Photo-crosslinked chitosan immobilized tannase, preparation method thereof and application of photo-crosslinked chitosan immobilized tannase in treatment of tea extract

Abstract

The invention relates to photo-crosslinking chitosan immobilized tannase, a preparation method thereof and application of the photo-crosslinking chitosan immobilized tannase in treatment of a tea extract. According to the invention, methacrylic anhydride is successfully grafted to the surface of chitosan to prepare UV cross-linked CS-MA, and then tannase is anchored on the CS-MA hydrogel through NHS to successfully complete immobilization. The immobilized tannase degrades methyl gallate, and the activity, stability and reusability of the enzyme can be improved. The immobilized tannase is more ideal for improving the pH value, color and clarity of tea soup compared with free enzyme, and the improvement on the pH value is particularly remarkable.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to photo-crosslinked chitosan immobilized tannase and a preparation method thereof and application thereof in processing tea extracts. Background Art

[0002] Tannase (enzyme code EC3.1.1.20) is a hydrolase mainly derived from microorganisms such as Aspergillus niger, Aspergillus oryzae, Penicillium and Kluyveromyces. It is widely used in food industry, beverage industry, pharmaceutical industry and other fields, and is an important industrial enzyme.

[0003] Tannase can hydrolyze the ester bonds and carboxylphenolic acid bonds in polyphenolic substances to generate gallic acid and corresponding alcohols, and can accelerate the oxidation reaction of polyphenolic substances such as caffeine, tea polyphenols, and resveratrol in an acidic environment, thereby affecting the color, aroma, taste, and shelf life of food and beverages. Tannase is used to decompose the ester bonds of certain tannins in tea soup, thereby preventing the reaction of tannins with caffeine and improving the stability of tea soup. The ester catechins in tea soup can be hydrolyzed into non-ester catechins such as epigallocatechin and epicatechin, making the tea soup sweet. After tannase treatment, the recovery rate of polyphenols in the tea extraction process is greatly improved, and the biological activity of tea extracts (such as antioxidant activity) is also improved. Shao et al. cloned a tannase from Aspergillus niger. This tannase can exert optimal catalytic activity at 80°C, and its enzymatic reaction significantly increases the total polyphenol content of green tea extract. Although enzymatic treatment of tea extract has many advantages, such as high catalytic efficiency, mild reaction conditions, and low pollution, its disadvantages are insufficient stability and difficulty in recycling the enzyme after use, which makes the process costly and difficult to implement. Therefore, people use various carriers and different fixation processes to immobilize tannase. Compared with traditional enzymatic treatment, immobilized tannase technology can significantly reduce process costs, improve production efficiency, and be more environmentally friendly.

[0004] Immobilized enzymes provide a new way for enzyme applications, effectively enhancing stability, improving catalytic efficiency, and reducing use costs. However, there are still some technical bottlenecks in immobilized enzymes, and further research is needed on carrier types and immobilization methods to improve immobilization efficiency, stability, and the number of reuses, and on this basis, explore new application areas to improve industrial production efficiency and product quality.

[0005] Methyl gallate in tea extract can be degraded into gallic acid and methanol by tannase. The obtained gallic acid has good biological activities such as antioxidant, anti-tumor and antiviral. However, the degradation of methyl gallate by free tannase is inefficient and costly. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a photo-crosslinked chitosan immobilized tannase and a preparation method thereof and an application thereof in treating tea extracts, so as to enable the tannase to achieve better catalytic activity and catalytic efficiency.

[0007] The present invention first provides a method for preparing photo-crosslinked chitosan immobilized tannase, comprising the following steps:

[0008] (1) dissolving chitosan in an acetic acid solution, dropping methacrylic anhydride into the solution, reacting at a constant temperature, adjusting the pH value to 7.0 after the reaction, dialyzing, adding a photoinitiator, stirring evenly, and then irradiating under ultraviolet light to solidify into a methacrylic anhydride-modified chitosan hydrogel;

[0009] Chitosan is shrimp shell chitosan;

[0010] The molar ratio of methacrylic anhydride to the amino group on chitosan is 2:1-6:1, preferably 4:1;

[0011] The reaction temperature is 30-80°C, preferably 60°C;

[0012] The reaction is carried out under stirring conditions, and the stirring rate is 100-400r / min;

[0013] The reaction time is 2-10 hours, preferably 6 hours;

[0014] (2) Tannase and N-hydroxysuccinimide ester are mixed in a citric acid buffer, and then added into a methacrylic anhydride-modified chitosan hydrogel, stirred and incubated to obtain immobilized tannase.

