Feed additive containing cellulase and preparation method thereof

Abstract

The invention relates to the technical field of feed, in particular to a feed additive containing cellulase and a preparation method thereof, and the preparation method comprises the following steps: preparation of a microgel carrier, preparation of alginic acid dialdehyde, preparation of a cellulase condensate, preparation of a polyphenol condensate and preparation of a preparation. The feed additive prepared by the preparation method has relatively good stability, and is beneficial to stable effectiveness of cellulase in poultry bodies.

Description

Technical Field

[0001] This invention relates to the field of feed technology, and in particular to a feed additive containing cellulase and its preparation method. Background Technology

[0002] Cellulase is a complex enzyme that degrades cellulose into glucose, and it has been widely used as a feed additive in poultry and livestock farming. Among farmed poultry and livestock, only ruminants can utilize rumen microorganisms symbiotically residing in their bodies to break down and utilize cellulose, while most other farmed poultry and livestock cannot utilize cellulose, leading to indigestion, diarrhea, and other problems. Therefore, it is necessary to artificially add cellulase to enhance the digestive capacity of poultry and livestock.

[0003] Although cellulase has been added to existing poultry and livestock feed to enhance their digestive capacity, the effect of adding cellulase alone is limited. The pH of poultry and livestock stomachs is usually 2-3, which is far from the optimal pH of 5-6 for cellulase. Moreover, during daily storage, the environmental temperature and moisture can easily deactivate cellulase, thereby reducing its ability to break down cellulose.

[0004] Therefore, there is a need for a feed additive containing cellulase, in which the cellulase is stable and can promote digestion in poultry and livestock, and enhance their ability to absorb and convert feed. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of current feed additives, such as instability and easy deactivation of cellulase, as described in the background art. Here, a new feed additive containing cellulase and its preparation method are proposed. The feed additive prepared by this method is not easily deactivated and can promote the absorption and conversion of feed by poultry and livestock.

[0006] The technical solution adopted by this invention to solve its technical problem is: a method for preparing a feed additive containing cellulase, the preparation method comprising the following steps:

[0007] S1. Preparation of microgel carriers:

[0008] Add 2% acetic acid aqueous solution to a reactor containing a gel matrix until the mass fraction of the gel matrix in the acetic acid aqueous solution is 4-6%. Add glutaraldehyde until the mass fraction of glutaraldehyde in the acetic acid aqueous solution is 1-2%. Heat to 40-50℃ and keep the temperature for 1-2 hours to precipitate solids. Filter, cool and dry to obtain a microgel carrier. The gel matrix is ​​a mixture of chitosan and sodium alginate.

[0009] Preparation of S2 and alginate dialdehyde:

[0010] Sodium alginate is oxidized by reacting sodium periodate to obtain dialgaldehyde;

[0011] S3. Preparation of cellulase immobilized product:

[0012] The microgel carrier was placed in a mixed enzyme solution, and glutaraldehyde was added until the mass fraction of glutaraldehyde in the mixed enzyme solution was 0.5-1%. The temperature was raised to 25-30℃ and mixed for 15-30 minutes to obtain the first mixture. The first mixture was sprayed into a calcium chloride aqueous solution, the temperature was lowered to 10-20℃, microwaved, filtered, and freeze-dried to obtain the cellulase immobilized product.

[0013] The mixed enzyme solution is a mixed aqueous solution of cellulase and alginate dialdehyde;

[0014] S4. Preparation of polyphenol cured products:

[0015] The microgel carrier was placed in a polyphenol extract solution, heated to 25-30℃, and mixed for 15-30 minutes to obtain a second mixture. The second mixture was then sprayed into a calcium chloride aqueous solution, the temperature was lowered to 10-20℃, microwaved, filtered, and freeze-dried to obtain the polyphenol solidified product.

[0016] The polyphenol extract is a mixture of green tea extract, grape extract, and onion extract;

[0017] S5, Preparation:

[0018] A feed additive containing cellulase is obtained by mixing cellulase-cured solids and polyphenol-cured solids.

