Method for extracting anthocyanin in blueberries by using sodium alginate immobilized enzyme

Abstract

The invention discloses the technical field of anthocyanin extraction, and particularly relates to a method for extracting anthocyanin in blueberries by using a sodium alginate immobilized enzyme. According to the method, blueberries are used as a raw material, sodium alginate is used as a carrier to immobilize cellulase, and process conditions for extracting anthocyanin are optimized, including sodium alginate concentration, enzymolysis temperature and time, enzyme concentration and enzyme proportion. The immobilized enzyme is applied to an anthocyanin extraction process, when the temperature is 50 DEG C and the reaction time is 3h, the yield of anthocyanin is improved by 18.7% compared with that of free enzyme, and the activity of the immobilized enzyme still keeps 75% or above after the immobilized enzyme is repeatedly used for five times. In addition, the sodium alginate immobilized enzyme shows good thermal stability.

Description

Technical Field

[0001] The invention relates to the technical field of anthocyanin extraction, and in particular to a method for extracting anthocyanin from blueberry by utilizing sodium alginate immobilized enzyme. Background Art

[0002] Anthocyanins are bioflavonoids with numerous health benefits, including antioxidant activity, lipid-lowering properties, cancer cell apoptosis promotion, liver protection, and intestinal flora regulation, making them highly valuable medicinally. Blueberry anthocyanins are the most potent of all plant anthocyanins, comprising 16 bioflavonoids. They possess superior physiological activity compared to typical plant anthocyanins, offering the widest range of applications and the lowest side effects.

[0003] The main methods for extracting anthocyanins include solvent extraction, microwave-assisted extraction, ultrasound-assisted extraction, and enzyme-assisted extraction. Microwave-assisted extraction requires expensive equipment and has certain limitations in solvent selection; ultrasound-assisted extraction requires high equipment requirements; and enzyme-assisted extraction is costly, has strict temperature requirements, and requires high extraction technology. Solvent extraction is the most common method for extracting anthocyanins. It is simple to operate and requires minimal equipment, but it carries safety risks, certain toxic side effects, long extraction times, low extraction yields, impurities, and high solvent consumption. Therefore, research on how to efficiently extract and purify blueberry anthocyanins is of great significance.

[0004] Immobilized enzyme technology involves fixing enzymes on inert or insoluble supports through adsorption or covalent bonding. This technology improves enzyme catalytic performance, enhances stability, and enables reuse, effectively overcoming the limitations of biological enzymes in their applications. Currently, there are no reports on the extraction of anthocyanins from blueberries using immobilized enzyme technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme, which solves the problems of low anthocyanin extraction efficiency and weak enzyme stability in the prior art.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] A method for extracting anthocyanins from blueberries using sodium alginate-immobilized enzyme, wherein the sodium alginate-immobilized enzyme is used to perform an enzymatic hydrolysis reaction on blueberry pulp, and the liquid filtered after the reaction is used as an anthocyanin extract;

[0008] The sodium alginate immobilized enzyme is prepared as follows: sodium alginate is mixed with a complex enzyme solution, and a calcium chloride solution containing glutaraldehyde is added dropwise to the mixed system to obtain the immobilized enzyme.

[0009] As a further improvement of the present invention, the temperature of the enzymatic hydrolysis reaction is 40-50° C., and the enzymatic hydrolysis time is 1.5-3 h.

[0010] As a further improvement of the present invention, the temperature of the enzymatic hydrolysis reaction is 45° C., and the enzymatic hydrolysis time is 2 hours.

[0011] As a further improvement of the present invention, the complex enzyme consists of pectinase, cellulase and hemicellulase, and the mass ratio of pectinase, cellulase and hemicellulase is (1-3): (1-2): (1-2).

[0012] As a further improvement of the present invention, the mass ratio of the pectinase, cellulase and hemicellulase is (2-3):1:1.

[0013] As a further improvement of the present invention, the system comprises, in terms of mass concentration, 0.08-2.0% sodium alginate, 0.05-0.2% complex enzyme, 0.05-0.2% glutaraldehyde, and 4-8% calcium chloride.

[0014] As a further improvement of the present invention, the system comprises, in terms of mass concentration, 1.6% sodium alginate, 0.1% complex enzyme, 0.1% glutaraldehyde concentration, and 6% calcium chloride concentration.

