation.

[0004] However, the stability of the directly prepared tea polysaccharide-chlorogenic acid complex is relatively poor, and its loading capacity needs to be improved. At present, there is no research in the existing literature on the use of surfactants to improve the structure of tea polysaccharides, and there is no report on the preparation of modified tea polysaccharide-chlorogenic acid complexes to improve the stability and utilization of chlorogenic acid. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides a preparation method of a modified tea polysaccharide-chlorogenic acid complex and its application.

[0006] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0007] The first aspect of the technical solution of the present invention is to provide a method for preparing a modified tea polysaccharide-chlorogenic acid complex, comprising:

[0008] S1: preparing a mixed solution of tea polysaccharides and nonionic surfactants, wherein the mass ratio of the nonionic surfactant to the tea polysaccharides is 0.4-0.5;

[0009] S2: adjusting the pH value of the mixed solution to 3-4, and freeze-drying to obtain modified tea polysaccharides;

[0010] S3: preparing a mixed solution of chlorogenic acid and modified tea polysaccharides, wherein the mass ratio of chlorogenic acid to modified tea polysaccharides is 3.8-4.2;

[0011] S4: adjusting the pH value of the mixed solution of chlorogenic acid and modified tea polysaccharide to 6-8, stirring and compounding in a water bath to obtain a composite solution;

[0012] S5: dialyzing the composite solution, and freeze-drying the retained solution obtained by the dialyzation to obtain a modified tea polysaccharide-chlorogenic acid complex.

[0013] The second aspect of the technical solution of the present invention provides a modified tea polysaccharide-chlorogenic acid complex prepared by the above-mentioned preparation method.

[0014] The third aspect of the technical solution of the present invention is to provide a use of the modified tea polysaccharide-chlorogenic acid complex as a preservative in the preservation of seafood.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention uses a surfactant to modify tea polysaccharides, and the modified tea polysaccharides are combined with chlorogenic acid to obtain a modified tea polysaccharide-chlorogenic acid complex. In the modified tea polysaccharide-chlorogenic acid complex, due to the effects of static electricity, dipole moment, hydrogen bond and hydrophobicity between the polymer and the surfactant, the association of the surfactant on the polysaccharide molecular chain in the water system is regulated, and the critical aggregation concentration, critical micelle concentration, binding amount and interfacial tension of the surfactant are changed. The multi-faceted effects make the final modified tea polysaccharide-chlorogenic acid complex more stable, and the encapsulation rate of chlorogenic acid is higher, and the application effect in the preservation of seafood such as fish is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of different Tween-80 concentrations, pH values, and vortex times on the particle size of the modified tea polysaccharide complex in one embodiment of the present invention;

[0018] Figure 2 The effect of different chlorogenic acid-tea polysaccharide mass ratios, pH, compounding temperature, and compounding time on the binding amount of the modified tea polysaccharide-chlorogenic acid complex in one embodiment of the present invention;

[0019] Figure 3 The scanning electron microscopy test results of chlorogenic acid (CA), tea polysaccharide (TPS), tea polysaccharide-chlorogenic acid complex (TC) and modified tea polysaccharide-chlorogenic acid complex (TTC) in one embodiment of the present invention;

[0020] Figure 4 The X-ray diffraction test results of TPS, TC and TTC in one embodiment of the present invention;

[0021] Figure 5 and Figure 6 The storage stability test results of TC and TTC in one embodiment of the present invention are shown;

[0022] Figure 7The thermal stability test results of TC and TTC in one embodiment of the present invention;

[0023] Figure 8 The results of the effects of TC and TTC on the total thiol content and surface hydrophobicity of surimi during freeze-thaw cycles in storage are shown in one embodiment of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0025] Surfactants are substances with a hydrophilic head and a hydrophobic tail that can reduce interfacial tension. Studies have shown that the modification of tea polysaccharides with surfactants can improve the physical and chemical properties of tea polysaccharides, and surfactants and tea polysaccharides can be used together to construct carriers of active substances, load chlorogenic acid and be used to preserve seafood such as surimi.

