A preparation method of whole soybean milk with low β-conglycinin allergenicity

Abstract

The present invention relates to the technical field of soymilk preparation, and discloses a method for preparing whole soybean milk with low β-conglycinin allergenicity. The preparation method of the present invention comprises the following steps: 1) adding glycosidase to the whole soybean milk obtained after soybean dehulling and grinding, and heating to the glycosidase activity temperature for enzymatic hydrolysis; 2) after enzymatic hydrolysis in step 1), heating to 45-55 °C, and adding flavor protease and composite cellulase for enzymatic hydrolysis for 20-30 min; 3) placing the whole soybean milk after enzymatic hydrolysis in step 2) under ultra-high pressure and continuing enzymatic hydrolysis for 10-20 min; 4) after enzymatic hydrolysis, boiling the soymilk and inactivating the enzyme activity to obtain whole soybean milk with low allergenicity. In the whole soybean milk prepared by the method of the present invention β β-conglycinin is basically completely degraded, the allergenicity reduction rate reaches more than 88%, and the whole soybean milk has a good taste. Moreover, the enzymatic hydrolysis time and the overall desensitization process time are greatly shortened, effectively reducing energy consumption, and being suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of soy milk preparation, and particularly relates to a method for preparing whole soy milk with low β-conglycinin allergenicity. Background Art

[0002] Soy milk is a traditional beverage obtained by cooking soybeans after grinding. It contains rich functional components such as protein, natural dietary fiber, soybean oligosaccharides, soy isoflavones, soybean lecithin, and various vitamins. However, soy milk prepared by traditional processes has allergenicity and is prone to allergic reactions such as respiratory, skin, and gastrointestinal symptoms. The allergenicity of soy milk is caused by soy allergenic proteins such as 15S, 11S, 7S, and 2S, such as soy globulin of the 11S class, β-conglycinin of the 7S class, Gly m Bd 30kDa (P34), and Gly m Bd 28kDa. β-conglycinin and soy globulin are two main allergenic proteins with strong allergenicity. In particular, the α subunit of β-conglycinin, namely Gly m Bd 60kDa, can cause allergic reactions in 25% of susceptible populations, making β-conglycinin have a higher allergenic probability than other allergenic proteins at the same content. Reducing the content of β-conglycinin is not only the key to reducing the allergenicity of soy protein but also conducive to improving the safety characteristics of soybeans. Therefore, when reducing the allergenicity of soybeans, not only should the overall low allergenicity of whole soy milk be maintained, but also the removal effect of β-conglycinin should be ensured.

[0003] Currently, methods for removing soy allergenic proteins include heat treatment, enzymatic hydrolysis, chemical reagent method, ultra-high pressure method, glycosylation method, etc. However, chemical reagent residues may pose new food safety hazards, and protein glycosylation may cause risks of complications in diabetic patients. More importantly, any of the above single methods has poor removal effects on soy allergenic proteins, especially cannot specifically reduce the allergenicity of β-conglycinin. Chinese patent application CN201110362795.7 discloses a method for reducing the allergenicity of soy protein isolate for soy-based infant formula, by combining ultra-high pressure and enzymatic hydrolysis to treat soy protein isolate, and the allergenicity of the soy protein isolate product is reduced by 88% - 90%. However, the object treated by this method is soy protein isolate, which has a large difference from whole soy milk and cannot be directly applied to whole soy milk. On the one hand, enzymatic hydrolysis will cause the soy milk to taste bitter and have a poor flavor. On the other hand, the components contained in whole soy milk are more complex than those of soy protein isolate, and this method cannot ensure the basic removal of β-conglycinin in whole soy milk. Moreover, this method requires long-term enzymatic hydrolysis after ultra-high pressure treatment, with high energy consumption and high cost. Summary of the Invention

[0004] Aiming at the problem that the soy allergenic protein in whole soybean milk is likely to cause allergic reactions, the purpose of the present invention is to provide a preparation method of whole soybean milk with low β-conglycinin allergenicity. By combining glycosidase treatment, enzymatic hydrolysis and ultra-high pressure, and screening suitable enzymes, the allergenicity in whole soybean milk can be significantly reduced, especially β-conglycinin is basically removed, and the obtained whole soybean milk has a good taste.

