CN101948820A - Acidic proteinase and preparation method thereof - Google Patents
Acidic proteinase and preparation method thereof Download PDFInfo
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- CN101948820A CN101948820A CN 201010265354 CN201010265354A CN101948820A CN 101948820 A CN101948820 A CN 101948820A CN 201010265354 CN201010265354 CN 201010265354 CN 201010265354 A CN201010265354 A CN 201010265354A CN 101948820 A CN101948820 A CN 101948820A
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Abstract
The invention relates to an acidic proteinase and a preparation method thereof and the acidic proteinase belongs to the microbial acidic proteinase. The invention is characterized in that 1) the enzymatic characteristics of the acidic proteinase are as follows: the optimum pH value is 2.5-3.5, the stable pH value is 2.5-6.0; the optimum temperature is 40-50 DEG C, the temperature stability range is 30-50 DEG C; and 2) the preparation method uses Aspergillus Niger which is stored in the China Center of Industrial Culture Collection (CICC) and has a preservation number of 2238, as the enzyme-producing strain and uses wheat bran as the raw material; the solid fermentation technology is adopted for preparation; the fermentation and enzyme-producing capability is not less than 47000u/g (dry yeast), the liquid enzyme yield is not less than 85% and the soid enzyme yield is not less than 80%, which all achieve the food grade sanitation standard. The invention provides the acidic proteinase and the preparation method thereof, wherein the utilization rate of the fermentation equipment is high, the byproduct of crop processing is used as the main raw material, the enzyme activity for fermentation is high, the extracting and purifying rate of enzyme is high and the production cost is low. The acidic proteinase of the invention is suitable to be used as the raw material or product additive of the products in the leather industry, the pharmaceutical industry, the brewing industry and the feed industry.
Description
Technical field
The present invention is a kind of aspartic protease and preparation method thereof.Belonging to the aspartic protease from microbial source, is the zymogenic bacteria kind with aspergillus niger (A.niger) particularly, adopts aspartic protease of solid fermentation process preparation and preparation method thereof.
Background technology
Aspartic protease can be in sour environment protein hydrolysate, be widely used in tanning industry, pharmaceutical sector, Brewing industry and fodder industry.Aspartic protease can obviously promote growing of young animal as a kind of novel fodder additives, reduces the stress effect that wean brings, and feeding effect highly significant is the boundless zymin of a class application prospect.
In the prior art, aspartic protease mainly adopts liquid-state fermentation technology, and the fermentation equipment utilization ratio is low, is carbon source with corn, soybean mainly, consumes a large amount of valuable grain resources; The fermenting enzyme vigor is low, enzyme extraction, purification yield are low; Cause the products production cost to rise.
A kind of fermentation equipment utilization ratio height is a main raw material with the grain processing by product, and fermenting enzyme vigor height, enzyme extraction purification yield height, aspartic protease that production cost is lower and preparation method thereof are that people expect.
Summary of the invention
The objective of the invention is to avoid above-mentioned weak point of the prior art, and provide a kind of fermentation equipment utilization ratio height, with the grain processing by product is main raw material, fermenting enzyme vigor height, enzyme extraction purification yield height, aspartic protease that production cost is lower and preparation method thereof.
Purpose of the present invention can reach by following measure:
Aspartic protease of the present invention is characterized in that:
A. enzymatic property: optimal pH 2.5-3.5, stablize pH2.5-6.0; Optimum temperature is 40 ℃-50 ℃, and the temperature stability scope is 30 ℃-50 ℃;
B. with aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238 and be the zymogenic bacteria kind, wheat bran is main raw material, adopts solid fermentation, immersion extraction, filtering and concentrating, dissolved purifying, ultrafiltration, the preparation of smart filtering technology, reaches following technical indicator:
1. enzymatic production ability: 〉=47000u/g (in dry medium),
2. soak extraction, ultrafiltration, smart filter, liquid enzymes yield 〉=85%, solid enzyme yield 〉=80% reaches the food grade hygienic standard.
The present inventor finds unexpectedly, by the cereal shell or not hulled grain participate in can making aspergillus niger (Aspergillus niger) as the solid fermentation main culture based raw material, the enzymatic productivity that is preserved in CICC numbering 2238 significantly improves.Wheat bran is as the by-product of whole meal flour processing industry, and the source is abundant.Selecting wheat bran for use is that main carbon source had both been saved valuable grain resource, has improved aspergillus niger (Aspergillus niger) again, is preserved in the enzymatic productivity of CICC numbering 2238.For productive rate and its production cost of reduction of improving aspartic protease of the present invention are made great contributions to.With aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238 and be the zymogenic bacteria kind, adopt the solid fermentation process method, improve the utilization ratio that feeds intake of fermentation equipment greatly, thereby improved the throughput of equipment.Soak the enforcement of extraction, ultrafiltration and concentration, dissolved purifying, ultrafiltration, smart filter, vacuum drying process, for the raising of enzyme extraction purification yield provides technical support.