[0019] In the above preparation method, chitosan-sodium alginate microgels are first prepared using chitosan and sodium alginate as raw materials. These microgels serve as a carrier, covalently binding and immobilizing subsequently added cellulase to enhance its stability. Throughout the preparation process, the microgel carrier reacts with calcium chloride to form a porous three-dimensional structure. The microgel carrier possesses carboxylic acid and primary amine functional groups, which can covalently bind to the amine functional groups on the cellulase under the influence of glyoxal, thus immobilizing the cellulase within the microgel carrier. Alglyoxal possesses carboxyl and aldehyde groups. The aldehyde groups on the alginate react with the cellulase via a Schiff base reaction, while the carboxyl groups react with calcium chloride, forming an anti-detachment layer on the microgel surface, further immobilizing the cellulase.

[0020] Preferably, in step S1, the mass ratio of chitosan to sodium alginate is 1:(1-10), and more preferably 1:5.

[0021] In the above technical solutions, both chitosan and sodium alginate can undergo ion exchange reactions with calcium chloride to form gels. Chitosan has poor acid resistance, which is not conducive to the stability of cellulase after entering poultry. However, the composite gel formed by the combination of chitosan and sodium alginate has better stability.

[0022] Preferably, step S2 is as follows: sodium alginate and sodium periodate are dissolved in water and reacted in the dark under inert gas protection for 16-20 hours. Ethylene glycol is added to terminate the reaction. Sodium chloride and ethanol are added to precipitate a white solid. The solid is filtered and freeze-dried to obtain alginate dialdehyde. For every 1g of sodium alginate added, the amount of sodium periodate added is 1-4g, the amount of ethylene glycol added is 0.1-1mL, the amount of sodium chloride added is 0.1-0.5g, and the amount of ethanol added is 200-300mL.

[0023] Preferably, in step S3, the mass ratio of the microgel carrier to the mixed enzyme solution is 1:(10-20), more preferably 1:15.

[0024] The volume ratio of the first mixture to the calcium chloride aqueous solution is 1:(2-3), more preferably 1:3.

[0025] The mass ratio of cellulase, alginate dialdehyde, and water is (5-10):(1-5):100, and more preferably 10:3:100.

[0026] Preferably, in step S3, the specific conditions for microwave mixing are: 500MHz for 5-10 minutes, 800MHz for 5-10 minutes, and 1000MHz for 5-10 minutes.

[0027] Preferably, in step S4, the mass ratio of the microgel carrier to the polyphenol extract solution is 1:(10-20), more preferably 1:15.

[0028] The volume ratio of the second mixture to the calcium chloride aqueous solution is 1:2-3, more preferably 1:3.

[0029] The mass ratio of the green tea extract, grape extract, and onion extract is 1:(0.5-1):(0.5-1), more preferably 1:0.5:0.5.

[0030] The concentration of the polyphenol extract solution is 1-6 wt%, more preferably 3 wt%.

[0031] Preferably, in steps S3 and S4, the concentration of the calcium chloride aqueous solution is 1-3 wt%, more preferably 2.5 wt%.

[0032] Preferably, in step S5, the mass ratio of the cellulase-cured product to the polyphenol-cured product is (5-10):1, and more preferably 5:1.

[0033] As a preferred technical solution, a method for preparing a feed additive containing cellulase includes the following steps:

[0034] S1. Preparation of microgel carriers:

[0035] Add 2% acetic acid aqueous solution to a reactor containing a gel matrix until the mass fraction of the gel matrix in the acetic acid aqueous solution is 4-6%. Add glutaraldehyde until the mass fraction of glutaraldehyde in the acetic acid aqueous solution is 1-2%. Heat to 40-50℃ and keep the temperature for 1-2 hours to precipitate solids. Filter, cool and dry to obtain a microgel carrier. The gel matrix is ​​a mixture of chitosan and sodium alginate, and the mass ratio of chitosan to sodium alginate is 1:5.