[0026] The tea polysaccharide of the present invention is a kind of complex polysaccharide extracted from tea leaves, including arabinose, galactose, mannose, rhamnose, ribose, xylose and the like.

[0027] The present invention is further described below in conjunction with various embodiments.

[0028] Example 1

[0029] In this embodiment, a method for preparing a modified tea polysaccharide-chlorogenic acid complex is provided, which specifically comprises the following steps:

[0030] Tea polysaccharides are modified by using surfactants: 0.25 g of tea polysaccharide solid powder is taken into a 50 mL centrifuge tube, and 25 mL of pure water is added to dissolve it to obtain a 10 mg / mL tea polysaccharide solution; an appropriate amount of surfactant is weighed, and the surfactant is dissolved by using the tea polysaccharide solution to make the concentration of the surfactant in the mixed solution greater than 3 mg / mL; the pH value of the system in the centrifuge tube is adjusted to 3-4 with a 1 moL / L hydrochloric acid solution, vortex mixed for 15s-20s, and then freeze-dried in a freeze dryer for 48h-50h to obtain a solid powder of modified tea polysaccharides.

[0031] Compounding the modified tea polysaccharide with chlorogenic acid: prepare several dialysis bags (500Da), boil them in boiling water for 10 minutes, and let them stand to cool to room temperature; take 0.22g of chlorogenic acid solid powder in a beaker, add 220mL of water to dissolve, and obtain a 1mg / mL chlorogenic acid solution; then take 0.060g of modified tea polysaccharide solid powder in a beaker, add 30mL of water to dissolve, and prepare a 2mg / mL modified tea polysaccharide solution; measure a certain amount of chlorogenic acid solution and modified tea polysaccharide solution at a phenol-sugar mass ratio of 3.8-4.2, quickly adjust the pH value of the system to 6-8 with 1moL / L NaOH solution, place it in a water bath at 35℃-38℃ and stir for 30min; then transfer 36mL of the complex sample solution to the pretreated dialysis bag, and dialyze it in 800mL of pure water with magnetic stirring for 6h to dialysis equilibrium; vacuum freeze-dry to obtain a solid modified tea polysaccharide-chlorogenic acid complex, which is sealed and stored.

[0032] In this example, based on the above preparation method, the modification effects of tea polysaccharides with different surfactants were tested. The particle size, PDI, potential, loading amount and encapsulation efficiency of chlorogenic acid after modification of tea polysaccharides with different surfactants are shown in Table 1.

[0033] Table 1 Effects of different surfactants on TPS particle size, PDI, potential, loading capacity and encapsulation efficiency

[0034]

[0035] The results showed that the chlorogenic acid loading and encapsulation efficiency of unmodified tea polysaccharides were 89.66 mg / g and 35.82%, respectively, and the loading and encapsulation efficiency of modified tea polysaccharides were significantly improved. Cationic surfactants had the best effect on the loading and encapsulation efficiency of chlorogenic acid. Nonionic surfactants had a greater effect on the particle size of polysaccharides, among which Tween-80 had the strongest effect, and the PDI was lower than that of the original. Therefore, Tween-80 was selected in the subsequent examples to prepare modified tea polysaccharides.

[0036] Example 2

[0037] In this example, the modified tea polysaccharide-chlorogenic acid complex was prepared by the above-mentioned preparation method, and different process parameters were selected for control experiments.

[0038] Figure 1 The effects of different Tween-80 concentrations, pH, and vortex time on the particle size of Tween 80-modified tea polysaccharides (Tween 80-TPS) were demonstrated. The results of the single factor test showed that as the concentration of Tween-80 increased, the particle size of TPS decreased significantly, but when its concentration exceeded 3 mg / mL, its effect on the particle size of TPS had no significant difference ( Figure 1As the pH increases, the particle size of TPS decreases first, and then increases when the pH is higher than 4 ( Figure 1 During vortexing, TPS is subjected to a certain shear force, resulting in a decrease in particle size. However, when the vortexing time exceeds 20 seconds, the stability of the system decreases, and aggregation leads to an increase in particle size ( Figure 1 Part c in the figure). Through modeling and simulation calculation, the optimal preparation process of modified tea polysaccharide Tween 80-TPS is Tween 80 concentration = 4.6 mg / mL, pH = 3.73, and vortex time of 16 s. Under this process, about 0.124 g of Tween-80 is added to the modified tea polysaccharide sample solution.

