CN115232806B - Neutral protease micropill and preparation method thereof - Google Patents
Neutral protease micropill and preparation method thereof Download PDFInfo
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- 108090000145 Bacillolysin Proteins 0.000 title claims abstract description 30
- 102000035092 Neutral proteases Human genes 0.000 title claims abstract description 30
- 108091005507 Neutral proteases Proteins 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 55
- 108090000790 Enzymes Proteins 0.000 claims abstract description 55
- 238000000855 fermentation Methods 0.000 claims abstract description 45
- 230000004151 fermentation Effects 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 238000001914 filtration Methods 0.000 claims abstract description 21
- 238000005469 granulation Methods 0.000 claims abstract description 12
- 230000003179 granulation Effects 0.000 claims abstract description 12
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
- 239000007921 spray Substances 0.000 claims abstract description 7
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- 238000005086 pumping Methods 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 17
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 14
- 240000008042 Zea mays Species 0.000 claims description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 10
- 235000005822 corn Nutrition 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- 238000000889 atomisation Methods 0.000 claims description 9
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 7
- 229920002261 Corn starch Polymers 0.000 claims description 7
- 239000004375 Dextrin Substances 0.000 claims description 7
- 229920001353 Dextrin Polymers 0.000 claims description 7
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 7
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 7
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 7
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 7
- 239000000919 ceramic Substances 0.000 claims description 7
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- 235000019425 dextrin Nutrition 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 238000001694 spray drying Methods 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 5
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 5
- 244000061458 Solanum melongena Species 0.000 claims description 5
- 235000002597 Solanum melongena Nutrition 0.000 claims description 5
- 235000019764 Soybean Meal Nutrition 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
- 239000004455 soybean meal Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000008188 pellet Substances 0.000 abstract description 15
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- 230000000694 effects Effects 0.000 abstract description 5
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- 102000035195 Peptidases Human genes 0.000 description 11
- 108091005804 Peptidases Proteins 0.000 description 11
- 239000004365 Protease Substances 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 9
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- 238000001514 detection method Methods 0.000 description 3
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D1/00—Evaporating
- B01D1/16—Evaporating by spraying
- B01D1/18—Evaporating by spraying to obtain dry solids
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
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Abstract
The invention discloses a neutral protease pellet and a preparation method thereof, belonging to the technical field of microbial fermentation and enzyme engineering; the preparation method sequentially comprises the steps of fermenting strains of bacillus subtilis into seed liquid, and preparing fermentation liquid with the enzyme activity of over 20000U/mL; and then, a series of operations such as centrifugal impurity removal, filtration and sterilization, ultrafiltration concentration, preparation of a granulating enzyme liquid, spray granulation and the like are carried out to prepare the neutral protease micropill with uniform granularity and uniform active ingredients, and the preparation method has the advantages of high production efficiency, low production cost, wide popularization and application prospect and higher economic benefit.
Description
Technical Field
The invention discloses a neutral protease pellet and a preparation method thereof, belonging to the technical field of fermentation technology and preparation.
Background
Protease, an endogenous enzyme secreted by animals, is an enzyme that catalyzes the hydrolysis of peptide bonds, and is found in animal viscera, plant stems and leaves, fruits and microorganisms, and has been widely used in leather, fur, silk, medicine, food, brewing and other aspects. Because of its specificity and complexity, it has been used as a component of complex enzymes. In recent years, due to development of maximum potential of animal production by breeders, rising price of protein raw materials and transition from simple daily ration to complex daily ration, research on single protease and complex protease by researchers is increasing. The protease hydrolyzes peptide bonds in protein, and the final product is amino acid, so that the premise of improving animal growth performance by adding the protease is mainly whether the protease can have favorable influence on the digestion and utilization rate of the amino acid of animals.
After the animal ingests the protein, the protein is converted into free amino acid through the synergistic effect of protease and other enzymes, so that the requirement of normal metabolism of the animal on the amino acid is met. Protease is mainly divided into three types of acid protease, neutral protease and alkaline protease according to the action pH range, wherein the neutral protease is widely applied to the fields of food, medicine, feed and the like due to mild action conditions. The protease is mainly produced by microbial metabolism at present, and the neutral protease preparation in the market is mainly powder type, so that when the protease preparation is used for animal feed, the protein utilization rate in the feed and the animal production performance can be obviously improved, and the cost is reduced.
