CN107488595A - A kind of ocean aspergillus niger ZJUBE2010 and its application - Google Patents
A kind of ocean aspergillus niger ZJUBE2010 and its application Download PDFInfo
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- CN107488595A CN107488595A CN201710689555.5A CN201710689555A CN107488595A CN 107488595 A CN107488595 A CN 107488595A CN 201710689555 A CN201710689555 A CN 201710689555A CN 107488595 A CN107488595 A CN 107488595A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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Abstract
The invention discloses a kind of ocean aspergillus niger ZJUBE 2010 and its application.Ocean aspergillus niger ZJUBE 2010 (Aspergillus sp.ZJUBE 2010) of the present invention is preserved in China typical culture collection center on June 2nd, 2010, and deposit number is CCTCC NO：M2010132.Ocean aspergillus niger ZJUBE 2010 of the present invention is used for fermenting and producing cellulase, and yield is high, and enzymatic activity is good；In addition, ocean aspergillus niger ZJUBE 2010 can be additionally used in the fermentative degradation in charcoal source, monose high conversion rate, fermentation process need not carry out the sterilization treatment of HTHP in itself to raw material fermentation culture medium, equipment, ferment effect is completely without any influence, equipment manufacturing costs height, production cost are effectively reduced, reduces energy consumption.
Description
Technical field
The invention belongs to technical field of microbial fermentation, more particularly to a kind of ocean aspergillus niger ZJUBE 2010 and its should
With.
Background technology
Aspergillus niger produces cellulase on the premise of using cellulose as inducer, is using list for degraded cellulose
Sugar.The most frequently used fermentation substrate is wheat bran, maize straw, rice straw powder in fermentation of Aspergillus niger, and primary carbon source can be used as simultaneous
Inducer.
The conventional fermentation process of aspergillus niger has two kinds of liquid state fermentation and solid state fermentation.Wherein, it is practically free of in solid state fermentation
Free state water, closer to microorganism nature growth conditions, enzyme activity is higher, and subsequent treatment is simple, and pollution is few, non-wastewater discharge；
It is easy to control without rigorous aseptic environment, living contaminants and solid state fermentation mode is more extensive.
However, solid state fermentation, which exists, sterilizes the problem of difficult, it is necessary to be passed through a large amount of steam, it is higher to equipment requirement.Therefore,
If fermentation substrate is unsterilised, directly using raw material fermentation, then it can reach simplified Zymolysis Equipment, save the energy, reduce production
The purpose of cost.
The content of the invention
It is an object of the invention to provide a kind of ocean aspergillus niger ZJUBE 2010 and its application, it is intended to solves existing solid-state
Needing to carry out fermentation substrate sterilizing in fermentation process causes that Zymolysis Equipment manufacturing cost is high, production cost is high, energy consumption consumption is big
The problem of.
The present invention is achieved in that a kind of (Aspergillus sp.ZJUBE of ocean aspergillus niger ZJUBE 2010
2010), the ocean aspergillus niger was preserved in China typical culture collection center, deposit number CCTCC on June 2nd, 2010
NO：M2010132.
The present invention further discloses above-mentioned ocean aspergillus niger ZJUBE 2010 answering in terms of fermenting and producing cellulase
With.
It is being monose side by fermentation process degraded charcoal source the present invention further discloses ocean aspergillus niger ZJUBE 2010
The application in face.
Preferably, the fermentation process comprises the following steps：
(1) strains of ocean aspergillus niger ZJUBE 2010 are seeded on Spore cultivation base and cultivate spore；
(2) spore suspension in step (1) is inoculated into seed culture medium and cultivates seed liquor；
(3) seed liquor in step (2) is inoculated into raw material fermentation culture medium and carries out Solid Fermentation of Uncooked Material；Wherein, raw material
Solid state fermentation is specially：It is that 20~50% strains are directly seeded in without high temperature by mass percent at a temperature of 35~40 DEG C
Cultivated 3~5 days in high pressure-treated raw material fermentation culture medium.
Preferably, in step (1), the Spore cultivation base potato dextrose agar, Spore cultivation temperature
For 30~35 DEG C, incubation time is 3~5 days.
Preferably, in step (2), the seed culture medium includes mass fraction as 1~5% using carbon source and matter
The organic nitrogen source that fraction is 0.25~3% is measured, the seed culture medium carries out conventional sterilant processing, the seed liquor before inoculation
Cultivation temperature is 30 DEG C, incubation time is 2 days.
Preferably, it is described to include slowly utilizing carbon source using carbon source；The organic nitrogen source includes peptone.
Preferably, in step (3), Solid Fermentation of Uncooked Material is carried out in tray, and the heap of the raw material fermentation culture medium
Product thickness is 3~5 centimetres；
Raw material fermentation culture medium includes described in 100ml：5~12g sodium chloride, the 1~6g carbon sources quickly utilized, slowly utilize
1~25g carbon sources, and 5~25g cellulase induction things；The material-water ratio of the raw material fermentation culture medium is 3：5.
Preferably, the carbon source quickly utilized includes grape sugar, and the carbon source slowly utilized includes corn stalk powder, rice
Grass meal；The cellulase induction thing is wheat bran.
