Blueberry extract efficient extraction method based on gradient temperature control synergistic enzymolysis

Abstract

The invention relates to an efficient blueberry extract extraction method based on gradient temperature control synergistic enzymolysis, and belongs to the technical field of natural product extraction. The method comprises the following steps: (1) raw material pretreatment: freezing blueberries to-18 DEG C or below-18 DEG C, and then crushing the blueberries to 20-40 meshes to obtain frozen and crushed blueberries; (2) gradient temperature control enzymolysis: the enzymolysis process is divided into three stages: in the initial stage, the temperature is controlled to be 38 + / -1 DEG C, and the enzymolysis time is 15-30 minutes; in the main reaction stage, the temperature is controlled to be 50 + / -1 DEG C, and the enzymolysis time is 45-90 minutes; in the final stage, the temperature is controlled to be 42 + / -1 DEG C, and the enzymolysis time is 20-30 minutes; and (3) eluting, purifying and drying to obtain a dried blueberry extraction product.

Description

Technical Field

[0001] This application relates to the technical field of natural product extraction, and particularly relates to an efficient extraction method for blueberry extracts based on gradient temperature control and enzymatic hydrolysis in cooperation. Background Art

[0002] Anthocyanidin, also known as anthocyanin, is a water-soluble natural pigment widely present in plants in nature. Anthocyanidin is mainly used for food coloring, and can also be used in dyes, medicine, cosmetics, etc. Research shows that anthocyanidin is a pure natural anti-aging nutritional supplement, and is the more effective antioxidant discovered by humans today. Its antioxidant performance is fifty times higher than that of vitamin E and two hundred times higher than that of vitamin C. The anthocyanidin content in blueberries far exceeds that of other fruits such as apples and grapes, so it is a better raw material object for anthocyanidin extraction.

[0003] The extraction methods of anthocyanidin generally include solvent method, carbon dioxide supercritical extraction method, biological complex enzymatic hydrolysis method, etc. For example, in the "Extraction, Separation and Purification Method of a Kind of Blueberry Anthocyanidin" with the publication number CN111978280B, the method includes washing and drying blueberry fruits, soaking them in brine, then washing and drying them with clean water, and performing crushing treatment to obtain blueberry pulp; adding mixed enzyme I and performing enzymatic hydrolysis to obtain pulp enzymatic hydrolysate I; adding an acidic ethanol solution as an extractant and placing it in a container for sealing treatment; performing ultrasonic extraction to obtain an ultrasonic extraction product, and performing vacuum filtration to obtain a crude extract; adding mixed enzyme II, standing for enzymatic hydrolysis, and vacuum concentrating the enzymatic hydrolysate; eluting and purifying the concentrated solution, and vacuum concentrating and freeze-drying the eluate to obtain a dried blueberry anthocyanidin extraction product. Although this method realizes the efficient extraction of anthocyanidin, not only does it need to add different mixed enzymes for enzymatic hydrolysis twice during the extraction process, but also ethanol extraction and ultrasonic extraction are required. The entire extraction process is cumbersome and has a long process.

[0004] Improving the extraction rate of blueberry anthocyanidin, shortening the process flow, expanding the types of enzymes used, and facilitating large-scale production have always been pursued by those skilled in the art. Summary of the Invention

[0005] To solve or partially solve the problems existing in the related technologies, this application provides an efficient extraction method for blueberry extracts based on gradient temperature control and enzymatic hydrolysis in cooperation, which expands the types of enzymes used, has a high extraction rate of anthocyanidin and a short extraction process, and this method is suitable for large-scale production.