[0036] Preparation of S2 and alginate dialdehyde:

[0037] Sodium alginate and sodium periodate were dissolved in water and reacted in the dark under an inert gas atmosphere for 16-20 hours. The reaction was terminated by adding ethylene glycol. Sodium chloride and ethanol were added to precipitate a white solid. The solid was filtered and freeze-dried to obtain alginate dialdehyde. For every 1g of sodium alginate added, the amount of sodium periodate added was 1-4g, the amount of ethylene glycol added was 0.1-1mL, the amount of sodium chloride added was 0.1-0.5g, and the amount of ethanol added was 200-300mL.

[0038] S3. Preparation of cellulase immobilized product:

[0039] The microgel carrier was placed in a mixed enzyme solution, and glutaraldehyde was added until the mass fraction of glutaraldehyde in the mixed enzyme solution was 0.5-1%. The temperature was raised to 25-30℃ and mixed for 15-30 minutes to obtain the first mixture. The first mixture was sprayed into a calcium chloride aqueous solution, the temperature was lowered to 10-20℃, microwaved, filtered, and freeze-dried to obtain the cellulase immobilized product.

[0040] The mass ratio of the microgel carrier to the mixed enzyme solution is 1:15.

[0041] The volume ratio of the first mixture to the calcium chloride aqueous solution is 1:3.

[0042] The mixed enzyme solution is a mixed aqueous solution of cellulase and dialgaldehyde, wherein the mass ratio of cellulase, dialgaldehyde and water is 10:3:100.

[0043] S4. Preparation of polyphenol cured products:

[0044] The microgel carrier was placed in a polyphenol extract solution, heated to 25-30℃, and mixed for 15-30 minutes to obtain a second mixture. The second mixture was then sprayed into a calcium chloride aqueous solution, the temperature was lowered to 10-20℃, microwaved, filtered, and freeze-dried to obtain the polyphenol solidified product.

[0045] The mass ratio of the microgel carrier to the polyphenol extract solution is 1:15.

[0046] The volume ratio of the second mixture to the calcium chloride aqueous solution is 1:3.

[0047] The polyphenol extract is a mixture of green tea extract, grape extract and onion extract, wherein the mass ratio of green tea extract, grape extract and onion extract is 1:0.5:0.5, and the concentration of the polyphenol extract solution is 3wt%.

[0048] S5, Preparation:

[0049] A cellulase-containing feed additive is obtained by mixing cellulase-cured material and polyphenol-cured material, wherein the mass ratio of cellulase-cured material to polyphenol-cured material is 5:1.

[0050] A feed additive containing cellulase prepared by the method described above.

[0051] The beneficial effects of this invention are:

[0052] 1. Compared to directly adding cellulase into microgels for fixation, the addition of alginate dialdehyde further improves the stability of cellulase.

[0053] 2. Compared with the addition of cellulase alone, the addition of polyphenol solids can delay the inactivation of cellulase and enhance the anti-interference and anti-acidity capabilities of cellulase. Detailed Implementation

[0054] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0055] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0056] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified).

[0057] Example 1:

[0058] A method for preparing a feed additive containing cellulase, the method comprising:

[0059] S1. Preparation of microgel carriers:

[0060] Chitosan and sodium alginate in a mass ratio of 1:5 were added to the reactor. 2% acetic acid aqueous solution was added until the mass fraction of chitosan in the acetic acid aqueous solution was 1%. Glutaraldehyde was then added until the mass fraction of glutaraldehyde in the acetic acid aqueous solution was 2%. After stirring and mixing, the mixture was heated to 45°C and kept at that temperature for 1 hour to precipitate solids. The solids were filtered, cooled and dried to obtain the microgel carrier.

[0061] Preparation of S2 and alginate dialdehyde:

[0062] Dissolve 1g of sodium alginate and 1g of sodium periodate separately in water. Mix the aqueous solutions of the two and react for 20 hours in the dark under nitrogen protection. Add 0.5mL of ethylene glycol to terminate the reaction. Then add 0.3g of sodium chloride and 250mL of ethanol. A white solid precipitates out. Filter and freeze dry to obtain alginate dialdehyde.