[0015] As a further improvement of the present invention, the system comprises, in terms of mass concentration, 2% sodium alginate, 0.1% complex enzyme, 0.05% glutaraldehyde, and 8% calcium chloride.

[0016] As a further improvement of the present invention, the following steps are included:

[0017] (1) Preparation of immobilized enzyme

[0018] Mix the sodium alginate and complex enzyme solution evenly, draw up the solution with a syringe, drop it into a calcium chloride solution containing glutaraldehyde from a height of 10 cm, fix it for 0.5 h, filter it, wash it with distilled water 2-3 times, and store it in a refrigerator;

[0019] (2) Anthocyanin extraction

[0020] Take blueberry pulp, add the immobilized enzyme, place it at 40-50°C for reaction for 1.5-3h, filter after enzymatic hydrolysis to obtain clear blueberry juice as anthocyanin extract, and wash the immobilized enzyme with distilled water for the next batch of reaction.

[0021] As a further improvement of the present invention, the activity of the immobilized enzyme is greater than 75% after being reused 5 times.

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

[0023] This invention uses blueberry pulp as raw material and sodium alginate as a carrier to immobilize cellulase, optimizing the process conditions for anthocyanin extraction. The immobilized enzyme, when applied to anthocyanin extraction at 50°C and a reaction time of 3 hours, increased anthocyanin yield by 18.7% compared to free enzyme. The immobilized enzyme retained over 75% activity after five reuses. Furthermore, the sodium alginate-immobilized enzyme exhibited good thermal stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a line graph of anthocyanin content extracted with different enzyme ratios;

[0025] Figure 2 is a bar graph of anthocyanin content at different enzyme concentrations;

[0026] Figure 3 is a bar graph of anthocyanin content at different sodium alginate concentrations;

[0027] Figure 4 is a bar graph of anthocyanin content at different calcium chloride concentrations;

[0028] Figure 5 is a bar graph of anthocyanin content at different glutaraldehyde concentrations;

[0029] Figure 6 is a histogram of anthocyanin content at different temperature concentrations;

[0030] Figure 7 It is a line graph of anthocyanin content at different temperature concentrations;

[0031] Figure 8 is the response surface model diagram of enzymatic hydrolysis temperature and time;

[0032] Figure 9 This is the response surface model diagram of enzymatic hydrolysis time and enzyme ratio;

[0033] Figure 10 This is the response surface model diagram of enzymatic hydrolysis temperature and enzyme ratio. DETAILED DESCRIPTION

[0034] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed solely to enable those skilled in the art to better understand and implement the subject matter described herein, and that changes may be made to the functions and arrangements of the elements discussed without departing from the scope of this specification.

[0035] At least one embodiment of the present invention discloses a method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme, comprising:

[0036] (1) Preparation of immobilized enzyme

[0037] Mix the sodium alginate and complex enzyme solution evenly, draw up the solution with a syringe, drop it into a calcium chloride solution containing glutaraldehyde from a height of 10 cm, fix it for 0.5 h, filter it, wash it with distilled water 2-3 times, and store it in a refrigerator;

[0038] The complex enzyme consists of pectinase, cellulase and hemicellulase, and the mass ratio of pectinase, cellulase and hemicellulase is (1-3): (1-2): (1-2);

[0039] In the mixed system: the mass concentration of sodium alginate is 0.08-2.0%, the mass concentration of complex enzyme is 0.05-0.2%, the mass concentration of glutaraldehyde is 0.05-0.2%, and the mass concentration of calcium chloride is 4-8%;

[0040] (2) Anthocyanin extraction

[0041] Take blueberry pulp, add immobilized enzyme, place it at 40-50℃ for reaction for 1.5-3h, filter after enzymatic hydrolysis to obtain clarified blueberry juice, which is used as anthocyanin extract. Wash the immobilized enzyme with distilled water for the next batch of reaction.

[0042] The recovery process of sodium alginate immobilized enzyme is as follows:

[0043] Termination reaction: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysis mixture is filtered using a 100-mesh filter to obtain a mixture of blueberry juice and blueberry immobilized enzyme.

[0044] Washing the immobilized enzyme: Add deionized water to the mixture of blueberries and immobilized enzyme, let it stand for 1 to 2 minutes, wait for the gel beads to settle, carefully pour off most of the supernatant (blueberry peel and pomace), and repeat several times until the washing liquid is clear.