[0039] Figure 2 The effects of different phenol-sugar mass ratios (CA:TPS), pH, compound temperature, and compound time on the binding amount of modified tea polysaccharide-chlorogenic acid complex (Tween 80-TPS-CA) were demonstrated. The results showed that with the decrease of the mass ratio of CA to TPS, the binding amount gradually decreased, and the highest binding amount was achieved when the mass ratio of the two was 4:1 (CA:TPS); polyphenols were easily degraded under superacid and superalkaline conditions, and the binding amount was the highest when pH was 8; with the increase of compound temperature, the binding amount increased, and the binding amount was the highest when the compound temperature was 35℃, and then the binding amount decreased; the compound time had little effect on the binding amount. According to modeling and simulation calculations, the optimal compounding process of Tween 80 modified tea polysaccharide-chlorogenic acid complex was a phenol-sugar mass ratio of 4, a pH of 7.83, and a compound temperature of 37.2℃.

[0040] Combining the above-mentioned optimal preparation process of Tween 80-TPS and the optimal composite process of Tween 80-TPS-CA, the optimal preparation process of Tween 80-TPS-CA can be obtained. Under this process condition, the expected particle size of the composite is 13.49nm. Experimental verification under the same conditions showed that there was no significant difference between the actual particle size and the predicted value (13.97±0.23nm).

[0041] Example 3

[0042] This embodiment provides a method for preparing tea polysaccharides, and the specific steps are as follows:

[0043] Tea polysaccharide crude preparation: After drying the fresh tea leaves, crush them, add 80% ethanol at a ratio of 1:20 (w / v) to the solid-liquid ratio, and heat in a 70℃ water bath for extraction for 1h, remove the supernatant, collect the residue, repeat the extraction twice, and place the residue in a 60℃ oven until it is fully dry for use. Subsequently, add 90℃ hot water for leaching for 2h at a ratio of 1:20 (w / v) to the solid-liquid ratio, repeat the leaching twice, combine the filtrate, place it in a rotary evaporator at 60℃ and concentrate it under reduced pressure to about 1 / 5 of the original volume to obtain a concentrated solution. Add 5 times the volume of anhydrous ethanol to the concentrated solution, place it in a 4℃ refrigerator and let it stand for precipitation overnight, collect the precipitate by centrifugation at 3000rpm for 15min, wash the precipitate with anhydrous ethanol several times, dissolve the washed precipitate with distilled water, and use the Sevage method to deproteinize the aqueous solution until no obvious protein layer is produced, and collect the supernatant. The supernatant is placed in a pretreated dialysis bag (3500Da) and dialyzed with deionized water for 48h. Then, 4 times the volume of anhydrous ethanol was added, stirred, and allowed to stand at 4°C for 24 hours. The precipitate was collected by centrifugation, washed with ethanol until the solution was colorless, the ethanol was removed by rotary evaporation, and freeze-dried to obtain crude polysaccharide.

[0044] The crude polysaccharide was purified by anion exchange chromatography using DEAE-52 cellulose, eluted with NaCl solution, concentrated, dialyzed, and vacuum freeze-dried. The purified polysaccharide was further separated and purified by gel column using G-100 dextran gel, concentrated after purification, dialyzed with running water for 48 hours, concentrated again, and vacuum freeze-dried to obtain tea polysaccharide separated and purified by gel column, which was sealed and stored.

[0045] The purity of the prepared tea polysaccharide is ≥95%.