[0006] An efficient extraction method for blueberry extracts based on gradient temperature control and enzymatic hydrolysis in cooperation in this application includes the following steps: (1) Raw material pretreatment: Freeze blueberries to -18°C or below and then crush them to 20 - 40 mesh to obtain frozen and crushed blueberries; (2) Gradient temperature-controlled enzymatic hydrolysis: Mix the freeze-crushed blueberries with the complex enzyme and ethanol solution and then carry out enzymatic hydrolysis; the mass ratio of the freeze-crushed blueberries, ethanol solution and complex enzyme is 1:5 - 10:0.3 - 0.6; Among them, the enzymatic hydrolysis process is divided into three stages: the initial stage controls the temperature at 38 ± 1 °C and enzymatic hydrolysis for 15 - 30 min; the main reaction stage controls the temperature at 50 ± 1 °C and enzymatic hydrolysis for 45 - 90 min; the final stage controls the temperature at 42 ± 1 °C and enzymatic hydrolysis for 20 - 30 min; (3) Elution, purification and drying: The enzymatic hydrolysate is concentrated, then eluted and purified, and the eluate is vacuum concentrated and freeze-dried to obtain the dried blueberry extract.

[0007] Further, the complex enzyme is: cellulase 3000 U / g, pectinase 5000 U / g and β-glucosidase 2000 U / g, and the mass ratio of the three is 3:5:2.

[0008] Further, the complex enzyme is: cellulase 3000 U / g, 5000 U / g pectinase, 3000 U / g papain, and the mass ratio of the three is 3:4:4.

[0009] Further, the complex enzyme is: 3000 U / g cellulase, 2500 U / g sucrase, 1000 U / g tannase, and the mass ratio of the three is 1:2:1.

[0010] Further, the volume fraction of ethanol in the ethanol solution is 60 - 80%.

[0011] Further, in the step (2), the mass ratio of the freeze-crushed blueberries, ethanol solution and complex enzyme is 1:8:0.5; the complex enzyme is: cellulase 3000 U / g, pectinase 8000 U / g and β-glucosidase 2000 U / g, and the mass ratio of the three is 3:5:2.

[0012] Further, in the initial stage of the step (2), the temperature is controlled at 38 ± 1 °C and enzymatic hydrolysis is carried out for 30 min; in the main reaction stage, the temperature is controlled at 50 ± 1 °C and enzymatic hydrolysis is carried out for 90 min; in the final stage, the temperature is controlled at 42 ± 1 °C and enzymatic hydrolysis is carried out for 20 min.

[0013] The beneficial effects of this application are: 1. When extracting anthocyanins from blueberries in this application, first, the blueberries are frozen. The frozen blueberries can fully retain volatile components during the crushing process. Second, the crushed blueberries are subjected to gradient temperature-controlled enzymatic hydrolysis. The complex enzyme is first activated at a relatively low temperature in the initial stage, then at a relatively high temperature in the main reaction stage to efficiently degrade the cell wall and release the components to be extracted. Finally, at a temperature between that of the initial stage and the main reaction stage in the final reaction stage, a certain activity of the complex enzyme is maintained to continuously release the intracellular components. This application creatively uses the combination of crushing after freezing treatment and gradient temperature-controlled synergistic enzymatic hydrolysis to extract anthocyanins from blueberries. Crushing after freezing treatment can, on the one hand, retain more anthocyanins, and on the other hand, it can better physically destroy the cell wall structure during crushing after freezing, facilitating the release of anthocyanins during subsequent enzymatic hydrolysis. Further, during the gradient temperature-controlled synergistic enzymatic hydrolysis process, through the cooperation of the complex enzyme with specific gradient temperature control, the enzyme activity is activated in the initial stage, the cell wall is efficiently degraded chemically in the main reaction stage, and the enzyme activity is maintained in the final stage to continuously release the intracellular components. By adopting the above method, the extraction rate of anthocyanins is significantly improved.