[0005] The present invention provides the following technical solutions:

[0006] A preparation method of low-allergenic whole soybean milk, comprising the following steps:

[0007] 1) Add glycosidase to the whole soybean milk obtained after soybean dehulling and grinding, and raise the temperature to the glycosidase activity temperature for enzymatic hydrolysis;

[0008] 2) After the enzymatic hydrolysis in step 1), raise the temperature to 45-55 °C, and add flavor protease and cellulase for enzymatic hydrolysis for 20-30 min;

[0009] 3) Place the whole soybean milk after the enzymatic hydrolysis in step 2) under ultra-high pressure and continue enzymatic hydrolysis for 10-20 min;

[0010] 4) After the enzymatic hydrolysis, cook the soybean milk and inactivate the enzyme activity to obtain low-allergenic whole soybean milk.

[0011] In the process of preparing whole soybean milk by the method of the present invention, glycosidase treatment, complex enzymatic hydrolysis method and ultra-high pressure treatment technology act on soybean allergens in a cross-coordinated manner. By screening suitable complex enzymes, antigenic epitopes related to allergy are directionally excised and changed, effectively reducing allergenicity and maintaining the product quality stability. Through SDS-PAGE analysis, β-conglycinin is basically completely degraded, and the reduction rate of allergenicity caused is 90-99%. The reduction rate of allergenicity of whole soybean milk reaches 88%-96%. Moreover, the whole soybean milk has a good taste, and the enzymatic hydrolysis time and the overall desensitization process time are significantly shortened compared with the prior art, effectively reducing energy consumption and being suitable for industrial production.

[0012] As a preference of the method of the present invention, in step 1), the glycosidase is one or two of endoglycosidase H and glycopeptidase A.

[0013] As a preference of the method of the present invention, in step 1), the addition amount of glycosidase is 0.008%-0.032% of the mass of whole soybean milk, and / or, the enzymatic hydrolysis temperature is 35-40 °C, and the enzymatic hydrolysis time is 20-30 min.

[0014] As a preference of the method of the present invention,

[0015] in step 2), the addition amount of flavor protease is 0.008%-0.032% of the mass of whole soybean milk;

[0016] And / or, the addition amount of cellulase is 0.008% - 0.032% of the mass of whole soybean milk. Cellulase is a complex enzyme system such as glucanase and β-glucosidase. Through screening, it is found that the combined use of cellulase and flavor protease in a certain amount can effectively enhance the directional removal of β-conglycinin in this application, etc., and improve the taste of whole soybean milk.

[0017] As a preference of the method of the present invention,

[0018] In step 3), the pressure of ultra-high pressure is 300 - 400 MPa.

[0019] As a preference of the method of the present invention,

[0020] In step 3), the enzymatic hydrolysis temperature under ultra-high pressure is 45 - 55 °C.

[0021] As a preference of the method of the present invention, in the process of peeling and grinding the whole soybean milk used in step 1): the soybeans are roasted, peeled and then finely ground, and then mixed with water for grinding, and after degassing, they are treated by high-pressure microfluidics to obtain whole soybean milk. The high-pressure microfluidics technology is used to extract whole-grain soybeans at low temperature, reducing the thermal loss of soybean components, solving the key problems in the traditional soy milk grinding process such as the need for slurry residue separation, resulting in a large loss of beneficial components such as dietary fiber and functional polysaccharides, a viscous and rough taste, and the generation of wastewater and waste residue.

[0022] As a preference of the method of the present invention,

[0023] The particle size after fine grinding is 80 - 100 mesh;

[0024] And / or, the mass ratio of soybean to water is 1:3 - 7;

[0025] And / or, the pressure of high-pressure microfluidics is 100 - 140 MPa.

[0026] As a preference of the method of the present invention, in step 4), instantaneous steam and material mixing is used to inactivate enzyme activity. An instantaneous steam and fluid food mixing heating device is used to solve the problems of untimely and incomplete enzyme inactivation, thermal damage to soybean nutrition, too long time for the enzyme inactivation process in soymilk boiling, unstable heating, and high energy consumption, which affect the product quality.

[0027] As a preference of the method of the present invention,

[0028] The steam temperature is 100 - 105 °C;

[0029] And / or, after enzyme inactivation, keep warm at 90 - 100 °C for 3 - 5 min.