[0045] Filtration method: transfer the washed sodium alginate immobilized enzyme to a sieve (100 mesh), rinse with deionized water, collect and dry with gauze.

[0046] Among them, since a complex enzyme was used, the activity of the immobilized enzyme was judged by the amount of blueberry anthocyanin extracted.

[0047] The present application will be described in further detail below in conjunction with experiments and accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0048] 1. Description

[0049] 1.1 Materials

[0050] Blueberry puree, rabbiteye blueberries pureed into pulp, was purchased from Longhai Xuyang Food Co., Ltd.

[0051] Pectinase, cellulase, and hemicellulase were purchased from Beijing Coolbo Technology Co., Ltd.;

[0052] Calcium chloride was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0053] Glutaraldehyde was purchased from Shanghai Adamas Reagent Co., Ltd.

[0054] Sodium alginate was purchased from Shanghai Adamas Reagent Co., Ltd.

[0055] 1.2 Methods

[0056] The anthocyanin content was determined by UV-visible spectroscopy at OD = 535 nm.

[0057]

[0058] Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art. The reagents and other materials used are commercially available products unless otherwise specified. The instruments used are conventional instruments known to those skilled in the art unless otherwise specified.

[0059] 2. Methods

[0060] 2.1 Immobilized enzyme for extraction of anthocyanins from blueberry

[0061] (1) Preparation of immobilized enzyme

[0062] 2% sodium alginate and 0.4% complex enzyme were diluted to 10 mL with distilled water and mixed thoroughly on a magnetic stirrer. Aspirate the solution using a 5 mL syringe and drip it into a 15 mL solution of 2% (m / v) CaCl2 containing 0.4% glutaraldehyde from a height of 10 cm. Fix the solution for 0.5 h. Filter the pellets, wash them 2-3 times with distilled water, and store them in a refrigerator at 4°C until use.

[0063] (2) Anthocyanin extraction

[0064] Prepare the blueberry jelly, thaw it in advance, and then blend it into a pulp. Accurately weigh 10g of blueberry pulp into a 25mL centrifuge tube, add the immobilized enzyme, and react in a water bath at 50°C for 2.5 hours. After enzymatic hydrolysis, filter through a double layer of gauze to obtain the clarified blueberry juice, and measure the volume. Determine the anthocyanin content at OD = 535nm. Obtain the sodium alginate-immobilized enzyme by filtration for the next batch of reactions.

[0065] Among them, the immobilized enzyme still maintained more than 75% of its activity after being reused 5 times.

[0066] 2.2 Extraction of blueberry anthocyanins using different immobilized enzymes

[0067] Gelatin (heated), agarose (heated), cellulose acetate (0.5% solvent added during preparation), and polyacrylamide were used to replace the sodium alginate in 2.1-(1), and the preparation conditions and dosages were adaptively adjusted. The enzyme ratio was 2:1:1, the enzyme concentration was 0.1%, the sodium alginate concentration was 1.6%, the glutaraldehyde concentration was 0.1%, and the calcium chloride concentration was 6%. Gelatin-immobilized enzyme, agarose-immobilized enzyme, cellulose acetate-immobilized enzyme, and polyacrylamide-immobilized enzyme were obtained.

[0068] Weigh 4 portions of 10g blueberries and perform enzymatic hydrolysis at 45°C for 2 hours. The anthocyanin extraction rate was used as the evaluation index. Each test was repeated 3 times. The results are shown in the following table:

[0069]

[0070] According to the results in the table above, sodium alginate immobilized enzyme has the best extraction effect. Gelatin immobilization and agarose immobilization require heating and dissolution. This process affects the activity of heat-sensitive enzymes, resulting in enzyme inactivation and poor immobilization effect. As a result, the anthocyanin extraction rate is extremely poor, which may be just the free anthocyanin content in the blueberry juice. Cellulose acetate immobilized enzyme requires 0.5% solvent and the extraction effect is also poor.

[0071] The polyacrylamide immobilized enzyme was recovered and found to retain only about 15% of its activity after being reused five times.