However, most of enzyme preparations are powder, so that the powder has poor temperature resistance and stability in different environments, dust pollution is serious during use, waste exists, and allergic damage to skin is easy to cause. In order to solve the above-mentioned drawbacks, researchers and practitioners prepare powder enzyme preparations into pellet granules, for example, chinese patent application No. 200410051993.1 discloses a multicolor pellet type feeding compound enzyme and a manufacturing method thereof, chinese patent application No. 201410638567.1 discloses a compound enzyme coated pellet and a manufacturing method thereof, and Chinese patent application No. 202111548278.9 discloses a granule neutral protease and a manufacturing method thereof; the preparation method for preparing the pellet type granule is mentioned, but the adopted method is extrusion type, swing type or fluidized bed for preparing the pellet. The traditional preparation methods such as swing granulation, extrusion granulation, roller granulation, cyclone fluidized bed granulation and the like have the defects of low yield per unit time, low efficiency, high cost, poor uniformity of pellet granularity, uneven active ingredients and the like, and cannot meet the actual production requirements.
Disclosure of Invention
First, the technical problem to be solved
The invention aims to solve the technical problems of time consumption, low yield, uneven granularity and uneven active ingredients of the traditional granulation process, which are caused by poor activity stability of the existing neutral protease powder preparation.
(II) technical scheme
In order to solve the technical problems, the invention provides a preparation method of neutral protease pellets, which comprises the following steps:
s1, inoculating bacillus subtilis strain into an LB culture medium of an eggplant bottle to prepare a fermentation strain;
s2, inoculating the fermentation strain into a culture medium in a seed tank, and controlling the air quantity to be 40-50 m at the temperature of 33-34 ℃ and the rotating speed of 180rpm 3 Culturing for 18-20 h in the environment of/h to obtain seed liquid;
s3, inoculating the seed liquid in the step S2 into a fermentation medium of an enzyme-producing fermentation tank, and culturing for 26-30 h at the temperature of 35-38 ℃ and the rotating speed of 140-160 rpm under the air volume of 1:0.8-1:1VVM environment to obtain fermentation liquid;
s4, centrifugal impurity removal: pumping the fermentation liquor into a horizontal decanter centrifuge, removing undissolved and insoluble culture medium particles, and collecting centrifugate;
s5, filtering and sterilizing: pumping the collected centrifugate into a ceramic membrane for filtration, and collecting filtered enzyme solution;
s6, ultrafiltration concentration: pumping the collected filtered enzyme solution into ultrafiltration concentration equipment for concentrating the enzyme solution, wherein the concentration multiple is 3-5 times to obtain concentrated enzyme solution;
s7, preparing a granulating enzyme solution: adding dextrin or carboxymethyl cellulose, light calcium carbonate and corn starch into the concentrated enzyme solution, and uniformly stirring;
s8, spray granulation: pumping the granulating enzyme liquid into a spray drying tower through a diaphragm pump for atomization, drying and granulating.
Further, the culture conditions in step S1 are culture at a temperature of 35℃for 28 to 36 hours.
Further, the culture medium in the seed tank in step S2 is: corn 20g/L, soybean meal 70g/L, na 2 HPO 4 4g/L，KH 2 PO 4 0.3g/L, and the inoculation amount of the fermentation strain is 2%.
Further, the fermentation medium in step S3 is: 58g/L corn, 42g/L bean pulp, 50g/L bran and Na 2 HPO 4 4.5g/L，KH 2 PO 4 0.3g/L, and natural pH.
Further, in the step S4, the rotating speed of the horizontal decanter centrifuge is 2900-3000 rpm, and the centrifuging time is 4-5 h.
Further, in the step S5, the filter pore diameter of the ceramic membrane is 500nm, and the filter time is 8-10 hours.
Further, the addition amounts of the components in step S7 are as follows: 8-12% of dextrin or carboxymethyl cellulose, 20-30% of light calcium carbonate and 10-20% of corn starch.
Further, the model of the atomizing nozzle in the step S8 is 1.8mm or 2.0mm; the diaphragm pump pressure is 40-45Hz; drying conditions: the inlet temperature is 150-200deg.C, the tower temperature is 70-80deg.C, and the outlet temperature is 70-75deg.C.
The invention also provides a neutral protease micropill which is prepared by adopting the preparation method of the neutral protease micropill.
(III) beneficial effects
The technical scheme of the invention has the following advantages: the neutral protease micropill and the preparation method thereof provided by the invention have the advantages of high production efficiency, uniform granularity, uniform active ingredients and low production cost. The fermentation process and other parameters in the production process of neutral protease are started, so that the culture medium is optimized, and meanwhile, the separation and concentration processes of fermentation liquid are increased; the formula of the protease micropills is optimized, and the neutral protease micropills with uniform granularity are prepared by one-step molding in the pressure spray drying process; the spray granulation is formed at one time, the yield per unit time is high, and the efficiency is high. Has good popularization and application prospect and market prospect.