[0030] The beneficial effects of the present invention are as follows:

[0031] The method for preparing low-allergenic whole soybean milk provided by the present invention: 1) The overall allergenicity of the whole soybean milk is low, reaching 88-96%, the contained β-conglycinin is basically degraded, and the reduction rate of the induced allergenicity reaches 90-99%; 2) The enzymolysis time is greatly shortened, effectively reducing energy consumption, and is suitable for industrial production; 3) The whole soybean milk has a good taste, no bitter taste, is delicate and smooth, is rich in high natural dietary fiber and high-quality protein, has a balanced nutrition, a strong sense of satiety, and is easily accepted by more consumers. Detailed implementation manners

[0032] The following further describes the detailed implementation manners of the present invention.

[0033] Unless otherwise specified, the raw materials used in the present invention can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are all conventional methods in the art.

[0034] Example 1

[0035] A method for preparing low-allergenic whole soybean milk, the steps are as follows:

[0036] 1) Select non-moldy soybeans, bake them in an oven at 70°C for 60 minutes, crush them to 40 meshes in a peeling machine, and then finely crush them to 100 meshes. Mix them with water at a mass ratio of 1:5 and grind them into slurry. After degassing the obtained soybean milk, process it in a high-pressure microfluidic device at a pressure of 100 MPa to obtain whole soybean milk. Add endoglycosidase H with a mass ratio of 0.02% to the whole soybean milk and enzymolyze it at 35°C for 30 minutes;

[0037] 2) Heat the whole soybean milk after enzymolysis in step 1) to 50°C, and add flavor protease and cellulase for enzymolysis for 25 minutes. The addition amount of flavor protease is 0.02% of the mass of the whole soybean milk; the addition amount of cellulase is 0.008% of the mass of the whole soybean milk;

[0038] 3) Place the whole soybean milk after enzymolysis in step 2) under ultra-high pressure and continue enzymolysis for 20 minutes. The ultra-high pressure is 400 MPa, and the enzymolysis temperature is 50°C;

[0039] 4) After enzymolysis, cook the soybean milk and instantaneously mix it with steam to inactivate the enzyme activity. The steam temperature is 100°C, and keep it at 100°C for 5 minutes after inactivating the enzyme activity to obtain low-allergenic whole soybean milk.

[0040] Example 2

[0041] A method for preparing low-allergenic whole soybean milk, the steps are as follows:

[0042] 1) Select soybeans without mildew and bake them in an oven at 80 °C for 30 min. Then place them in a peeling machine and crush them to 20 mesh, and then finely crush them to 80 mesh. Then mix them with water at a mass ratio of 1:3 and grind them into a slurry. Degas the obtained soy milk and then process it in a high-pressure microfluidic device at a pressure of 140 MPa to obtain whole soybean milk. Add glycopeptidase A accounting for 0.008% by mass of the whole soybean milk and enzymatically hydrolyze for 30 min;

[0043] 2) Heat the whole soybean milk after enzymatic hydrolysis in step 1) to 45 °C, and add flavor protease and cellulase for enzymatic hydrolysis for 30 min. The addition amount of flavor protease is 0.008% of the mass of the whole soybean milk, and the addition amount of cellulase is 0.032% of the mass of the whole soybean milk;

[0044] 3) Place the whole soybean milk after enzymatic hydrolysis in step 2) under ultra-high pressure and continue enzymatic hydrolysis for 15 min. The ultra-high pressure is 300 MPa, and the enzymatic hydrolysis temperature is 45 °C;

[0045] 4) After enzymatic hydrolysis, cook the slurry and instantaneously mix it with steam to inactivate the enzyme activity. The steam temperature is 105 °C, and keep it at 100 °C for 3 min after inactivating the enzyme activity to obtain low-allergenic whole soybean milk.