[0063] S3. Preparation of cellulase immobilized product:

[0064] A mixed enzyme solution was prepared by mixing cellulase, alginate dialdehyde, and water at a mass ratio of 10:3:100. A microgel carrier at a mass ratio of 1:15 was then mixed with the mixed enzyme solution, followed by the addition of glutaraldehyde until the glutaraldehyde concentration in the mixed enzyme solution reached 1%. The mixture was heated to 25°C and kept at this temperature for 30 minutes to obtain a first mixed solution. This first mixed solution was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution. The temperature was then lowered to 15°C, and the mixture was microwaved, filtered, and freeze-dried to obtain the cellulase solidified product.

[0065] The specific conditions for microwave mixing are: 500MHz for 10 minutes, 800MHz for 10 minutes, and 1000MHz for 10 minutes.

[0066] S4. Preparation of polyphenol cured products:

[0067] A polyphenol extract was obtained by mixing green tea extract (mainly tea polyphenols), grape extract (mainly grape polyphenols), and onion extract (mainly onion polyphenols) in a mass ratio of 1:0.5:0.5. The polyphenol extract was then mixed with water in a mass ratio of 3:100 to obtain a polyphenol extract solution. A second mixture was prepared by mixing a microgel carrier in a mass ratio of 1:15 with the polyphenol extract solution. This second mixture was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution, cooled to 15°C, microwaved, filtered, and freeze-dried to obtain a cellulase-cured product.

[0068] The specific conditions for microwave mixing are: 300MHz for 30 minutes.

[0069] S5, Preparation:

[0070] A feed additive containing cellulase was obtained by mixing cellulase solids and polyphenol solids at a mass ratio of 5:1.

[0071] Example 2:

[0072] A method for preparing a feed additive containing cellulase, the method comprising:

[0073] S1. Preparation of microgel carriers:

[0074] Chitosan and sodium alginate in a mass ratio of 1:10 were added to the reactor. 2% acetic acid aqueous solution was added until the mass fraction of chitosan in the acetic acid aqueous solution was 1%. Glutaraldehyde was then added until the mass fraction of glutaraldehyde in the acetic acid aqueous solution was 2%. After stirring and mixing, the mixture was heated to 45°C and kept at that temperature for 1 hour to precipitate solids. The solids were filtered, cooled and dried to obtain the microgel carrier.

[0075] Preparation of S2 and alginate dialdehyde:

[0076] Dissolve 1g of sodium alginate and 1g of sodium periodate separately in water. Mix the aqueous solutions of the two and react for 20 hours in the dark under nitrogen protection. Add 0.5mL of ethylene glycol to terminate the reaction. Then add 0.3g of sodium chloride and 250mL of ethanol. A white solid precipitates out. Filter and freeze dry to obtain alginate dialdehyde.

[0077] S3. Preparation of cellulase immobilized product:

[0078] A mixed enzyme solution was prepared by mixing cellulase, alginate dialdehyde, and water at a mass ratio of 5:1:100. A microgel carrier at a mass ratio of 1:15 was then mixed with the mixed enzyme solution, followed by the addition of glutaraldehyde until the glutaraldehyde concentration in the mixed enzyme solution reached 1%. The mixture was heated to 25°C and kept at this temperature for 30 minutes to obtain a first mixed solution. This first mixed solution was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution. The temperature was then lowered to 15°C, and the mixture was microwaved, filtered, and freeze-dried to obtain the cellulase solidified product.

[0079] The specific conditions for microwave mixing are: 500MHz for 10 minutes, 800MHz for 10 minutes, and 1000MHz for 10 minutes.

[0080] S4. Preparation of polyphenol cured products:

[0081] A polyphenol extract was obtained by mixing green tea extract (mainly tea polyphenols), grape extract (mainly grape polyphenols), and onion extract (mainly onion polyphenols) in a mass ratio of 1:0.5:0.5. The polyphenol extract was then mixed with water at a mass ratio of 1:100 to obtain a polyphenol extract solution. A second mixture was prepared by mixing a microgel carrier and the polyphenol extract solution in a mass ratio of 1:15. This second mixture was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution, the temperature was lowered to 15°C, and the mixture was microwaved, filtered, and freeze-dried to obtain a cellulase-cured product.