In addition to the technical problems, features of the constituent technical solutions and advantages brought by the technical features of the technical solutions described above, other technical features of the present invention and advantages brought by the technical features of the technical solutions will be further described with reference to the embodiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment discloses a neutral protease pellet and a preparation method thereof, wherein the preparation method of the neutral protease pellet comprises the following steps:
s1, inoculating bacillus subtilis strain into an LB culture medium of an eggplant bottle, and culturing for 28 hours at the temperature of 35 ℃ to obtain a fermentation strain;
s2, inoculating the fermentation strain into a seed tank with the inoculum size of 2%, wherein the formula of a culture medium in the seed tank is 20g/L of corn, 70g/L of soybean meal and Na 2 HPO 4 4g/L，KH 2 PO 4 0.3g/L; culture time 18h, culture environment: the temperature is 33 ℃, the rotating speed is 180rpm, and the air quantity is 40m 3 /h; preparing seed liquid;
s3, inoculating the seed liquid in the step S2 into a fermentation medium of an enzyme production fermentation tank, wherein the formula of the fermentation medium is as follows: 58g/L corn, 42g/L bean pulp, 50g/L bran and Na 2 HPO 4 4.5g/L，KH 2 PO 4 0.3g/L, natural pH, 26h of culture time, culture environment: the temperature is 35 ℃, the rotating speed is 140rpm, and the air volume is 1:0.8VVM; preparing fermentation liquor, wherein the enzyme activity of the fermentation liquor is more than 20000U/mL, and the detection standard is as follows: GB/T23527;
s4, centrifugal impurity removal: pumping the fermentation liquor into a horizontal decanter centrifuge, wherein the rotation speed is 2900rpm, and the centrifugation time is 4 hours; removing undissolved and insoluble culture medium particles, and collecting centrifugate;
s5, filtering and sterilizing: pumping the collected centrifugate into a ceramic membrane for filtration, wherein the filtration pore diameter is 500nm, and the filtration time is 8 hours; collecting and filtering enzyme liquid;
s6, ultrafiltration concentration: pumping the collected filtered enzyme solution into ultrafiltration concentration equipment for concentrating the enzyme solution, wherein the concentration multiple is 3 times to obtain concentrated enzyme solution;
s7, preparing a granulating enzyme solution: adding 8% dextrin or carboxymethyl cellulose, 20% light calcium carbonate and 10% corn starch into the concentrated enzyme solution, and uniformly stirring to obtain a granulating enzyme solution;
s8, spray granulation: pumping the granulating enzyme liquid into a spray drying tower through a diaphragm pump for atomization, drying and granulating, wherein the model of an atomization nozzle is as follows: 1.8mm; the diaphragm pump pressure was 40Hz; drying conditions: inlet temperature 150 ℃, tower temperature 70 ℃, and outlet temperature 70 ℃.
The neutral protease micropill disclosed in this example is prepared by the preparation method of the neutral protease micropill disclosed in this example.
Example 2
The embodiment discloses a neutral protease pellet and a preparation method thereof, wherein the preparation method of the neutral protease pellet comprises the following steps:
s1, inoculating bacillus subtilis strain into an LB culture medium of an eggplant bottle, and culturing for 36 hours at the temperature of 35 ℃ to obtain a fermentation strain;
s2, inoculating the fermentation strain into a seed tank with the inoculum size of 2%, wherein the formula of a culture medium in the seed tank is 20g/L of corn, 70g/L of soybean meal and Na 2 HPO 4 4g/L，KH 2 PO 4 0.3g/L; culture time 20h, culture environment: the temperature is 34 ℃, the rotating speed is 180rpm, and the air quantity is 50m 3 /h; preparing seed liquid;
s3, inoculating the seed liquid in the step S2 into a fermentation medium of an enzyme production fermentation tank, wherein the formula of the fermentation medium is as follows: 58g/L corn, 42g/L bean pulp, 50g/L bran and Na 2 HPO 4 4.5g/L，KH 2 PO 4 0.3g/L, natural pH, 30h of culture time, culture environment: the temperature is 38 ℃, the rotating speed is 140-160 rpm, the air quantity is 1:0.8-1:1VVM; preparing fermentation liquor, wherein the enzyme activity of the fermentation liquor is more than 20000U/mL, and the detection standard is as follows: GB/T23527;
s4, centrifugal impurity removal: pumping the fermentation liquor into a horizontal decanter centrifuge, and centrifuging at 3000rpm for 5h; removing undissolved and insoluble culture medium particles, and collecting centrifugate;
s5, filtering and sterilizing: pumping the collected centrifugate into a ceramic membrane for filtration, wherein the filtration pore diameter is 500nm, and the filtration time is 10 hours; collecting and filtering enzyme liquid;
s6, ultrafiltration concentration: pumping the collected filtered enzyme solution into ultrafiltration concentration equipment for concentrating the enzyme solution, wherein the concentration multiple is 5 times, so as to obtain concentrated enzyme solution;
s7, preparing a granulating enzyme solution: adding 12% dextrin or carboxymethyl cellulose, 30% light calcium carbonate and 20% corn starch into the concentrated enzyme solution, and uniformly stirring to obtain a granulating enzyme solution;
s8, spray granulation: pumping the granulating enzyme liquid into a spray drying tower through a diaphragm pump for atomization, drying and granulating, wherein the model of an atomization nozzle is as follows: 2.0mm; the diaphragm pump pressure was 45Hz; drying conditions: inlet temperature is 200 ℃, tower temperature is 80 ℃, and outlet temperature is 75 ℃.