[0014] 2. During the process of producing anthocyanins using the enzymatic hydrolysis method, the activity and stability of the enzyme are crucial, directly related to the yield of anthocyanins and the efficiency of the entire anthocyanin extraction. The complex enzyme and gradient temperature control in this application can stably and efficiently complete enzymatic hydrolysis in a short time based on the frozen crushing of blueberries, and this method is suitable for large-scale production. Specific Embodiments

[0015] The embodiments of the present application will be described in more detail below with reference to the examples. Although the embodiments show the embodiments of the present application, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art. Example 1

[0016] A high-efficiency extraction method for blueberry extract based on gradient temperature-controlled synergistic enzymatic hydrolysis, comprising the following steps: (1) Raw material pretreatment: Freeze the picked blueberry fruits to -18°C and then crush them to 20 - 40 meshes to obtain frozen and crushed blueberries; (2) Gradient temperature-controlled enzymatic hydrolysis: The freeze - pulverized blueberries are mixed with a complex enzyme and an ethanol solution with a volume fraction of 60% and then enzymolyzed; the mass ratio of the freeze - pulverized blueberries, the ethanol solution and the complex enzyme is 1:10:0.3; the complex enzyme is: cellulase 3000 U / g, pectinase 8000 U / g and β - glucosidase 2000 U / g, and the mass ratio of the three is 3:5:2; Among them, the enzymolysis process is divided into three stages: the initial stage controls the temperature at 38±1°C and enzymolyzes for 20 min; the main reaction stage controls the temperature at 50±1°C and enzymolyzes for 90 min; the final stage controls the temperature at 42±1°C and enzymolyzes for 20 min; (3)Elution, purification, drying: After the enzymolysis solution is vacuum - concentrated at 45°C to one - fifth of the original volume, it is loaded onto an AB - 8 macroporous resin column. The loading volume is 0.5 times the resin column volume, adsorbed for 8 h, rinsed with distilled water, the eluate is discarded, and then eluted with an ethanol solution with a volume fraction of 60% for 3 times the resin volume and the eluate is collected. The eluate is vacuum - concentrated at 45°C and freeze - dried at - 40°C for 24 h to obtain blueberry anthocyanin extract. Example 2

[0017] An efficient extraction method of blueberry extract based on gradient temperature control and synergistic enzymolysis, comprising the following steps: (1)Raw material pretreatment: The picked blueberry fruits are frozen to - 30°C and then pulverized to 20 - 40 mesh to obtain freeze - pulverized blueberries; (2)Gradient temperature - controlled enzymolysis: The freeze - pulverized blueberries are mixed with a complex enzyme and an ethanol solution with a volume fraction of 75% and then enzymolyzed; the mass ratio of the freeze - pulverized blueberries, the ethanol solution and the complex enzyme is 1:8:0.2; the complex enzyme is: cellulase 3000 U / g, pectinase 5000 U / g, papain 3000 U / g, and the mass ratio of the three is 3:4:4; Among them, the enzymolysis process is divided into three stages: the initial stage controls the temperature at 38±1°C and enzymolyzes for 30 min; the main reaction stage controls the temperature at 50±1°C and enzymolyzes for 45 min; the final stage controls the temperature at 42±1°C and enzymolyzes for 30 min; (3)Elution, purification, drying: After the enzymolysis solution is vacuum - concentrated at 50°C to one - fifth of the original volume, it is loaded onto an AB - 8 macroporous resin column. The loading volume is 0.5 times the resin column volume, adsorbed for 6 h, rinsed with distilled water, the eluate is discarded, and then eluted with an ethanol solution with a volume fraction of 75% for 3 times the resin volume and the eluate is collected. The eluate is vacuum - concentrated at 50°C and freeze - dried at - 40°C for 36 h to obtain blueberry anthocyanin extract. Example 3

[0018] An efficient extraction method of blueberry extract based on gradient temperature control and coenzymolysis, comprising the following steps: (1) Raw material pretreatment: Freeze the picked blueberries to -18°C and then crush them to 20-40 meshes to obtain frozen and crushed blueberries; (2) Gradient temperature control enzymolysis: Mix the frozen and crushed blueberries with a complex enzyme and an ethanol solution with a volume fraction of 80% and then carry out enzymolysis; the mass ratio of the frozen and crushed blueberries, the ethanol solution and the complex enzyme is 1:6:0.5; the complex enzyme is: 3000 U / g cellulase, 2500 U / g sucrase, 1000 U / g tannase, and the mass ratio of the three is 1:2:1; Among them, the enzymolysis process is divided into three stages: the initial stage controls the temperature at 38±1°C and enzymolyzes for 20 min; the main reaction stage controls the temperature at 50±1°C and enzymolyzes for 90 min; the final stage controls the temperature at 42±1°C and enzymolyzes for 20 min; (3) Elution, purification, and drying: After the enzymolysis solution is vacuum concentrated at 45°C to one-fifth of the original volume, it is loaded onto a column filled with AB-8 macroporous resin. The loading amount is 0.5 times the resin column volume, adsorbed for 8 h, washed with distilled water, and the eluate is discarded. Then, it is eluted with an ethanol solution with a volume fraction of 60% for 3 times the resin volume and the eluate is collected. The eluate is vacuum concentrated at 45°C and freeze-dried at -40°C for 30 h to obtain blueberry anthocyanin extract. Example 4