[0082] The specific conditions for microwave mixing are: 300MHz for 30 minutes.

[0083] S5, Preparation:

[0084] A feed additive containing cellulase was obtained by mixing cellulase solids and polyphenol solids at a mass ratio of 10:1.

[0085] Example 3:

[0086] A method for preparing a feed additive containing cellulase, the method comprising:

[0087] S1. Preparation of microgel carriers:

[0088] Chitosan and sodium alginate in a mass ratio of 1:1 were added to the reactor. 2% acetic acid aqueous solution was added until the mass fraction of chitosan in the acetic acid aqueous solution was 1%. Glutaraldehyde was then added until the mass fraction of glutaraldehyde in the acetic acid aqueous solution was 2%. After stirring and mixing, the mixture was heated to 45°C and kept at that temperature for 1 hour to precipitate solids. The solids were filtered, cooled and dried to obtain the microgel carrier.

[0089] Preparation of S2 and alginate dialdehyde:

[0090] Dissolve 1g of sodium alginate and 1g of sodium periodate separately in water. Mix the aqueous solutions of the two and react for 20 hours in the dark under nitrogen protection. Add 0.5mL of ethylene glycol to terminate the reaction. Then add 0.3g of sodium chloride and 250mL of ethanol. A white solid precipitates out. Filter and freeze dry to obtain alginate dialdehyde.

[0091] S3. Preparation of cellulase immobilized product:

[0092] A mixed enzyme solution was prepared by mixing cellulase, alginate dialdehyde, and water at a mass ratio of 10:1:100. A microgel carrier at a mass ratio of 1:15 was then mixed with the mixed enzyme solution, followed by the addition of glutaraldehyde until the glutaraldehyde concentration in the mixed enzyme solution reached 1%. The mixture was heated to 25°C and kept at this temperature for 30 minutes to obtain a first mixed solution. This first mixed solution was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution. The temperature was then lowered to 15°C, and the mixture was microwaved, filtered, and freeze-dried to obtain the cellulase solidified product.

[0093] The specific conditions for microwave mixing are: 500MHz for 10 minutes, 800MHz for 10 minutes, and 1000MHz for 10 minutes.

[0094] S4. Preparation of polyphenol cured products:

[0095] A polyphenol extract was obtained by mixing green tea extract (mainly tea polyphenols), grape extract (mainly grape polyphenols), and onion extract (mainly onion polyphenols) in a mass ratio of 1:1:1. The polyphenol extract was then mixed with water at a mass ratio of 3:100 to obtain a polyphenol extract solution. A second mixture was prepared by mixing a microgel carrier and the polyphenol extract solution in a mass ratio of 1:15. This second mixture was then sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution, cooled to 15°C, microwaved, filtered, and freeze-dried to obtain a cellulase-cured product.

[0096] The specific conditions for microwave mixing are: 300MHz for 30 minutes.

[0097] S5, Preparation:

[0098] A feed additive containing cellulase was obtained by mixing cellulase solids and polyphenol solids at a mass ratio of 5:1.

[0099] Comparative Example 1:

[0100] A method for preparing a feed additive containing cellulase, the method comprising:

[0101] S1. Preparation of microgel carriers:

[0102] Chitosan and sodium alginate in a mass ratio of 1:5 were added to the reactor. 2% acetic acid aqueous solution was added until the mass fraction of chitosan in the acetic acid aqueous solution was 1%. Glutaraldehyde was then added until the mass fraction of glutaraldehyde in the acetic acid aqueous solution was 2%. After stirring and mixing, the mixture was heated to 45°C and kept at that temperature for 1 hour to precipitate solids. The solids were filtered, cooled and dried to obtain the microgel carrier.

[0103] S2. Preparation of cellulase immobilized product:

[0104] A cellulase solution was prepared by mixing cellulase and water at a mass ratio of 1:10. A microgel carrier at a mass ratio of 1:15 was then mixed with the cellulase solution, and glutaraldehyde was added until the mass fraction of glutaraldehyde in the cellulase solution reached 1%. The mixture was heated to 25°C and kept at this temperature for 30 minutes to obtain a first mixture. This first mixture was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution. The temperature was then lowered to 15°C, and the mixture was microwaved, filtered, and freeze-dried to obtain the cellulase solidified product.