The neutral protease micropill disclosed in this example is prepared by the preparation method of the neutral protease micropill disclosed in this example.
Example 3
The embodiment discloses a neutral protease pellet and a preparation method thereof, wherein the preparation method of the neutral protease pellet comprises the following steps:
s1, inoculating bacillus subtilis strain into an LB culture medium of an eggplant bottle, and culturing for 32 hours at the temperature of 35 ℃ to obtain a fermentation strain;
s2, inoculating the fermentation strain into a seed tank with the inoculum size of 2%, wherein the formula of a culture medium in the seed tank is 20g/L of corn, 70g/L of soybean meal and Na 2 HPO 4 4g/L，KH 2 PO 4 0.3g/L; culture time 19h, culture environment: the temperature is 34 ℃, the rotating speed is 180rpm, and the air quantity is 50m 3 /h; preparing seed liquid;
s3, inoculating the seed liquid in the step S2 into a fermentation medium of an enzyme production fermentation tank, wherein the formula of the fermentation medium is as follows: 58g/L corn, 42g/L bean pulp, 50g/L bran and Na 2 HPO 4 4.5g/L，KH 2 PO 4 0.3g/L, natural pH, culture time of 28h, culture environment: the temperature is 36 ℃, the rotating speed is 160rpm, and the air quantity is 1:1VVM; preparing fermentation liquor, wherein the enzyme activity of the fermentation liquor is more than 20000U/mL, and the detection standard is as follows: GB/T23527;
s4, centrifugal impurity removal: pumping the fermentation liquor into a horizontal decanter centrifuge, wherein the rotating speed is 2900-3000 rpm, and the centrifugation time is 5h; removing undissolved and insoluble culture medium particles, and collecting centrifugate;
s5, filtering and sterilizing: pumping the collected centrifugate into a ceramic membrane for filtration, wherein the filtration pore diameter is 500nm, and the filtration time is 10 hours; collecting and filtering enzyme liquid;
s6, ultrafiltration concentration: pumping the collected filtered enzyme solution into ultrafiltration concentration equipment for concentrating the enzyme solution, wherein the concentration multiple is 4 times, so as to obtain concentrated enzyme solution;
s7, preparing a granulating enzyme solution: adding 12% dextrin or carboxymethyl cellulose, 28% light calcium carbonate and 18% corn starch into the concentrated enzyme solution, and uniformly stirring to obtain a granulating enzyme solution;
s8, spray granulation: pumping the granulating enzyme liquid into a spray drying tower through a diaphragm pump for atomization, drying and granulating, wherein the model of an atomization nozzle is as follows: 2.0mm; the diaphragm pump pressure was 45Hz; drying conditions: inlet temperature 180 deg.c, tower temperature 80 deg.c and outlet temperature 75 deg.c.
The neutral protease micropill disclosed in this example is prepared by the preparation method of the neutral protease micropill disclosed in this example.