[0015] The mass ratio of tannase to N-hydroxysuccinimide ester is (2-20):15;

[0016] The ratio of tannase to methacrylic anhydride modified chitosan hydrogel is 0.1-1 mg / mL, preferably 0.5 mg / mL;

[0017] The pH of the citrate buffer is 4-8, preferably pH 7.

[0018] The incubation time is 2-12 hours, preferably 9 hours.

[0019] The photo-cross-linked chitosan-immobilized tannase prepared by the preparation method is disclosed.

[0020] Application of tannin immobilized on photocrosslinked chitosan in the degradation of methyl gallate. The degradation conditions were pH 6 and temperature 50℃.

[0021] Application of tannase immobilized on photocross-linked chitosan in processing tea soup.

[0022] The present invention successfully grafts methacrylic anhydride onto the surface of chitosan to prepare CS-MA that can be cross-linked by ultraviolet light, and then anchors tannase on the CS-MA hydrogel through NHS to successfully complete immobilization. The optimal pH for the degradation of methyl gallate by immobilized tannase is 6.0, and its stability between pH 4-8 is better than that of free tannase. Through testing at an ambient temperature between 30°C and 80°C, it was found that its optimal reaction temperature is 50°C. The thermal stability of immobilized tannase at 50°C is slightly stronger than that of free tannase, but its thermal stability at 70°C is greatly improved compared to free tannase. It can be seen that immobilization treatment can improve the activity, stability and reusability of the enzyme. The improvement of pH, color and clarity of tea soup by immobilized tannase is also more ideal than that of free enzyme, among which the improvement of pH is particularly significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :BCA protein curve;

[0024] Figure 2 : UV full spectrum of methyl gallate;

[0025] Figure 3 : Standard curve for enzyme activity determination;

[0026] Figure 4 : Relative enzyme activities of free tannase and immobilized tannase at different pH;

[0027] Figure 5 : Relative enzyme activities of free tannase and immobilized tannase at different temperatures;

[0028] Figure 6 : Residual activity of free tannase and immobilized tannase after different time at 50℃ and 70℃;

[0029] Figure 7 : The results of tea soup treated by free tannase and immobilized tannase. Tea soup G1 is the initial green tea soup; G2 is the green tea soup treated by free tannase; G3 is the green tea soup treated by immobilized tannase; B1 is the initial black tea soup; B2 is the black tea soup treated by free tannase; B3 is the black tea soup treated by immobilized tannase. DETAILED DESCRIPTION

[0030] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0031] Example 1

[0032] A method for preparing photo-crosslinked chitosan immobilized tannase comprises the following steps:

[0033] Take a 100mL beaker and prepare a 1% (v / v) acetic acid solution. Use an electronic balance to weigh 0.5g CS and add it to the acetic acid solution. Stir it at room temperature on a magnetic stirrer until it is completely dissolved. After dissolution, use a 1000μL pipette to draw 1.75mL of MA according to the molar ratio of MA to the amino group on CS of 4:1, and slowly drip it into the beaker. Set the magnetic stirrer temperature to 60℃ and keep the above conditions at 200r / min for 6h. After the reaction stops, use saturated NaHCO 3 The solution was adjusted to pH 7.0 to obtain CS-MA. CS-MA was transferred to a dialysis bag with a molecular weight cutoff of 3500D and dialyzed in deionized water for 3 days. Then, 0.05% (w / v) photoinitiator was added, mixed evenly with a magnetic stirrer, and irradiated under a UV lamp for 10 minutes to cure into methacrylic acid to successfully modify chitosan hydrogel.