[0019] The difference between Example 4 and Example 1 lies in that during the gradient temperature control enzymolysis process: the initial stage controls the temperature at 38±1°C and enzymolyzes for 30 min; the main reaction stage controls the temperature at 50±1°C and enzymolyzes for 90 min; the final stage controls the temperature at 42±1°C and enzymolyzes for 20 min; the rest are the same.

[0020] Comparative Example 1 Group 1: In Group 1, it was not frozen to -18°C and was directly crushed to 20-40 meshes at room temperature of 20°C, and the rest were the same as in Example 1.

[0021] Group 2: In Group 2, it was not frozen to -18°C, and the blueberries were frozen to 0°C and then crushed to 20-40 meshes, and the rest were the same as in Example 1.

[0022] Comparative Example 2 Group 1: The extraction method of Example 1 was adopted, and the difference was that: in Group 1, gradient temperature control enzymolysis was not carried out, but the whole enzymolysis process was carried out under the condition of controlling the temperature at 50±1°C, and the enzymolysis time was 130 min.

[0023] Group 2: The extraction method of Example 1 was adopted, with the difference that in Group 2, gradient temperature-controlled enzymolysis was not carried out, but the whole enzymolysis process was carried out under the condition of controlling the temperature at 50±1°C for 480 min.

[0024] Group 3: The extraction method of Example 2 was adopted, with the difference that in Group 3, gradient temperature-controlled enzymolysis was not carried out, but the whole enzymolysis process was carried out under the condition of controlling the temperature at 50±1°C for 480 min.

[0025] Group 4: The extraction method of Example 3 was adopted, with the difference that in Group 4, gradient temperature-controlled enzymolysis was not carried out, but the whole enzymolysis process was carried out under the condition of controlling the temperature at 50±1°C for 480 min.

[0026] Comparative Example 3 Investigate the effect of different gradient enzymolysis times on the extraction rate of anthocyanins Group 1: The extraction method of Example 1 was adopted, with the difference that in Group 1, the temperature was controlled at 38±1°C for 10 min in the initial stage of the enzymolysis process; the temperature was controlled at 50±1°C for 90 min in the main reaction stage; the temperature was controlled at 42±1°C for 20 min in the final stage. Group 2: The extraction method of Example 1 was adopted, with the difference that in Group 1, the temperature was controlled at 38±1°C for 60 min in the initial stage of the enzymolysis process; the temperature was controlled at 50±1°C for 90 min in the main reaction stage; the temperature was controlled at 42±1°C for 20 min in the final stage. Group 3: The extraction method of Example 1 was adopted, with the difference that in Group 1, the temperature was controlled at 38±1°C for 20 min in the initial stage of the enzymolysis process; the temperature was controlled at 50±1°C for 30 min in the main reaction stage; the temperature was controlled at 42±1°C for 20 min in the final stage. Group 4: The extraction method of Example 1 was adopted, with the difference that in Group 1, the temperature was controlled at 38±1°C for 20 min in the initial stage of the enzymolysis process; the temperature was controlled at 50±1°C for 120 min in the main reaction stage; the temperature was controlled at 42±1°C for 20 min in the final stage. Group 5: The extraction method of Example 1 was adopted, with the difference that in Group 1, the temperature was controlled at 38±1°C for 20 min in the initial stage of the enzymolysis process; the temperature was controlled at 50±1°C for 90 min in the main reaction stage; the temperature was controlled at 42±1°C for 10 min in the final stage. Group Six: The extraction method of Example 1 was adopted, except that in Group One, the temperature was controlled at 38±1°C in the initial stage of the enzymatic hydrolysis process for 10 min, at 50±1°C in the main reaction stage for 90 min, and at 42±1°C in the final stage for 60 min. The extraction rates of blueberry anthocyanins in the products obtained in Examples 1-4 and Comparative Examples 1-3 were measured. It should be noted that the extraction rate of blueberry anthocyanins was measured by the method in the existing technology. For the specific measurement method, reference can be made to the dual-wavelength differential method in Zhang Panpan, Wang Li, Shi Zhijun, Optimization of Ultrasonic-Assisted Extraction Process of Anthocyanins from Blueberry Pomace [J]. Food & Machinery, 2017(2) to measure the extraction rates of three groups of blueberry anthocyanins.