[0046] Example 4

[0047] Prepare chlorogenic acid with a purity of ≥98%; prepare tea polysaccharides according to the method described in Example 3; use the obtained tea polysaccharides and chlorogenic acid to prepare tea polysaccharide-chlorogenic acid complex (TC) and modified tea polysaccharide-chlorogenic acid complex (TTC) according to the method described in Example 1. In the process of preparing TC, the tea polysaccharide is not modified, but the tea polysaccharide and chlorogenic acid are directly compounded by the composite method described in Example 1. In the process of preparing TTC, the non-ionic surfactant is Tween 80.

[0048] The obtained chlorogenic acid (CA), tea polysaccharides (TPS), tea polysaccharide-chlorogenic acid complex (TC) and modified tea polysaccharide-chlorogenic acid complex (TTC) were subjected to structural characterization tests, including scanning electron microscopy tests and X-ray diffraction tests.

[0049] The results of SEM test are as follows Figure 3The results showed that chlorogenic acid (CA) was needle-shaped crystals; tea polysaccharides (TPS) were regular flakes with a smooth surface; after modification with Tween 80 surfactant, the surface morphology of modified tea polysaccharides (TT) changed, showing an uneven, passivated surface with larger gaps; the composites (TC and TTC) both showed amorphous flake structures of varying sizes, partially rough surfaces, and relatively compact textures.

[0050] X-ray diffraction test results are as follows Figure 4 As shown. As can be seen from the figure, TPS has six sharp and narrow crystal diffraction peaks, with the two characteristic peaks at 19.1° and 29.9° being particularly prominent, indicating that it has a high degree of crystallinity. The diffraction peak intensities of the two complexes (TC and TTC) are greatly reduced, and some characteristic peaks disappear. Both have broad and diffuse diffraction peaks at 19.6°, indicating that the crystallinity of the complex is low. This may be because the addition of polyphenols leads to an increase in the amorphous structure of tea polysaccharides, which is of positive significance for improving the solubility of the complex and increasing its utilization rate. At the same time, the effect of the complex made from modified tea polysaccharides is more obvious.

[0051] The storage stability test of the prepared composites TC and TTC was performed. Specifically, TC and TTC were dissolved to obtain TC sample solutions and TTC sample solutions of equal concentrations, and the TC sample solutions and TTC sample solutions were placed under different environmental conditions for 20 days, respectively, and the changes in the appearance of the solutions were observed, and the polyphenol content in the solutions was detected every 5 days.

[0052] Appearance changes such as Figure 5 By comparing the colors of the two complex solutions, it can be found that under the same storage conditions, the chlorogenic acid in the modified complex TTC is more stable. Figure 6 After 20 days of storage at low temperature and in the dark, the polyphenol content of TTC was slightly higher than that of TC at multiple stages. This indicates that the polyphenol content of TC was lost more during the storage of the complex, and the polyphenols in the improved complex TTC were more stable. In general, the storage stability of TTC is better than that of TC.

[0053] The thermal stability test of the composites TC and TTC prepared above was carried out. The test results are as follows Figure 7 The results showed that as the temperature increased, the color of the composite sample liquid gradually darkened; with the increase of temperature, the polyphenol loss rate of TC was higher than that of TTC. The above results show that the thermal stability of TTC is better than that of TC.

[0054] In summary, the modified tea polysaccharide proposed in the present invention has a higher loading capacity and encapsulation efficiency for chlorogenic acid, and the modified tea polysaccharide-chlorogenic acid complex has better storage stability and thermal stability than the unmodified tea polysaccharide-chlorogenic acid complex.