Purpose of the present invention can also reach by following measure:
Aspartic protease of the present invention, it is characterized in that the aspergillus niger described in the b (Aspergillus niger), be preserved in CICC and number 2238 zymogenic bacteria kinds with cobalt 60 rays, the processing of excess ultraviolet mutagenesis, give and produce aspartic protease bacterial strain (Aspergillus niger), be preserved in CICC and number good characteristics such as 2238 stabilization characteristics of genetics are good, product enzyme speed is fast, product enzyme level height.The performance and the productive rate that improve aspartic protease of the present invention have been brought into play active effect.
Aspartic protease of the present invention is characterized in that moisture content in the solid fermentation raw material between 40%-50%, is an optimized technical scheme.
The preparation method of aspartic protease of the present invention is characterized in that comprising the steps:
A. liquid spawn is cultivated in advance: substratum 10-25 weight part, add water to 100 weight parts, and regulate pH to 4.6-5.0, sterilized 10-35 minute for 121 ℃, be cooled to 30 ℃-35 ℃, insert bacterial classification aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 30 ℃-40 ℃ of controlled temperature, ventilation 45-60m
3/ h, pressure 0.05Mpa-0.08Mpa cultivated 28-32 hour, got liquid spawn, was equipped with solid state fermentation and used;
B. solid state fermentation: 120 ℃-123 ℃ of raw materials boiling 50-60 minute, be cooled to 30 ℃-35 ℃, insert the liquid spawn of dilution, stir evenly, make material initial water content 40-50%, ventilation: 50-60m
3/ h, culture cycle 5.0 days; Fermentation level 〉=47000u/g (in dry medium);
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, pH to 4.3 ± 0.1, liquid enzymes yield 〉=85%, solid enzyme yield 〉=80% reaches the food grade hygienic standard.
Aspartic protease of the present invention is used, and it is characterized in that being applicable to preparation raw material or product additive as tanning industry, pharmaceutical sector, Brewing industry and fodder industry product.
Technical scheme of the present invention has outstanding substantive distinguishing features and obvious improvement compared to existing technology, implements the back and produces following positively effect:
1. a kind of fermentation equipment utilization ratio height is provided, and is main raw material with the grain processing by product, fermenting enzyme vigor height, enzyme extraction purification yield height, aspartic protease that production cost is lower and preparation method thereof.
2. selecting wheat bran for use is that main raw material had both been saved valuable grain resource, has improved aspergillus niger (Aspergillus niger) again, is preserved in the enzymatic productivity of CICC numbering 2238.
3. with aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238 and be the zymogenic bacteria kind, adopt the solid fermentation process method, improved the utilization ratio that feeds intake of fermentation equipment greatly, thereby improved the throughput of equipment.
4. soak the enforcement of extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filtering technology, for the raising of enzyme extraction purification yield provides technical support.
5. aspartic protease is applicable to preparation raw material or the product additive as tanning industry, pharmaceutical sector, Brewing industry and fodder industry product.For the user has won benefit.
Embodiment
The present invention is described in further detail below in conjunction with embodiment:
Embodiment 1
A. liquid spawn is cultivated in advance: with 100 kilograms in dextrin, and 10 kilograms of peptones, 5 kilograms of yeast extract pastes, 20 kilograms of corn steep liquors drop in the 1000L reactor, add water to 1000 kilograms of total charging capacitys, and regulate pH to 5.0; Be warming up to 121 ℃, insulated sterilizing 35 minutes.Be cooled to 30 ℃, insert aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 30 ℃ of controlled temperature, ventilation 60m
3/ h, jar internal pressure 0.05Mpa-0.08Mpa, culture cycle 32 hours gets liquid spawn, is equipped with solid fermentation and uses;
B. solid fermentation: 3000 kilograms in wheat bran, under 121 ℃ of temperature, boiling 60 minutes; Be cooled to 35 ℃; Stir the liquid spawn of inserting down through water-reducible step a preparation, stir, the initial water content that makes fermentation raw material is at 40%-50%; At 35 ℃ of temperature, ventilation 60m
3/ h, fermentation culture 5.0 days, fermentation level: 〉=47000u/g (in dry medium);
C. adopt and soak extraction, ultrafiltration and concentration, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.3 ± 0.1, the allotment can, liquid enzymes yield 〉=85% reaches the food grade hygienic standard.