[0105] The specific conditions for microwave mixing are: 500MHz for 10 minutes, 800MHz for 10 minutes, and 1000MHz for 10 minutes.

[0106] S3. Preparation of polyphenol cured products:

[0107] A polyphenol extract was obtained by mixing green tea extract (mainly tea polyphenols), grape extract (mainly grape polyphenols), and onion extract (mainly onion polyphenols) in a mass ratio of 1:0.5:0.5. The polyphenol extract was then mixed with water in a mass ratio of 3:100 to obtain a polyphenol extract solution. A second mixture was prepared by mixing a microgel carrier in a mass ratio of 1:15 with the polyphenol extract solution. This second mixture was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution, cooled to 15°C, microwaved, filtered, and freeze-dried to obtain a cellulase-cured product.

[0108] The specific conditions for microwave mixing are: 300MHz for 30 minutes.

[0109] S4, Preparation:

[0110] A feed additive containing cellulase was obtained by mixing cellulase solids and polyphenol solids at a mass ratio of 5:1.

[0111] Comparative Example 2:

[0112] A method for preparing a feed additive containing cellulase, the method comprising:

[0113] S1. Preparation of microgel carriers:

[0114] Chitosan and sodium alginate in a mass ratio of 1:5 were added to the reactor. 2% acetic acid aqueous solution was added until the mass fraction of chitosan in the acetic acid aqueous solution was 1%. Glutaraldehyde was then added until the mass fraction of glutaraldehyde in the acetic acid aqueous solution was 2%. After stirring and mixing, the mixture was heated to 45°C and kept at that temperature for 1 hour to precipitate solids. The solids were filtered, cooled and dried to obtain the microgel carrier.

[0115] Preparation of S2 and alginate dialdehyde:

[0116] Dissolve 1g of sodium alginate and 1g of sodium periodate separately in water. Mix the aqueous solutions of the two and react for 20 hours in the dark under nitrogen protection. Add 0.5mL of ethylene glycol to terminate the reaction. Then add 0.3g of sodium chloride and 250mL of ethanol. A white solid precipitates out. Filter and freeze dry to obtain alginate dialdehyde.

[0117] S3. Preparation of cellulase immobilized product:

[0118] A mixed enzyme solution was prepared by mixing cellulase, alginate dialdehyde, and water in a mass ratio of 10:3:100. A microgel carrier in a mass ratio of 1:15 was then mixed with the mixed enzyme solution, and glutaraldehyde was added until the mass fraction of glutaraldehyde in the mixed enzyme solution was 1%. The mixture was heated to 25°C and kept at that temperature for 30 minutes to obtain a first mixed solution. The first mixed solution was sprayed into a 2.5 wt% calcium chloride aqueous solution at a volume ratio of 3. The temperature was then lowered to 15°C, mixed, filtered, and freeze-dried to obtain the cellulase solidified product.

[0119] S4. Preparation of polyphenol cured products:

[0120] A polyphenol extract was obtained by mixing green tea extract (mainly tea polyphenols), grape extract (mainly grape polyphenols), and onion extract (mainly onion polyphenols) in a mass ratio of 1:0.5:0.5. The polyphenol extract was then mixed with water in a mass ratio of 3:100 to obtain a polyphenol extract solution. A second mixture was obtained by mixing a microgel carrier in a mass ratio of 1:15 with the polyphenol extract solution. The second mixture was then sprayed into a 2.5 wt% calcium chloride aqueous solution at three times its volume, the temperature was lowered to 15°C, mixed, filtered, and freeze-dried to obtain a cellulase-cured product.

[0121] S5, Preparation:

[0122] A feed additive containing cellulase was obtained by mixing cellulase solids and polyphenol solids at a mass ratio of 5:1.