[0034] 10 mg of tannase and 15 mg of NHS were simply mixed in 2 mL of citric acid buffer (pH 5.0) at room temperature, and then the above solution was added to 20 mL of CS-MA, that is, the ratio of enzyme feed to carrier was 0.5 mg / mL. The mixture was incubated at 30°C and 150 r / min for 9 hours. Finally, immobilized tannase was obtained.

[0035] Embodiment 2-5

[0036] The dosage of tannase was adjusted to 2, 5, 15, and 20 mg, that is, the ratio of enzyme dosage to carrier was 0.1 mg / mL, 0.25 mg / mL, 0.75 mg / mL, and 1 mg / mL, and the rest was the same as in Example 1.

[0037] Embodiment 6-9

[0038] The pH value of the citric acid buffer was adjusted to 4, 6, 7, and 8, and the rest was the same as in Example 1.

[0039] Examples 10-13

[0040] The incubation time was adjusted to 2, 4, 6, and 12 hours, and the rest was the same as in Example 1.

[0041] The fixed yield and enzyme activity of the products prepared in Examples 1-13 were determined under the following conditions:

[0042] The free enzyme content in the supernatant after immobilization of enzyme was determined using the BCA protein detection kit to calculate the immobilization yield. Bovine serum albumin (BSA) solutions with concentrations of 1.0 mg / mL, 0.8 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.2 mg / mL, and 0.1 mg / mL were prepared, and PBS buffer was used as the blank group. A powder of BCA reagent 1 was dissolved and mixed with reagent 1 to prepare reagent 1 application solution, and the working solution was prepared at a ratio of reagent 1 application solution: reagent 2 = 50:1. 200 μL of working solution and 10 μL of BSA solution of different concentrations were added to a 96-well plate, and then the plate was left to stand at room temperature of 30°C for 30 minutes, and the absorbance was measured at 570 nm using an enzyme reader and the data was recorded. The protein concentration-OD value standard curve was fitted using Origin software.

[0043] Soak 1g of CS-MA with fixed tannase in citric acid-sodium citrate buffer and centrifuge at 10000rpm for 5min. Take the supernatant and use BCA kit to determine the enzyme content. Perform three parallel experiments. Calculate the fixed yield using the standard curve:

[0044] Y = 100 × [(R0-R1) / R0];

[0045] Where R0 is the total amount of enzyme added, and R1 is the amount of enzyme in the supernatant after immobilization.

[0046] Gallic acid can be complexed with rhodanine and develop color under the action of KOH, so the tannase activity is measured by measuring the amount of gallic acid produced by methyl gallate. Weigh 0.092g (5μmol) of methyl gallate and dissolve it in 100mL of citric acid-sodium citrate buffer to prepare a 0.5μmol / mL methyl gallate solution, and use this as the mother solution to prepare methyl gallate solutions with concentrations of 0.4μmol / mL, 0.25μmol / mL, 0.1μmol / mL, and 0.05μmol / mL. Use a pipette to draw 0.5mL of the above methyl gallate solutions of each concentration, then weigh 10mg of tannase and add it to the substrate. Incubate the mixture solution at 50℃ for a sufficient time to completely degrade methyl gallate. Subsequently, add 0.3mL of rhodanine methanol (5.5mg / mL) solution and incubate at 30℃ for 5 minutes. Then add 0.2mL KOH solution (0.5mol / L) to the above mixture and incubate for further 5 minutes at 30°C. Finally, dilute the total solution to 4mL, aspirate 200μL of each sample in a 96-well plate, and measure the absorbance at 520nm using a microplate reader. Use Origin software to fit the methyl gallate concentration-OD value standard curve. One unit of enzyme activity is defined as the amount of enzyme required to catalyze 1μmol of methyl gallate per minute at 30°C.

[0047] The immobilization efficiency of tannase was determined by measuring the enzyme activity:

[0048] E = (A1 / A0) × 100%;

[0049] A0 is the free enzyme activity, and A1 is the immobilized enzyme activity.