[0055] Example 5

[0056] After removing the head, tail, skin and other waste materials of the silver carp, cut the fish meat into pieces. Prepare tea polysaccharide-chlorogenic acid complex (TC) and modified tea polysaccharide-chlorogenic acid complex (TTC) according to the method described in Example 1. Add 2.5% sodium chloride and 3% TC or TTC to the fish meat as cryoprotectants, and grind it in a blender to obtain fish paste. Add 2.5% sodium chloride and a mixture of 4% sucrose and 4% sorbitol to the fish meat as a positive control for cryoprotectants (PCG), and the group without adding protectants is used as a control. Store the fish paste in a refrigerator, freeze it at -20°C for 2 days, and refrigerate it at 4°C for 1 day, which is recorded as one freeze-thaw cycle. The total thiol content and surface hydrophobicity of the fish paste are analyzed after 0, 2, 4, and 6 cycles, respectively. The test results are as follows Figure 8 After adding PCG, TC, and TTC, the total thiol content of surimi in each group decreased, with the TTC group decreasing the least. In addition, the surface hydrophobicity of each group increased, among which the increase in the surface hydrophobicity of the TTC group was lower than that of the TC group and close to that of the PCG group.

[0057] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a modified tea polysaccharide-chlorogenic acid complex, characterized in that: include: S1: preparing a mixed solution of tea polysaccharides and nonionic surfactants, wherein the mass ratio of the nonionic surfactant to the tea polysaccharides is 0.4-0.5; S2: adjusting the pH value of the mixed solution to 3-4, and freeze-drying to obtain modified tea polysaccharides; S3: preparing a mixed solution of chlorogenic acid and modified tea polysaccharides, wherein the mass ratio of chlorogenic acid to modified tea polysaccharides is 3.8-4.2; S4: adjusting the pH value of the mixed solution of chlorogenic acid and modified tea polysaccharide to 6-8, stirring and compounding in a water bath to obtain a composite solution; S5: dialyzing the composite solution, and freeze-drying the retained solution obtained by the dialyzation to obtain a modified tea polysaccharide-chlorogenic acid complex.

2. The method for preparing the modified tea polysaccharide-chlorogenic acid complex according to claim 1, characterized in that: The S1 is specifically as follows: preparing a tea polysaccharide solution with a concentration of 9 mg / mL-11 mg / mL, and dissolving a nonionic surfactant with the tea polysaccharide solution to obtain a mixed solution of tea polysaccharide and nonionic surfactant, wherein the concentration of the nonionic surfactant in the mixed solution is greater than 3 mg / mL, and the mixing method is vortex mixing for 15s-20s.

3. The method for preparing the modified tea polysaccharide-chlorogenic acid complex according to claim 1, characterized in that: The nonionic surfactant is Tween-80.

4. The method for preparing the modified tea polysaccharide-chlorogenic acid complex according to claim 1, characterized in that: The pH value in S2 is adjusted by using a 1 mol / L hydrochloric acid solution; the pH value in S4 is adjusted by using a 1 mol / L NaOH solution.

5. The method for preparing the modified tea polysaccharide-chlorogenic acid complex according to claim 1, characterized in that: The S3 specifically comprises: preparing a 0.9 mg / mL-1.1 mg / mL chlorogenic acid solution, using the modified tea polysaccharide obtained in S2 to prepare a 1.8 mg / mL-2.1 mg / mL modified tea polysaccharide solution, mixing the chlorogenic acid solution with the modified tea polysaccharide solution, so that the mass ratio of chlorogenic acid to modified tea polysaccharide in the mixed solution is 3.8-4.

2.

6. The method for preparing the modified tea polysaccharide-chlorogenic acid complex according to claim 1, characterized in that: The temperature of the water bath in S4 is 35° C.-38° C., and the stirring and compounding time is 20 min-50 min.

7. The method for preparing the modified tea polysaccharide-chlorogenic acid complex according to claim 1, characterized in that: The dialysis of the composite solution described in S5 is specifically as follows: prepare a 500Da dialysis bag, boil it in boiling water for 10 minutes or more, and let it stand to cool to room temperature; transfer the composite solution to the dialysis bag, and then dialyze the dialysis bag in pure water with magnetic stirring until the dialysis equilibrium is reached.

8. The modified tea polysaccharide-chlorogenic acid complex prepared by the method of claim 1.

9. Use of the modified tea polysaccharide-chlorogenic acid complex according to claim 8 as a preservative in the preservation of seafood.

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