While the present invention has been described in detail with reference to the specific embodiments thereof, the present invention is not limited to the above embodiments, and various changes may be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (2)
1. The preparation method of the neutral protease micropill is characterized by comprising the following steps:
s1, inoculating bacillus subtilis strain into an LB culture medium of an eggplant bottle to prepare a fermentation strain;
s2, inoculating fermentation strains to a culture medium in a seed tank, wherein the culture medium in the seed tank is as follows: corn 20g/L, soybean meal 70g/L, na 2 HPO 4 4g/L，KH 2 PO 4 0.3g/L, the inoculum size of the fermentation strain is 2%, the rotation speed is 180rpm at the temperature of 33-34 ℃ and the air quantity is 40-50 m 3 Culturing for 18-19 h in a/h environment to obtain seed liquid;
s3, inoculating the seed liquid in the step S2 into a fermentation medium of an enzyme-producing fermentation tank, and controlling the air volume to be 1:0 at the temperature of 38 ℃ and the rotating speed of 140-160 rpmCulturing for 26-28 h in an 8-1:1VVM environment to obtain fermentation broth, wherein the fermentation medium is as follows: 58g/L corn, 42g/L bean pulp, 50g/L bran and Na 2 HPO 4 4.5g/L，KH 2 PO 4 0.3g/L, natural PH;
s4, centrifugal impurity removal: pumping the fermentation liquor into a decanter centrifuge, collecting the centrifugate, wherein the rotating speed of the decanter centrifuge is 2900-3000 rpm, and the centrifugation time is 4-5 hours;
s5, filtering and sterilizing: pumping the collected centrifugal liquid into a ceramic membrane for filtration, collecting a filtered enzyme liquid, wherein the pore diameter of the ceramic membrane is 500nm, and the filtration time is 8-10 hours;
s6, ultrafiltration concentration: pumping the collected filtered enzyme solution into ultrafiltration concentration equipment for concentrating the enzyme solution, wherein the concentration multiple is 3-5 times to obtain concentrated enzyme solution;
s7, preparing a granulating enzyme solution: adding dextrin or carboxymethyl cellulose, light calcium carbonate and corn starch into the concentrated enzyme solution, and uniformly stirring, wherein the adding amount of each component is as follows: 8-12% of dextrin or carboxymethyl cellulose, 20-30% of light calcium carbonate and 10-20% of corn starch;
s8, spray granulation: pumping the granulating enzyme liquid into a spray drying tower through a diaphragm pump for atomization drying and granulating, wherein the model of an atomization nozzle is 1.8mm or 2.0mm; the diaphragm pump pressure is 40-45Hz; drying conditions: the inlet temperature is 150-200deg.C, the tower temperature is 70-80deg.C, and the outlet temperature is 70-75deg.C.
2. The method for preparing the neutral protease micropill according to claim 1, wherein: and in the step S1, the culture condition is that the culture is carried out for 28-36 hours at the temperature of 35 ℃.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040045238A (en) * | 2002-11-23 | 2004-06-01 | 한국유나이티드제약 주식회사 | Pellet preparations and methods for improved solubility of itraconazole |
| US6924133B1 (en) * | 1999-10-01 | 2005-08-02 | Novozymes A/S | Spray dried enzyme product |
| CN102174503A (en) * | 2010-11-29 | 2011-09-07 | 天津市诺奥科技发展有限公司 | Spray drying micro-capsulation process for high-yield alkaline protease |
| CN107410710A (en) * | 2017-09-12 | 2017-12-01 | 昆明佰佳益康生物科技有限公司 | A kind of preparation method of phytase preparation |
| CN114317503A (en) * | 2021-12-17 | 2022-04-12 | 江苏习习生物科技有限公司 | Granular neutral protease and preparation method thereof |
-
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- 2022-08-30 CN CN202211049779.7A patent/CN115232806B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6924133B1 (en) * | 1999-10-01 | 2005-08-02 | Novozymes A/S | Spray dried enzyme product |
| KR20040045238A (en) * | 2002-11-23 | 2004-06-01 | 한국유나이티드제약 주식회사 | Pellet preparations and methods for improved solubility of itraconazole |
| CN102174503A (en) * | 2010-11-29 | 2011-09-07 | 天津市诺奥科技发展有限公司 | Spray drying micro-capsulation process for high-yield alkaline protease |
| CN107410710A (en) * | 2017-09-12 | 2017-12-01 | 昆明佰佳益康生物科技有限公司 | A kind of preparation method of phytase preparation |
| CN114317503A (en) * | 2021-12-17 | 2022-04-12 | 江苏习习生物科技有限公司 | Granular neutral protease and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 中兽药四黄止痢缓释微丸的研制;赖建彬等;《今日畜牧兽医》;第35卷(第3期);第14-15页 * |
| 张华等主编.《制剂单元操作及仿真实训》.上海交通大学出版社,2021,(第1版),第54页第3段. * |
| 托普司他缓释微丸胶囊的研究;岳珍;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》(第1期);第E079-54页 * |
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