[0050] The immobilization yields and immobilization efficiencies of Examples 1-13 are shown in Table 1.

[0051] Table 1 Immobilization yield and immobilization efficiency of Examples 1-13

[0052] Fixed yield / % Fixed efficiency / % Example 1 32.5 87.1 Example 2 15.0 78.2 Example 3 28.3 81.4 Example 4 25.1 72.6 Example 5 21.7 60.5 Example 6 22.1 58.3 Example 7 34.8 89.2 Example 8 35.2 91.8 Example 9 25.7 62.1 Example 10 12.3 92.1 Embodiment 11 19.7 89.4 Example 12 28.5 84.1 Embodiment 13 32.5 77.9

[0053] It can be seen from Table 1 that both the fixation yield and the fixation efficiency are the highest when the enzyme feed amount is 0.5 mg / mL, so the optimal enzyme feed amount is 0.5 mg / mL. The fixation yields at pH 6 and 7 are not much different, but the fixation efficiency of the immobilized enzyme at pH 7 is slightly higher than that at pH 6, so the optimal pH for immobilization is 7. The fixation yield increases with time, but there is no significant increase at 9h, which may be due to the saturation of the enzyme adsorbed by the carrier. The fixation efficiency slowly decreases with time. Overall, the optimal immobilization time is 9h.

[0054] Protein concentration standard curve: Prepare BSA solutions with concentrations of 1.0 mg / mL, 0.8 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, and use PBS buffer as the blank group. Use the BCA kit to detect and measure the absorbance of each concentration of BSA at 570 nm on an ELISA reader to fit the protein concentration standard curve. The experimental results are as follows: Figure 1 According to the protein concentration standard curve, the regression equation of protein concentration and absorbance was obtained, Y = 0.306X + 0.142, and the correlation coefficient R2 = 0.9901, indicating that the protein concentration and absorbance have a good linear relationship.

[0055] Enzyme activity standard curve: Tannase activity is measured by measuring the amount of gallic acid produced by the degradation of methyl gallate. First, prepare methyl gallate solutions with concentrations of 0.5μmol / mL, 0.4μmol / mL, 0.25μmol / mL, 0.1μmol / mL, and 0.05μmol / mL. After adding an equal amount of tannase to fully degrade, use 5.5mg / mL rhodanine methanol solution and 0.5mol / L KOH solution for color development. Then, use citric acid-sodium citrate buffer as a blank, use a UV-Vis spectrophotometer to measure, and then draw the UV full spectrum of the above solutions. The results are as follows Figure 2 ,according to Figure 2It was found that the absorbance of each solution was the highest at 520nm, and the absorbance change trend was consistent with the concentration gradient change trend. Therefore, the absorbance was measured at 520nm using an enzyme reader to draw a standard curve for enzyme activity determination. Methyl gallate solution was prepared again according to the above concentration, and the absorbance was measured at 520nm using a microplate reader after color development using rhodanine methanol solution and KOH solution. The standard curve for enzyme activity determination was fitted. The experimental results are shown in Figure 2. Figure 3 According to the standard curve, the regression equation of methyl gallate concentration and absorbance was obtained: Y = 1.156X + 0.201 correlation coefficient R2 = 0.9921, indicating that the concentration of methyl gallate and absorbance have a good linear relationship.

[0056] Relative activities of free tannase and immobilized tannase prepared in Example 1 under different pH conditions:

[0057] Prepare buffer solutions with pH values ​​of 4, 5, 6, 6.5, 7, and 8, control the reaction temperature at 30°C, add free tannase and immobilized tannase to 1 μmol / mL methyl gallate solution and incubate for 5 min, then use rhodanine methanol solution and KOH solution for color development, measure the absorbance at 520 nm, and repeat the experiment three times. Figure 3 The concentration of degraded methyl gallate was calculated using the standard curve. The optimal free enzyme activity was selected as 100% relative enzyme activity. The experimental results after data conversion are as follows: Figure 4 shown.