[0027] The specific measurement method of the blueberry anthocyanin extraction rate is as follows: Take 1 g of the product and dissolve it in 1 ml of 60% acidic ethanol (containing 0.02% glacial acetic acid). Add 9 ml of pH 4.5 buffer solution (0.4 mol / L sodium acetate) and pH 1.0 buffer solution (0.25 mol / L potassium chloride) respectively, shake well, let stand for 15 min, transfer it into a cuvette with an optical path length of 1 cm, and use 60% acidic ethanol (containing 0.02% glacial acetic acid) to replace the sample solution as the blank control, and measure the absorbance at wavelengths of 520 nm and 700 nm respectively.

[0028]

[0029] The measurement results are shown in Tables 1, 2 and 3. Among them, Table 1 shows the anthocyanin extraction rates of the products obtained in Examples 1-4, Table 2 shows the anthocyanin extraction rates of the products obtained in Comparative Examples 1-2, and Table 3 shows the anthocyanin extraction rates of the products obtained in Comparative Example 3. Table 1 Anthocyanin extraction rates of the products obtained in Examples 1-4 Serial number Example 1 Example 2 Example 3 Example 4 Extraction rate (mg / 100g) 652.25 649.41 642.36 655.58 Table 2 Anthocyanin extraction rates of the products obtained in Comparative Examples 1-2 Serial number Group 1 in Comparative Example 1 Group 2 in Comparative Example 1 Group 1 in Comparative Example 2 Group 2 in Comparative Example 2 Group 3 in Comparative Example 2 Group 4 in Comparative Example 2 Extraction rate (mg / 100g) 457.79 535.18 393.65 462.77 447.28 473.33 Table 3 Serial number Group 1 in Comparative Example 3 Group 2 in Comparative Example 3 Group 3 in Comparative Example 3 Group 4 in Comparative Example 3 Group 5 in Comparative Example 3 Group 6 in Comparative Example 3 Extraction rate (mg / 100g) 237.37 641.25 496.27 654.58 618.33 656.67 Result description: As can be seen from Table 1, by using the method in the present application, enzymatic hydrolysis was carried out with some commonly used complex enzymes in the existing technology, combined with the post-freezing crushing treatment and gradient temperature-controlled enzymatic hydrolysis in the present application, good anthocyanin extraction effects can be achieved, and the anthocyanin extraction effect of the above single complex enzyme is better than that of enzymatic hydrolysis in the case of using two complex enzymes disclosed in the existing technology. The results in Table 1 fully illustrate that the method in the present application effectively broadens the application of the types of enzymes.

[0030] As can be seen from Table 2, the pretreatment of blueberries before enzymatic hydrolysis in Comparative Example 1 has an important impact on the extraction of anthocyanins. Compared with enzymatic hydrolysis of blueberries crushed at room temperature and 0 °C, enzymatic hydrolysis after freezing blueberries to -18 °C and below can significantly improve the extraction effect of anthocyanins.