[0072] 2.3 Effect of immobilized enzyme preparation on the content of extracted anthocyanins

[0073] 2.3.1 Effect of the ratio of complex enzymes on the content of anthocyanins extracted by immobilized enzymes

[0074] Nine (10g) portions of blueberries were weighed and subjected to enzymatic hydrolysis at 50°C for 2.5 hours using an immobilized enzyme (0.4% complex enzyme, 2% sodium alginate, 0.4% glutaraldehyde, 2% calcium chloride). The effects of different complex enzyme ratios (1:1:1, 1:1:2, 1:2:1, 2:1:1, 2:1:2, 2:2:1, 3:1:1, 3:2:1, and 3:1:2) on anthocyanin extraction were investigated. Anthocyanin extraction rate was used as the evaluation metric, and each experiment was repeated three times to determine the optimal enzyme ratio for enzymatic hydrolysis.

[0075] like Figure 1 As shown, enzyme ratios of 2:1:1 and 3:1:1 both achieved good enzyme activity and high anthocyanin content. These enzyme ratios enhanced cell wall degradation efficiency, effectively disrupted plant cell wall structure, and increased anthocyanin dissolution. The lowest anthocyanin content was achieved at a concentration of 2:2:1, likely due to an inadequately coordinated enzyme ratio, which increased enzyme steric hindrance and reduced immobilized enzyme activity.

[0076] 2.3.2 Effect of enzyme concentration on anthocyanin content extracted by immobilized enzyme

[0077] Six (10g) portions of blueberries were weighed and enzymatically digested for 2.5 hours at a 2:1:1 enzyme ratio (pectinase: cellulase: hemicellulase) at 50°C. The enzymes used were immobilized (2% sodium alginate, 0.4% glutaraldehyde, 2% calcium chloride). The effects of different enzyme complex concentrations (0.05%, 0.1%, 0.2%, 0.4%, 0.6%, and 0.8%) on anthocyanin extraction were investigated. Anthocyanin extraction yield was used as the evaluation metric. Each experiment was repeated three times to determine the optimal enzyme complex concentration.

[0078] like Figure 2 As shown in the figure, as the enzyme concentration increases from 0.05% to 0.8%, the anthocyanin extraction rate shows a trend of first increasing and then decreasing. At an enzyme concentration of 0.05%, the anthocyanin dissolution efficiency is low due to insufficient enzyme active sites and limited reaction rate. When the enzyme concentration reaches the optimal value of 0.1%, the enzyme and substrate are fully combined, the hydrolysis efficiency is the highest, and the anthocyanin yield reaches its peak. The anthocyanin content decreases from 0.2%, which may be due to excessive enzyme concentration leading to increased steric hindrance between enzyme molecules, saturation of immobilized carrier adsorption, and even substrate inhibition, which in turn reduces the extraction efficiency.

[0079] 2.3.3 Effect of sodium alginate concentration on anthocyanin content extracted by immobilized enzyme

[0080] Seven (10g) portions of blueberries were weighed and enzymatically hydrolyzed for 2.5 hours at a 2:1:1 enzyme ratio (pectinase: cellulase: hemicellulase) at 50°C. The enzymes used were immobilized enzymes (0.4% complex enzyme concentration, 0.4% glutaraldehyde concentration, and 2% calcium chloride concentration). The effects of various sodium alginate concentrations (0.08%, 1.2%, 1.6%, 2.0%, 2.4%, 2.8%, and 3.2%) on anthocyanin extraction were investigated. Anthocyanin extraction yield was used as the evaluation metric. Each experiment was repeated three times to determine the optimal sodium alginate concentration for enzymatic hydrolysis.

[0081] like Figure 3As shown in the figure, as the sodium alginate concentration increases from 0.08% to 3.2%, the anthocyanin content shows a trend of first increasing and then decreasing. The anthocyanin content is highest at a sodium alginate concentration of 1.6%. At this concentration, a stable gel network can be formed, effectively fixing the enzyme molecules, thereby promoting the release and extraction of anthocyanins. The anthocyanin content at 0.08% is very low, which may be because when the concentration is too low, the gel strength is insufficient and the enzyme is prone to leakage, resulting in a decrease in immobilization efficiency and a decrease in anthocyanin yield. When the sodium alginate concentration is greater than 2.0%, it can be seen that the anthocyanin content decreases significantly. The reason may be that the concentration is too high, the gel pore size is too small or the mass transfer resistance is increased, which limits the contact between the substrate and the enzyme, and inhibits the dissolution of anthocyanins.