[0058] Depend on Figure 4 It can be seen that pH has a great influence on the activity of tannase. The activity of free tannase reaches the maximum value at pH 5.0, while the activity of immobilized tannase reaches the maximum at pH 6.0. When the environmental pH is too high or too low, the catalytic ability of tannase will be significantly reduced. At pH 4-8, the deviation between the maximum and minimum values ​​of the relative enzyme activity of immobilized tannase is only 23.4%, while the deviation between the maximum and minimum values ​​of the relative enzyme activity of free enzyme is 41.5%. These results may be caused by the buffering effect of the immobilized carrier on the microenvironment of the catalytic reaction. In short, compared with free tannase, CS-MA immobilized tannase is less sensitive to pH changes and has better adaptability to different pH.

[0059] Relative activities of free tannase and immobilized tannase prepared in Example 1 under different temperature conditions:

[0060] The pH condition was set to 5.5. Both free tannase and immobilized tannase had good catalytic activity at this pH. Five temperature gradients were set: 30°C, 40°C, 50°C, 60°C, and 70°C. Free tannase and immobilized tannase were added to 1 μmol / mL methyl gallate solution and incubated for 5 min. After color development, the absorbance was measured at 520 nm. Three sets of repeated experiments were performed. The optimal free enzyme activity was selected as 100% relative enzyme activity. The experimental results are shown in Figure 2. Figure 5 shown.

[0061] Depend on Figure 5 It can be seen that under the optimum pH conditions, the best enzyme activity of both free tannase and immobilized tannase is around 50°C, indicating that the immobilized enzyme did not affect the optimal reaction temperature of the enzyme in this experiment. However, when the ambient temperature was between 30°C and 70°C, the deviation between the maximum and minimum relative enzyme activity of the immobilized tannase was 21.9%, while the deviation between the maximum and minimum relative enzyme activity of the free enzyme was 37.5%. It can be inferred that although the CS-MA hydrogel carrier did not change the optimal temperature of the enzyme catalytic reaction, it still protected the active site of the tannase, preventing it from losing too much activity at higher or lower temperatures.

[0062] Thermal stability analysis of free tannase and immobilized tannase prepared in Example 1:

[0063] The conditions of the thermal stability experiment were set at 50℃ and 70℃, and the changes in the enzyme activity of free tannase and immobilized tannase within 3 hours were measured. The pH was controlled at 5.5, and the enzyme activity was measured at 5min, 30min, 60min, 90min, 120min, 150min, and 180min. Three sets of repeated experiments were performed, and the experimental results are shown in the figure. Figure 6 shown.

[0064] from Figure 6 It can be seen that the activity of free tannase decreased by 75.1% after only 30 minutes at 70℃, and basically lost its activity after being placed at 70℃ for 180 minutes, while the activity of immobilized tannase decreased by 41.7% after being placed at 70℃ for 30 minutes, and there was still 36.3% residual activity after 180 minutes. At the optimal temperature of 50℃, the activity of free tannase and immobilized tannase did not decrease much. The activity of free tannase decreased by 21.2% after being placed at 50℃ for 30 minutes, and the activity of immobilized tannase decreased by 19.9% ​​after being placed at 50℃ for 30 minutes. After 180 minutes, the remaining activities of free tannase and immobilized tannase were 45.9% and 55.7%, respectively. From this analysis, it can be concluded that the use of CS-MA to immobilize tannase can effectively improve the thermal stability of tannase.

[0065] Embodiment 14

[0066] Weigh 4 grams of green tea powder and black tea powder respectively, soak them in 100 mL of distilled water, place them in a magnetic stirrer and stir them at 300 r / min at 100°C for 0.5 h. After cooling to room temperature, filter with filter paper and collect the filtrate in a 200 mL conical flask.

[0067] 20 mL of the filtered tea infusion was mixed with 10 mg of free or immobilized tannase prepared in Example 1, and then incubated at the optimal temperature with stirring for 4 hours. After the reaction was completed, the sample was heated at 100° C. for 5 minutes to denature the enzyme, and centrifuged at 10,000 rpm for 5 minutes to collect the supernatant.