[0123] Comparative Example 3:

[0124] A method for preparing a feed additive containing cellulase, the method comprising:

[0125] S1. Preparation of microgel carriers:

[0126] Chitosan and sodium alginate in a mass ratio of 1:5 were added to the reactor. 2% acetic acid aqueous solution was added until the mass fraction of chitosan in the acetic acid aqueous solution was 1%. Glutaraldehyde was then added until the mass fraction of glutaraldehyde in the acetic acid aqueous solution was 2%. After stirring and mixing, the mixture was heated to 45°C and kept at that temperature for 1 hour to precipitate solids. The solids were filtered, cooled and dried to obtain the microgel carrier.

[0127] Preparation of S2 and alginate dialdehyde:

[0128] Dissolve 1g of sodium alginate and 1g of sodium periodate separately in water. Mix the aqueous solutions of the two and react for 20 hours in the dark under nitrogen protection. Add 0.5mL of ethylene glycol to terminate the reaction. Then add 0.3g of sodium chloride and 250mL of ethanol. A white solid precipitates out. Filter and freeze dry to obtain alginate dialdehyde.

[0129] S3. Preparation of cellulase immobilized product:

[0130] A mixed enzyme solution was prepared by mixing cellulase, alginate dialdehyde, and water at a mass ratio of 10:3:100. A microgel carrier at a mass ratio of 1:15 was then mixed with the mixed enzyme solution, followed by the addition of glutaraldehyde until the glutaraldehyde concentration in the mixed enzyme solution reached 1%. The mixture was heated to 25°C and kept at this temperature for 30 minutes to obtain a first mixed solution. This first mixed solution was sprayed into three volumes of a 2.5 wt% calcium chloride aqueous solution. The temperature was then lowered to 15°C, and the mixture was microwaved, filtered, and freeze-dried to obtain a cellulase-based solidified product (a feed additive containing cellulase).

[0131] The specific conditions for microwave mixing are: 500MHz for 10 minutes, 800MHz for 10 minutes, and 1000MHz for 10 minutes.

[0132] Enzyme activity assay: The cellulase-containing feed additives (10 mg) prepared in Examples 1-3 and Comparative Examples 1-3 were placed in 1 ml of citrate-sodium citrate buffer at pH 4.8, and 50 mg of filter paper was added. The mixture was incubated at 50 °C for 1 h, and then 3 ml of DNS colorimetric solution was added. The mixture was placed in a boiling water bath for 10 min, cooled, and the absorbance was measured at 540 nm. The enzyme activity was determined by using a standard curve.

[0133] The cellulase-containing feed additives prepared in Examples 1-3 and Comparative Examples 1-3 were placed in a citrate-sodium citrate buffer solution at pH 3.5 and allowed to stand for 12 hours. The enzyme activity of the acid-treated cellulase-containing feed additives was repeatedly tested. The specific test results are shown in Table 1. The enzyme activity without acid treatment as measured in Example 1 was used as the baseline and recorded as 100%.

[0134] Table 1: Enzyme Activity Detection Table for Feed Additives Containing Cellulase

[0135] project Normal enzyme activity (%) Enzyme activity (%) after acid treatment Example 1 100 86 Example 2 98 84 Example 3 96 82 Comparative Example 1 89 71 Comparative Example 2 92 80 Comparative Example 3 99 80

[0136] As can be seen from Table 1, Example 1 and Comparative Example 1, the activity of cellulase decreased when no alginate dialdehyde was added during the preparation stage of the cellulase solidified product. This may be because the lack of alginate dialdehyde connecting the microgel and cellulase caused some cellulase to detach and become free, resulting in a significant decrease in cellulase activity after acid treatment. As can be seen from the comparison between Example 1 and Comparative Example 2, microwave mixing is beneficial to the formation of cellulase solidified product and increases the loading of cellulase on the microgel. As can be seen from Example 1 and Comparative Example 3, polyphenol solidified product is beneficial to the preservation of cellulase activity under acidic conditions.

[0137] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to in the method section.

[0138] The foregoing has provided a detailed description of a cellulase-containing feed additive and its preparation method. Specific examples have been used to illustrate the principles and implementation methods of the invention. These examples are merely illustrative and are intended to aid in understanding the method and core concepts of the invention. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims.