[0046] Example 3

[0047] A preparation method of low-allergenic whole soybean milk is as follows:

[0048] 1) Select soybeans without mildew and bake them in an oven at 75 °C for 50 min. Then place them in a peeling machine and crush them to 30 mesh, and then finely crush them to 90 mesh. Then mix them with water at a mass ratio of 1:7 and grind them into a slurry. Degas the obtained soy milk and then process it in a high-pressure microfluidic device at a pressure of 120 MPa to obtain whole soybean milk. Add endoglycosidase H and enzymatically hydrolyze at 38 °C for 25 min. The addition amount of endoglycosidase H is 0.032% of the mass of the whole soybean milk;

[0049] 2) Heat the whole soybean milk after enzymatic hydrolysis in step 1) to 55 °C, and add flavor protease and cellulase (brand number) for enzymatic hydrolysis for 20 min. The addition amount of flavor protease is 0.032% of the mass of the whole soybean milk; the addition amount of cellulase is 0.02% of the mass of the whole soybean milk;

[0050] 3) Place the whole soybean milk after enzymatic hydrolysis in step 2) under ultra-high pressure and continue enzymatic hydrolysis for 10 min. The ultra-high pressure is 350 MPa, and the enzymatic hydrolysis temperature is 55 °C;

[0051] 4) After enzymatic hydrolysis, cook the slurry and instantaneously mix it with steam to inactivate the enzyme activity. The steam temperature is 100 °C, and keep it at 90 °C for 5 min after inactivating the enzyme activity to obtain low-allergenic whole soybean milk.

[0052] Control group

[0053] The whole soybean milk prepared in step 1) of Example 1 was first maintained at 352 MPa of ultra-high pressure for 16 min (the pressure increasing rate was 250 MPa / min, and the pressure releasing rate was 300 MPa / min), then enzymatically hydrolyzed with a complex enzyme of flavor protease and cellulase for 2.5 h at a temperature of 50 °C, the mass ratio of flavor protease to cellulase was 1:1, and the total addition amount was 2.49%. After enzymatic hydrolysis, the enzyme activity was inactivated by heating to obtain whole soybean milk.

[0054] Comparative Example 1

[0055] The difference from Example 1 was that the whole soybean milk obtained after high-pressure microfluidization treatment in step 1) was heated to 50 °C, first treated at 400 MPa of ultra-high pressure for 20 min, then flavor protease and cellulase were added and enzymatically hydrolyzed at 50 °C for 25 min. The addition amount of flavor protease was 0.02% of the mass of the whole soybean milk, and the addition amount of cellulase was 0.008% of the mass of the whole soybean milk. After the enzymatic hydrolysis, the steps of boiling the soybean milk and instantaneously inactivating the enzyme activity in step 4) were carried out.

[0056] Comparative Example 2

[0057] The difference from Comparative Example 1 was that endoglycosidase H / glycopeptidase A with a mass ratio of 0.02% was first added to the whole soybean milk obtained after high-pressure microfluidization treatment in step 1), enzymatically hydrolyzed at 35 °C for 30 min, then heated to 50 °C, treated at 400 MPa of ultra-high pressure for 20 min, then flavor protease and cellulase were added and enzymatically hydrolyzed at 50 °C for 25 min. The addition amount of flavor protease was 0.02% of the mass of the whole soybean milk, and the addition amount of cellulase was 0.008% of the mass of the whole soybean milk. After the enzymatic hydrolysis, the steps of boiling the soybean milk and instantaneously inactivating the enzyme activity in step 4) were carried out.

[0058] Comparative Example 3

[0059] The difference from Comparative Example 1 was that flavor protease and cellulase were added and enzymatically hydrolyzed under the same conditions first, and then continued to be enzymatically hydrolyzed under the same ultra-high pressure conditions for 20 min.

[0060] Comparative Example 4

[0061] The difference from Example 1 was that after enzymatic hydrolysis in step 2), the enzyme activity was inactivated first, and then the ultra-high pressure treatment in step 3) was carried out. There was no enzymatic hydrolysis during the ultra-high pressure treatment process.

[0062] Comparative Example 5

[0063] The difference from Example 1 is that in step 1), the whole soybean milk after glycosidase treatment is heated to 50°C, flavor protease and cellulase are added. The addition amount of flavor protease is 0.02% of the mass of the whole soybean milk, and the addition amount of cellulase is 0.008% of the mass of the whole soybean milk. Enzymatic hydrolysis is carried out at a super high pressure of 400 MPa for 20 min. The pressure of the super high pressure is [pressure value not provided in the original], and the enzymatic hydrolysis temperature is 50°C. Then, the steps of cooking the soybean milk and instantaneously steaming to inactivate the enzyme activity in step 4) are carried out.