[0068] Using a UV-Vis spectrophotometer, with distilled water as blank, the OD value was measured at 640nm as the basis for judging the transmittance of the tea soup, and the OD value was measured at 420nm as the basis for judging the color of the tea soup. A pH meter was used to detect the change in pH of the tea soup before and after the reaction.

[0069] Table 2 Changes of green tea soup before and after tannase treatment

[0070]

[0071] Table 3 Changes of black tea soup before and after tannase treatment

[0072]

[0073] According to Table 1 and Table 2, it can be seen that after treatment with immobilized tannase, the pH and color of both green tea and black tea soup have been improved, and the effect is better than that of free tannase. The improvement of pH by immobilized enzyme is particularly obvious, which may be because the amino groups on the surface of CS-MA react with substances with carboxylic acid groups in the tea soup, resulting in the improvement of pH by immobilized tannase better than free tannase. In the treatment of black tea soup, immobilized tannase greatly improved the transmittance of tea soup, that is, improved the clarity of tea soup, but the transmittance of tea soup after free enzyme treatment is not much different. This is most likely because the CS-MA hydrogel as an immobilized carrier has a certain adsorption effect on impurities in black tea soup.

[0074] Figure 7 The results of free tannase and immobilized tannase on tea soup are shown in Figure 2. It can be seen that the improvement of pH, color and clarity of tea soup by immobilized tannase is more ideal than that by free enzyme, and the improvement of pH is particularly significant.

[0075] The principles, steps, etc. not explicitly described in the present invention are all obtainable by those skilled in the art through conventional technical means, and therefore will not be described in detail. Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing photo-crosslinked chitosan immobilized tannase, characterized in that: The following steps are involved: (1) dissolving chitosan in an acetic acid solution, dropping methacrylic anhydride into the solution, reacting at a constant temperature, adjusting the pH value to 7.0 after the reaction, dialyzing, adding a photoinitiator, stirring evenly, and then irradiating under ultraviolet light to solidify into a methacrylic anhydride-modified chitosan hydrogel; (2) Tannase and N-hydroxysuccinimide ester are mixed in a citric acid buffer, and then added into a methacrylic anhydride-modified chitosan hydrogel, stirred and incubated to obtain immobilized tannase.

2. The method for preparing a photo-crosslinked chitosan-immobilized tannase according to claim 1, characterized in that: In step (1): Chitosan is shrimp shell chitosan; The molar ratio of methacrylic anhydride to the amino group on chitosan is 2:1-6:1; The reaction temperature is 30-80°C; The reaction is carried out under stirring conditions; The reaction time is 2-10 hours.

3. The method for preparing a photo-crosslinked chitosan-immobilized tannase according to claim 2, characterized in that: In step (1): The molar ratio of methacrylic anhydride to the amino groups on chitosan is 4:1; The reaction temperature is 60°C; The reaction is carried out under stirring conditions, and the stirring rate is 100-400r / min; The reaction time is 6h.

4. The method for preparing a photo-crosslinked chitosan-immobilized tannase according to claim 1, characterized in that: In step (2): The mass ratio of tannase to N-hydroxysuccinimide ester is (2-20):15; The ratio of tannase to methacrylic anhydride-modified chitosan hydrogel was 0.1-1 mg / mL; The pH of the citric acid buffer is 4-8; The incubation time is 2-12h.

5. The method for preparing a photo-crosslinked chitosan-immobilized tannase according to claim 4, characterized in that: In step (2): The mass ratio of tannase to N-hydroxysuccinimide ester was 10:15; The ratio of tannase to methacrylic anhydride-modified chitosan hydrogel was 0.5 mg / mL; The pH of the citric acid buffer is 7; The incubation time is 9h.

6. The photo-cross-linked chitosan-immobilized tannase prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the photo-cross-linked chitosan-immobilized tannase according to claim 6 in the degradation of methyl gallate.

8. The use according to claim 7, wherein the degradation conditions are pH 6 and temperature 50°C.

9. Use of the photo-cross-linked chitosan-immobilized tannase according to claim 6 in treating tea soup.

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