Embodiment 2
A. liquid spawn is cultivated in advance: with 100 kilograms of starch, and 10 kilograms of peptones, 5 kilograms of yeast extract pastes, 20 kilograms of corn steep liquors, alpha-amylase 1000ml, 0.5 kilogram in calcium chloride drops in the 1000L reactor, adds water to 1000 kilograms of total charging capacitys, and regulates pH to 5.0; Be warming up to 121 ℃, insulated sterilizing 35 minutes.Be cooled to 30 ℃, insert aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 30 ℃ of controlled temperature, ventilation 60m
3/ h, jar internal pressure 0.05Mpa-0.08Mpa, culture cycle 35 hours gets liquid spawn, is equipped with solid fermentation and uses;
B. solid fermentation: 3000 kilograms in wheat bran, under 123 ℃ of temperature, boiling 50 minutes; Be cooled to 35 ℃; Stir the liquid spawn of inserting down through water-reducible step a preparation, stir, the initial water content that makes fermentation raw material is 50%; At 30 ℃ of temperature, ventilation 50m
3/ h, fermentation culture 5.0 days, fermentation level: 〉=47000u/g (in dry medium);
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, regulate pH to 4.3 ± 0.1, the allotment can, liquid enzymes yield 〉=85% reaches the food grade hygienic standard.
Embodiment 3
A. liquid spawn is cultivated in advance: with 100 kilograms of malt syrups, and 10 kilograms of peptones, 5 kilograms of yeast extract pastes, 20 kilograms of corn steep liquors, 12 kilograms of soybean cake powder drop in the 1000L reactor, add water to 1000 kilograms of total charging capacitys, and regulate pH to 4.8; Be warming up to 121 ℃, insulated sterilizing 35 minutes.Be cooled to 30 ℃, insert aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 30 ℃ of controlled temperature, ventilation 60m
3/ h, jar internal pressure 0.05Mpa-0.08Mpa, culture cycle 28 hours gets liquid spawn, is equipped with solid fermentation and uses;
B. solid fermentation: 3000 kilograms in wheat bran, under 123 ℃ of temperature, boiling 50 minutes; Be cooled to 32 ℃; Stir the liquid spawn of inserting down through water-reducible step a preparation, stir, the initial water content that makes fermentation raw material is 40%; At 32 ℃ of temperature, ventilation 50m
3/ h, fermentation culture 5.0 days, fermentation level: 〉=47000u/g (in dry medium);
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.3 ± 0.1, vacuum-drying, packing.Solid enzyme yield 〉=80% reaches the food grade hygienic standard.
Claims (5)
1. aspartic protease is characterized in that:
A. enzymatic property: optimal pH 2.5-3.5, stablize pH2.5-6.0; Optimum temperature is 40 ℃-50 ℃, and the temperature stability scope is 30 ℃-50 ℃;
B. with aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238 and be the zymogenic bacteria kind, wheat bran is main raw material, adopts solid fermentation, immersion extraction, filtering and concentrating, dissolved purifying, ultrafiltration, the preparation of smart filtering technology, reaches following technical indicator:
1. enzymatic production ability: 〉=47000u/g (in dry medium),
2. soak extraction, ultrafiltration, smart filter, liquid enzymes yield 〉=85%, solid enzyme yield 〉=80% reaches the food grade hygienic standard.
2. according to the aspartic protease of claim 1, it is characterized in that the aspergillus niger described in the b (Aspergillus niger), be preserved in CICC and number 2238 zymogenic bacteria kinds with cobalt 60 rays, the processing of excess ultraviolet mutagenesis, give and produce aspartic protease bacterial strain (Aspergillus niger), be preserved in CICC and number good characteristics such as 2238 stabilization characteristics of genetics are good, product enzyme speed is fast, product enzyme level height.
3. according to the aspartic protease of claim 1, it is characterized in that moisture content in the solid fermentation raw material is between 40%-50%.
4. the preparation method of the aspartic protease of a claim 1 is characterized in that comprising the steps:
A. liquid spawn is cultivated in advance: substratum 10-25 weight part, add water to 100 weight parts, and regulate pH to 4.6-5.0, sterilized 10-35 minute for 121 ℃, be cooled to 30 ℃-35 ℃, insert bacterial classification aspergillus niger (Aspergillus niger), be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 30 ℃-40 ℃ of controlled temperature, ventilation 45-60m
3/ h, pressure 0.05Mpa-0.08Mpa cultivated 28-32 hour, got liquid spawn, was equipped with solid state fermentation and used;
B. solid state fermentation: 120 ℃-123 ℃ of raw materials boiling 50-60 minute, be cooled to 30 ℃-35 ℃, insert the liquid spawn of dilution, stir evenly, make material initial water content 40%-50%, ventilation: 50-60m
3/ h, culture cycle 5.0 days; Fermentation level 〉=47000u/g (in dry medium);
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, pH to 4.3 ± 0.1, liquid enzymes yield 〉=85%, solid enzyme yield 〉=80% reaches the food grade hygienic standard.