[0064] Comparative Example 6

[0065] The difference from Example 1 is that the whole soybean milk after glycosidase treatment in step 1) is subjected to enzymatic hydrolysis at a super high pressure of 400 MPa for 20 min, and then the steps of cooking the soybean milk and instantaneously steaming to inactivate the enzyme activity in step 4) are carried out.

[0066] Comparative Example 7

[0067] The difference from Example 1 is that after adding flavor protease and cellulase to the whole soybean milk after glycosidase treatment in step 1), it is first treated under the same super high pressure conditions, then the pressure is released and enzymatic hydrolysis is continued for 25 min. After the enzymatic hydrolysis is completed, the steps of cooking the soybean milk and instantaneously steaming to inactivate the enzyme activity in step 4) are carried out.

[0068] Comparative Example 8

[0069] The difference from Example 1 is that in step 2), an equal amount of pectinase is used to replace cellulase.

[0070] Comparative Example 9

[0071] The difference from Example 1 is that in step 2), an equal amount of neutral protease is used to replace cellulase.

[0072] Comparative Example 10

[0073] The difference from Example 1 is that in step 2), an equal amount of α - amylase is used to replace flavor protease.

[0074] Comparative Example 11

[0075] The difference from Example 1 is that in step 2), an equal amount of glucose enzyme is used to replace flavor protease.

[0076] Comparative Example 12

[0077] The difference from Example 1 is that in step 2), the dosage of cellulase is 0.002%, and the dosage of flavor protease remains unchanged.

[0078] The full soybean milk obtained from the above-mentioned examples, control group, and comparative examples was filtered through a double-stage filter with a filtration mesh size of 120 meshes, followed by UHT sterilization (temperature 140 °C, time 2 s), and then PET bottling to obtain the finished product. The degree of reduction in the allergenicity of the full soybean milk was analyzed by SDS-PAGE, and the taste of the full soybean milk was recorded. The results are shown in Table 1 below.

[0079] Table 1 Test Results

[0080]

[0081] As can be seen from the above table, the full soybean milk prepared by the method of the present application has lower allergenicity. As shown in Examples 1 to 3, not only is the overall allergenicity reduction rate of the full soybean milk reduced to 88% - 96%, but also β-conglycinin can be basically removed, with an allergenicity reduction rate of 90 - 99%. There is no bitter taste, the taste is good, and the entire allergenicity removal time is only 50 - 80 minutes.

[0082] It can be seen from the control group and Comparative Example 1 that when the enzyme system based on the present application adopts the ultra-high pressure + enzymatic hydrolysis method in the prior art, on the one hand, if the enzymatic hydrolysis time is short, the ideal effect cannot be achieved. For example, in Comparative Example 1, the comprehensive reduction rate is only 59%, and the reduction rate of β-conglycinin allergenicity is 63%. On the other hand, even if the enzymatic hydrolysis time is extended to 2.5 h, the comprehensive allergenicity reduction rate can be increased to 87%. However, the reduction rate of β-conglycinin is only 85%, indicating that there is still a large room for improvement. At the same time, it should be noted that the comprehensive allergenicity reduction rate is higher than that of glycinin and β-conglycinin. This may be because other allergenic components in whole soybean milk are removed more effectively as the enzymatic hydrolysis time prolongs, which indicates that a high comprehensive allergenicity reduction does not mean the basic removal of β-conglycinin and glycinin. It can be seen from the analysis of Comparative Example 2 that even if glycosidase treatment is further introduced on the basis of ultra-high pressure + enzymatic hydrolysis in Comparative Example 1, due to the lack of synchronous and synergistic operation of ultra-high pressure and enzymatic hydrolysis, and the short enzymatic hydrolysis time, the overall effect is not ideal. This can also be seen from Comparative Example 7, indicating the importance of the synchronous and synergistic operation of ultra-high pressure and enzymatic hydrolysis in the desensitization of whole soybean milk. As shown in Comparative Example 3, the effect is also not good when the glycosidase treatment process is omitted on the basis of Example 1, because glycosidase performs deglycosylation treatment and cleaves the peptide chain, creating conditions for enzymatic hydrolysis and ultra-high pressure / enzymatic hydrolysis. As shown in Comparative Example 4, when the enzymatic hydrolysis operation in the ultra-high pressure process is omitted, due to the short enzymatic hydrolysis time, the removal effect is mainly achieved by the ultra-high pressure process. As shown in Comparative Example 5, if there is no intermediate enzymatic hydrolysis as a transition and direct glycoside treatment is followed by ultra-high pressure / enzymatic hydrolysis, the activity of the enzyme cannot be fully exerted during the ultra-high pressure process. As shown in Comparative Example 6, the effect of using glycosidase + ultra-high pressure / glycosidase operation is also inferior to that of Comparative Example 3, indicating the importance of enzymatic hydrolysis. The above Comparative Examples 1-7 demonstrate the cross-synergistic effect among glycosidase, enzymatic hydrolysis, and synchronous and synergistic operation of ultra-high pressure / enzymatic hydrolysis in the method of the present invention. Each process is indispensable and the order cannot be adjusted. They jointly constitute the whole method of the present invention as an inseparable part.