[0031] As can be seen from Table 3, the time control at each stage of gradient temperature-controlled enzymatic hydrolysis affects the enzymatic hydrolysis effect. If the time for the activation enzyme process in the initial stage is too short, the activity of the enzyme cannot be effectively stimulated, resulting in a low extraction rate of anthocyanins. At the same time, if the time in the main reaction stage is too short, the extraction rate of anthocyanins will be reduced, and when the reaction has proceeded for a certain time, extending the time in the main reaction stage basically has no great promoting effect on the extraction of anthocyanins; in addition, extending the reaction time in the final stage also basically has no great promoting effect on the extraction of anthocyanins. Considering the extraction rate and extraction efficiency, the reaction times selected for each stage in this application are optimal.

[0032] The embodiments of the present application have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, the practical application, or the improvement of the technology in the market, or to enable other ordinary skill in the art in the technical field to understand the disclosed embodiments.

Claims

1. An efficient extraction method of blueberry extract based on gradient temperature control and enzymatic hydrolysis in coordination, characterized in that, It includes the following steps: (1) Raw material pretreatment: Freeze blueberries to -18°C or below and then crush them to 20 - 40 mesh to obtain frozen and crushed blueberries; (2) Gradient temperature-controlled enzymatic hydrolysis: Mix the frozen and crushed blueberries with a complex enzyme and an ethanol solution and then carry out enzymatic hydrolysis; the mass ratio of the frozen and crushed blueberries, the ethanol solution and the complex enzyme is 1:5 - 10:0.3 - 0.6; Among them, the enzymatic hydrolysis process is divided into three stages: control the temperature at 38 ± 1°C in the initial stage and carry out enzymatic hydrolysis for 15 - 30 min; control the temperature at 50 ± 1°C in the main reaction stage and carry out enzymatic hydrolysis for 45 - 90 min; control the temperature at 42 ± 1°C in the final stage and carry out enzymatic hydrolysis for 20 - 30 min; (3) Elution, purification, and drying: Concentrate the enzymatic hydrolysis solution, then carry out elution and purification, and vacuum-concentrate and freeze-dry the eluate to obtain the dried blueberry extract.

2. The high-efficiency extraction method of blueberry extract based on gradient temperature control and enzymatic hydrolysis according to claim 1, characterized in that, The complex enzyme is: cellulase 3000 U / g, pectinase 5000 U / g, and β-glucosidase 2000 U / g, and the mass ratio of the three is 3:5:

2.

3. The high-efficiency extraction method of blueberry extract based on gradient temperature control and enzymatic hydrolysis according to claim 1, characterized in that, The complex enzyme is: cellulase 3000 U / g, pectinase 5000 U / g, papain 3000 U / g, and the mass ratio of the three is 3:4:

4.

4. The efficient extraction method of blueberry extract based on gradient temperature control and coenzymatic hydrolysis according to claim 1, characterized in that, The complex enzyme is: cellulase 3000 U / g, invertase 2500 U / g, tannase 1000 U / g, and the mass ratio of the three is 1:2:

1.

5. The high-efficiency extraction method of blueberry extract based on gradient temperature control and enzymatic hydrolysis according to claim 1, characterized in that, The volume fraction of ethanol in the ethanol solution is 60 - 80%.

6. The high-efficiency extraction method of blueberry extract based on gradient temperature control and enzymatic hydrolysis according to claim 1, characterized in that In the step (2), the mass ratio of the frozen and crushed blueberries, the ethanol solution and the complex enzyme is 1:8:0.5; the complex enzyme is: cellulase 3000 U / g, pectinase 8000 U / g, and β-glucosidase 2000 U / g, and the mass ratio of the three is 3:5:

2.

7. The high-efficiency extraction method of blueberry extract based on gradient temperature control and synergistic enzymatic hydrolysis according to claim 1, characterized in that, In the step (2), control the temperature at 38 ± 1°C in the initial stage and carry out enzymatic hydrolysis for 30 min; control the temperature at 50 ± 1°C in the main reaction stage and carry out enzymatic hydrolysis for 90 min; control the temperature at 42 ± 1°C in the final stage and carry out enzymatic hydrolysis for 20 min.

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