5. the aspartic protease of claim 1 is used, and it is characterized in that being applicable to preparation raw material or product additive as tanning industry, pharmaceutical sector, Brewing industry and fodder industry product.
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| CN 201010265354 CN101948820A (en) | 2009-08-27 | 2010-08-27 | Acidic proteinase and preparation method thereof |
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| CN200910017983.9 | 2009-08-27 | ||
| CN200910017983A CN101638647A (en) | 2009-08-27 | 2009-08-27 | Acid protease and preparation method thereof |
| CN 201010265354 CN101948820A (en) | 2009-08-27 | 2010-08-27 | Acidic proteinase and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994485A (en) * | 2012-12-24 | 2013-03-27 | 湖南鸿鹰生物科技有限公司 | Method for preparing acid proteinase |
| CN105199969A (en) * | 2015-10-19 | 2015-12-30 | 山东隆科特酶制剂有限公司 | Aspergillus niger strain capable of highly producing acidic protease and method for fermenting aspergillus niger strain liquid to produce enzymes |
| CN105316308A (en) * | 2014-08-04 | 2016-02-10 | 湖南新鸿鹰生物工程有限公司 | High-activity acid protease |
| CN112940943A (en) * | 2019-12-11 | 2021-06-11 | 安琪酵母股份有限公司 | Bacterial strain for producing acid protease by liquid fermentation and application |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638647A (en) * | 2009-08-27 | 2010-02-03 | 山东隆科特酶制剂有限公司 | Acid protease and preparation method thereof |
| MD4186C1 (en) * | 2012-02-20 | 2013-06-30 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Strain of fungus Fusarium gibbosum - producer of acid and neutral proteases, xylanases and b-glucosidases |
| CN105950607A (en) * | 2016-05-25 | 2016-09-21 | 浙江工业大学 | Selecting method of acid proteinase high-yield strains obtained through induced mutation by 60Co-gamma rays |
| CN107384900B (en) * | 2017-08-01 | 2019-08-27 | 中国农业科学院饲料研究所 | The acid protease 6749 and its gene of a kind of originated from fungus and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101638647A (en) * | 2009-08-27 | 2010-02-03 | 山东隆科特酶制剂有限公司 | Acid protease and preparation method thereof |
| CN101671659A (en) * | 2009-09-25 | 2010-03-17 | 山东隆科特酶制剂有限公司 | Pectase and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638647A (en) * | 2009-08-27 | 2010-02-03 | 山东隆科特酶制剂有限公司 | Acid protease and preparation method thereof |
| CN101671659A (en) * | 2009-09-25 | 2010-03-17 | 山东隆科特酶制剂有限公司 | Pectase and preparation method thereof |
Non-Patent Citations (2)
| Title |
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| 《中国调味品》 20090331 姚菁华等 酱油固态发酵生产中黑曲霉产酶条件的优化研究 第34卷, 第3期 2 * |
| 《食品科学》 20031231 袁康培等 黑曲霉HU53 菌株产酸性蛋白酶的条件和酶学性质 第24卷, 第8期 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994485A (en) * | 2012-12-24 | 2013-03-27 | 湖南鸿鹰生物科技有限公司 | Method for preparing acid proteinase |
| CN102994485B (en) * | 2012-12-24 | 2016-08-24 | 湖南鸿鹰生物科技有限公司 | A kind of method producing acid protease |
| CN105316308A (en) * | 2014-08-04 | 2016-02-10 | 湖南新鸿鹰生物工程有限公司 | High-activity acid protease |
| CN105199969A (en) * | 2015-10-19 | 2015-12-30 | 山东隆科特酶制剂有限公司 | Aspergillus niger strain capable of highly producing acidic protease and method for fermenting aspergillus niger strain liquid to produce enzymes |
| CN105199969B (en) * | 2015-10-19 | 2019-05-14 | 山东隆科特酶制剂有限公司 | The Aspergillus niger strain and its liquid fermentation enzyme producing method of one plant height production acid protease |
| CN112940943A (en) * | 2019-12-11 | 2021-06-11 | 安琪酵母股份有限公司 | Bacterial strain for producing acid protease by liquid fermentation and application |
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| CN101638647A (en) | 2010-02-03 |
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Application publication date: 20110119 |