[0083] However, only considering from the operation process, the expected effect still cannot be achieved. This is because through the above analysis, it can be found that enzymatic hydrolysis plays an important role, and an important factor affecting the enzymatic hydrolysis effect is the degrading enzyme used. It can be known from the comparison between Comparative Examples 8-12 and Example 1 that for the whole soybean milk treated based on the present invention, the composite system of cellulase and flavor protease is the most suitable. Under this system, the dosage of the enzyme is controlled, and combined with the above treatment process, thus on the basis of efficiently reducing the comprehensive allergenicity, β-conglycinin is basically removed directionally, and the taste is good without a bitter taste, and the comprehensive dosage of the enzyme is also small.

Claims

1. A preparation method of full soybean milk with low β-conglycinin allergenicity, characterized in that, It includes the following steps: 1) Add glycosidase to the whole soybean milk obtained after soybean peeling and grinding, heat it to the glycosidase activity temperature for enzymatic hydrolysis; 2) After the enzymatic hydrolysis in step 1), heat it to 45 - 55 °C, and add flavor protease and cellulase for enzymatic hydrolysis for 20 - 30 min; 3) Place the whole soybean milk after the enzymatic hydrolysis in step 2) under ultra-high pressure for continuous enzymatic hydrolysis for 10 - 20 min; 4) After the enzymatic hydrolysis, cook the soybean milk and inactivate the enzyme activity to obtain low-allergenic whole soybean milk.

2. The method for preparing whole soybean milk according to claim 1, wherein in step 1), the glycosidase is one or both of endoglycosidase H and glycopeptidase A.

3. The method for preparing whole soybean milk according to claim 1, wherein the addition amount of the glycosidase in step 1) is 0.008% - 0.032% of the mass of the whole soybean milk; and / or, the enzymatic hydrolysis temperature is 35 - 40 °C, and the enzymatic hydrolysis time is 20 - 30 min.

4. The method for preparing whole soybean milk according to claim 1, wherein the addition amount of the flavor protease in step 2) is 0.008% - 0.032% of the mass of the whole soybean milk; and / or, the addition amount of the cellulase is 0.008% - 0.032% of the mass of the whole soybean milk.

5. The method for preparing whole soybean milk according to claim 1, wherein the ultra-high pressure in step 3) is 300 - 400 MPa.

6. The method for preparing whole soybean milk according to claim 1 or 5, wherein the enzymatic hydrolysis temperature under ultra-high pressure in step 3) is 45 - 55 °C.

7. The method for preparing whole soybean milk according to claim 1, wherein the process of peeling and grinding the whole soybean milk used in step 1) is: Roast and peel the soybeans, finely grind them, then mix with water for grinding, and after degassing, treat them by high-pressure microfluidization to obtain whole soybean milk.

8. The method for preparing whole soybean milk according to claim 7, wherein the particle size after fine grinding is 80 - 100 mesh; and / or, the mass ratio of soybean to water for mixing is 1:3 - 7; and / or, the pressure of high-pressure microfluidization is 100 - 140 MPa.

9. The method for preparing whole soybean milk according to claim 1, wherein in step 4), instantaneous steam-material mixing is used to inactivate the enzyme activity.

10. The method for preparing whole soybean milk according to claim 9, wherein the steam temperature is 100 - 105 °C; and / or, keep it at 90 - 100 °C for 3 - 5 min after inactivating the enzyme activity.

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