[0082] 2.3.4 Effect of calcium chloride on anthocyanin content extracted by immobilized enzyme

[0083] Four 5g portions of blueberries were weighed and enzymatically digested for 2.5 hours at a 2:1:1 enzyme ratio (pectinase: cellulase: hemicellulase) at 50°C. The enzymes used were immobilized enzymes (0.4% complex enzyme concentration, 0.4% glutaraldehyde concentration, and 2% sodium alginate concentration). The effects of calcium chloride concentrations (0.5%, 1%, 2%, 4%, 6%, and 8%) on anthocyanin extraction were investigated. Anthocyanin extraction rate was used as the evaluation metric. Each experiment was repeated three times to determine the optimal calcium chloride concentration for enzymatic hydrolysis.

[0084] like Figure 4 As shown in the figure, the anthocyanin extraction rate was the highest at a calcium chloride concentration of 6%. This concentration can effectively improve the stability and loading rate of the immobilized enzyme, promoting the release and extraction efficiency of anthocyanins. The anthocyanin content showed a downward trend from 4% to 0.5%, which may be due to insufficient cross-linking of the carrier, resulting in enzyme leakage or decreased activity. The low extraction rate at 8% concentration may be due to excessive cross-linking, which hinders substrate diffusion and reduces the binding ability of the enzyme to anthocyanins.

[0085] 2.3.5 Effect of glutaraldehyde on anthocyanin content extracted by immobilized enzyme

[0086] Seven (10g) portions of blueberries were weighed and enzymatically digested for 2.5 hours at a 2:1:1 enzyme ratio (pectinase: cellulase: hemicellulase) at 50°C. The enzymes used were immobilized enzymes (0.4% complex enzyme concentration, 2% calcium chloride concentration, and 2% sodium alginate concentration). The effects of glutaraldehyde concentrations (0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) on anthocyanin extraction were investigated. Anthocyanin extraction yield was used as the evaluation metric. Each experiment was repeated three times to determine the optimal glutaraldehyde concentration for enzymatic digestion.

[0087] like Figure 5As shown, there is a clear trend in the effect of glutaraldehyde concentrations between 0.05% and 1% on anthocyanins. The highest anthocyanin content was achieved at a concentration of 0.1%, indicating that this concentration effectively enhances enzyme covalent cross-linking, improving immobilization efficiency and stability, thereby promoting anthocyanin extraction. The slightly lower anthocyanin content at 0.05% may be due to insufficient cross-linking at such a low concentration, leading to easy enzyme shedding or reduced activity. Concentrations greater than 0.2% may cause excessive cross-linking, masking the enzyme's active center, reducing catalytic efficiency, and potentially destroying the anthocyanin structure, resulting in a decrease in content.

[0088] 2.4 Effect of enzymatic hydrolysis conditions on the content of extracted anthocyanins

[0089] 2.4.1 Enzymatic hydrolysis temperature

[0090] Temperature affects the enzymatic hydrolysis of blueberry anthocyanins. A 5% temperature gradient was used. Five (10g) portions of blueberries were weighed, and the hydrolysis time was 2.5 hours. The enzyme ratio was 2:1:1 (pectinase:cellulase:hemicellulase). The immobilized enzymes (0.4% complex enzyme concentration, 2% sodium alginate concentration, 0.4% glutaraldehyde concentration, and 2% calcium chloride concentration) were used. The effects of different hydrolysis temperatures (40, 45, 50, 55, and 60°C) on anthocyanin extraction yield were investigated. Using anthocyanin extraction yield as the evaluation metric, each experiment was repeated three times to determine the optimal temperature for the hydrolysis.

[0091] like Figure 6 As shown in the figure, it can be found that the anthocyanin content is the highest at 45°C. At this temperature, enzyme activity can be increased, promoting the release of anthocyanins from the plant matrix. The anthocyanin content is lower at 40°C, which may be because the temperature is too low, the enzyme reaction rate is reduced, and the anthocyanin dissolution efficiency decreases; and when the temperature exceeds 45°C, the content has been showing a downward trend, which may be because as the temperature continues to rise, the enzyme will denature and inactivate, and may destroy the anthocyanin structure, causing it to degrade or oxidize.

[0092] 2.4.2 Enzymatic hydrolysis time

[0093] Five (10g) portions of blueberries were weighed and subjected to enzymatic hydrolysis at 50°C, using a 2:1:1 enzyme ratio (pectinase:cellulase:hemicellulase). Immobilized enzymes (0.4% complex enzyme, 2% sodium alginate, 0.4% glutaraldehyde, 2% calcium chloride) were used. The effects of different enzymatic hydrolysis times (1.0h, 1.5h, 2.0h, 2.5h, and 3.0h) on anthocyanin extraction yield were investigated. Anthocyanin extraction yield was used as the evaluation metric, and each experiment was repeated three times to determine the optimal enzymatic hydrolysis temperature.