Claims

1. A method for preparing a feed additive containing cellulase, characterized in that: The preparation method includes the following steps: S1. Preparation of microgel carriers: Add 2% acetic acid aqueous solution to a reactor containing a gel matrix until the mass fraction of the gel matrix in the acetic acid aqueous solution is 4-6%. Add glutaraldehyde until the mass fraction of glutaraldehyde in the acetic acid aqueous solution is 1-2%. Heat to 40-50℃ and keep the temperature for 1-2 hours to precipitate solids. Filter, cool and dry to obtain a microgel carrier. The gel matrix is ​​a mixture of chitosan and sodium alginate. Preparation of S2 and alginate dialdehyde: Sodium alginate is oxidized by reacting sodium periodate to obtain dialgaldehyde; S3. Preparation of cellulase immobilized product: The microgel carrier was placed in a mixed enzyme solution, and glutaraldehyde was added until the mass fraction of glutaraldehyde in the mixed enzyme solution was 0.5-1%. The temperature was raised to 25-30℃ and mixed for 15-30 minutes to obtain the first mixture. The first mixture was sprayed into a calcium chloride aqueous solution, the temperature was lowered to 10-20℃, microwaved, filtered, and freeze-dried to obtain the cellulase immobilized product. The mixed enzyme solution is a mixed aqueous solution of cellulase and alginate dialdehyde; S4. Preparation of polyphenol cured products: The microgel carrier was placed in a polyphenol extract solution, heated to 25-30℃, and mixed for 15-30 minutes to obtain a second mixture. The second mixture was then sprayed into a calcium chloride aqueous solution, the temperature was lowered to 10-20℃, microwaved, filtered, and freeze-dried to obtain the polyphenol solidified product. The polyphenol extract is a mixture of green tea extract, grape extract, and onion extract; S5, Preparation: A feed additive containing cellulase is obtained by mixing cellulase-cured solids and polyphenol-cured solids.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of chitosan to sodium alginate is 1:(1-10).

3. The preparation method according to claim 1, characterized in that: Step S2 is as follows: Sodium alginate and sodium periodate are dissolved in water and reacted in the dark under an inert gas atmosphere for 16-20 hours. Ethylene glycol is added to terminate the reaction. Sodium chloride and ethanol are added to precipitate a white solid. The solid is filtered and freeze-dried to obtain alginate dialdehyde. For every 1g of sodium alginate added, the amount of sodium periodate added is 1-4g, the amount of ethylene glycol added is 0.1-1mL, the amount of sodium chloride added is 0.1-0.5g, and the amount of ethanol added is 200-300mL.

4. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of the microgel carrier to the mixed enzyme solution is 1:(10-20). The volume ratio of the first mixture to the calcium chloride aqueous solution is 1:(2-3). The mass ratio of cellulase, alginate dialdehyde, and water is (5-10):(1-5):

100.

5. The preparation method according to claim 1, characterized in that: In step S3, the specific conditions for microwave mixing are: 500MHz for 5-10 minutes, 800MHz for 5-10 minutes, and 1000MHz for 5-10 minutes.

6. The preparation method according to claim 1, characterized in that: In step S4, the mass ratio of the microgel carrier to the polyphenol extract solution is 1:(10-20). The volume ratio of the second mixture to the calcium chloride aqueous solution is 1:2-3. The mass ratio of the green tea extract, grape extract, and onion extract is 1:(0.5-1):(0.5-1). The concentration of the polyphenol extract solution is 1-6 wt%.

7. The preparation method according to claim 1, characterized in that: In step S4, the specific conditions for microwave mixing are: 300MHz for 10-30 minutes.

8. The preparation method according to claim 1, characterized in that: In steps S3 and S4, the concentration of the calcium chloride aqueous solution is 1-3 wt%.

9. The preparation method according to claim 1, characterized in that: In step S5, the mass ratio of the cellulase-cured product to the polyphenol-cured product is (5-10):

1.

10. A feed additive containing cellulase prepared by any one of the preparation methods of claims 1-9.