[0094] like Figure 7As shown in the figure, the highest content was obtained at a reaction time of 2 h, indicating that this time is the optimal reaction time. During this time, the enzyme and substrate are fully in contact, improving the anthocyanin extraction efficiency. The lowest content was obtained at 1 h, which may be due to the fact that the time is too short, the enzymatic reaction is not sufficient, and the anthocyanin release is incomplete. The content also showed a significant downward trend at 2.5 h and 3 h. Excessive reaction time may lead to oxidative degradation of anthocyanins, and the activity of the immobilized enzyme may decrease due to long reaction time.

[0095] 3. Conclusion

[0096] In the single-factor experiments in 2.3 and 2.4, the optimal conditions for each factor were: an enzyme ratio of 2:1:1, an enzyme concentration of 0.1%, a sodium alginate concentration of 1.6%, a glutaraldehyde concentration of 0.1%, a calcium chloride concentration of 6%, a temperature of 45°C, and a time of 2 hours. Regardless of the influence of each factor, these conditions yielded the best extraction results for blueberry anthocyanins.

[0097] Through orthogonal and response surface comprehensive analysis, after considering the interaction between various factors, such as Figure 8-10 As shown in the figure, based on the enzyme ratio of 2:1:1, temperature of 45°C and time of 2h, the optimal extraction process conditions are: enzyme concentration of 0.2%, sodium alginate concentration of 2%, glutaraldehyde concentration of 0.05% and calcium chloride concentration of 8%.

[0098] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme, characterized in that: The blueberry pulp was enzymatically hydrolyzed using sodium alginate-immobilized enzyme, and the filtered liquid after the reaction was used as anthocyanin extract; The sodium alginate immobilized enzyme is prepared as follows: sodium alginate is mixed with a complex enzyme solution, and a calcium chloride solution containing glutaraldehyde is added dropwise to the mixed system to obtain the immobilized enzyme.

2. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 1, characterized in that: The enzymatic hydrolysis reaction temperature is 40-50° C., and the enzymatic hydrolysis time is 1.5-3 hours.

3. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 2, characterized in that: The temperature of the enzymatic hydrolysis reaction is 45° C., and the enzymatic hydrolysis time is 2 h.

4. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 1, characterized in that: The complex enzyme consists of pectinase, cellulase and hemicellulase, and the mass ratio of the pectinase, cellulase and hemicellulase is (1-3): (1-2): (1-2).

5. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 4, characterized in that: The mass ratio of the pectinase, cellulase and hemicellulase is (2-3):1:

1.

6. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 1, characterized in that: Calculated by mass concentration, the system comprises: 0.08-2.0% sodium alginate, 0.05-0.2% complex enzyme, 0.05-0.2% glutaraldehyde, and 4-8% calcium chloride.

7. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 6, characterized in that: Calculated by mass concentration, the system includes: 1.6% sodium alginate, 0.1% complex enzyme, 0.1% glutaraldehyde, and 6% calcium chloride.

8. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 6, characterized in that: Calculated by mass concentration, the system includes: sodium alginate concentration of 2%, complex enzyme concentration of 0.1%, glutaraldehyde concentration of 0.05%, and calcium chloride concentration of 8%.

9. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 1, characterized in that: The following steps are involved: (1) Preparation of immobilized enzyme Mix the sodium alginate and complex enzyme solution evenly, draw up the solution with a syringe, drop it into a calcium chloride solution containing glutaraldehyde from a height of 10 cm, fix it for 0.5 h, filter it, wash it with distilled water 2-3 times, and store it in a refrigerator; (2) Anthocyanin extraction Take blueberry pulp, add the immobilized enzyme, place it at 40-50°C for reaction for 1.5-3h, filter after enzymatic hydrolysis to obtain clear blueberry juice as anthocyanin extract, and wash the immobilized enzyme with distilled water for the next batch of reaction.

10. The method for extracting anthocyanins from blueberries using sodium alginate immobilized enzyme according to claim 9, characterized in that: The activity of the immobilized enzyme is greater than 75% after being reused 5 times.

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