Overcome the deficiencies in the prior art of the present invention, there is provided a kind of ocean aspergillus niger ZJUBE 2010 and its application.The present invention
Ocean aspergillus niger ZJUBE 2010 (Aspergillus sp.ZJUBE 2010) or ocean aspergillus niger ZJUBE 2010
(Aspergillus niger.ZJUBE 2010), China typical culture collection center is preserved on June 2nd, 2010, protected
Hiding address is：Wuhan University of Wuhan, China city, deposit number are CCTCC NO：M2010132.The ocean aspergillus niger of the present invention
ZJUBE 2010 can be used for solid state fermentation degraded charcoal source for the application in terms of monose.
The shortcomings that compared to prior art and deficiency, the invention has the advantages that：
(1) ocean aspergillus niger ZJUBE 2010 of the present invention can be used for fermenting and producing cellulase, and yield is high, and enzymatic activity is good；
(2) ocean aspergillus niger ZJUBE 2010 of the present invention is applied to Solid Fermentation of Uncooked Material method, and cellulose fermentation is degraded to
The high conversion rate of monose；
(3) during ocean aspergillus niger ZJUBE 2010 is used for Solid Fermentation of Uncooked Material method, without to raw material fermentation
Culture medium, equipment carry out the sterilization treatment of HTHP in itself, and ferment effect without any influence, is effectively reduced and set completely
Standby manufacturing cost is high, production cost, reduces energy consumption.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
(1) preparation of spore cultures：
Spore cultivation base：Using potato dextrose agar PDA (200 grams of potato, 20 grams of glucose, agar
20 grams of powder, distilled water adds to 1 liter), the culture medium is dispensed into test tube with 5 milliliters of amount, will after 121 DEG C sterilize 20 minutes
Test tube is paved into inclined-plane to cooled and solidified.
Ocean aspergillus niger ZJUBE 2010 is inoculated on Spore cultivation base with oese, cultivated 5 days under the conditions of 30 DEG C.
The spores of ocean aspergillus niger ZJUBE 2010 are rinsed with 50ml sterile distilled waters and are transferred to equipped with bead
In 150ml triangular flasks, shaken 1 hour on 180~200rpm shaking table, spore is uniformly dispersed.
(2) preparation of seed
Seed culture medium：Glucose 1%, yeast extract 0.25%, peptone 0.25%, 1% using carbon source and
Mass fraction be 0.25% organic nitrogen source, natural pH.
Inoculation：Spore suspension concentration is adjusted to 106Level, inoculum concentration 3%, i.e., connect in every 100 milliliters of seed culture mediums
Enter 3 milliliters of spore suspension.
Culture：Seed culture is using deep layer liquid sinking cultivation, and in 1000 milliliters of triangular flasks, (liquid amount is 500 millis
Rise) in carry out, shaking table hunting speed be 200 revs/min, cultivation temperature keep 30 DEG C, cultivate 48 hours.
(3) Solid Fermentation of Uncooked Material
The preparation of fermentation medium：Wheat bran 20g, maize straw 5g, rice straw powder 5g, glucose 5g, NH4Cl 0.6g,
KH2PO40.3g, FeSO4·7H2O 0.1g, NaCl 5g, deionized water 50ml；After culture medium is mixed evenly, load shallow
Disk keeps thickness in 3cm or so.
Inoculation：The seed liquor that kind of age is 48 hours is inoculated into fermentation medium, inoculum concentration 30ml,
After seed liquor and culture medium are mixed evenly, sealed using 8 layers of gauze.
Culture：Tray is put into constant incubator, fermented and cultured terminates after 5 days under the conditions of 37 DEG C.
Enzyme activity determination：By culture medium and running water with 1 after fermentation ends：10 ratio mixing, shaking table vibrate 1 hour, from
The heart removes culture medium and thalline spore, obtains the zymotic fluid of cellulase, measure filter paper enzyme activity is 14.32U/g.
Embodiment 2
(1) preparation of spore cultures：
Spore cultivation base：Using potato dextrose agar PDA (200 grams of potato, 20 grams of glucose, agar
20 grams of powder, distilled water adds to 1 liter), the culture medium is dispensed into test tube with 5 milliliters of amount, will after 121 DEG C sterilize 20 minutes
Test tube is paved into inclined-plane to cooled and solidified.
Ocean aspergillus niger ZJUBE 2010 is inoculated on Spore cultivation base with oese, cultivated 3 days under the conditions of 35 DEG C.
The spores of ocean aspergillus niger ZJUBE 2010 are rinsed with 50ml sterile distilled waters and are transferred to equipped with bead
In 150ml triangular flasks, shaken 1 hour on 180~200rpm shaking table, spore is uniformly dispersed.
(2) preparation of seed
Seed culture medium：Glucose 1%, yeast extract 0.25%, peptone 0.25%, 5% using carbon source and
Mass fraction be 3% organic nitrogen source, natural pH.
Inoculation：Spore suspension concentration is adjusted to 106Level, inoculum concentration 3%, i.e., connect in every 100 milliliters of seed culture mediums
Enter 3 milliliters of spore suspension.
Culture：Seed culture is using deep layer liquid sinking cultivation, and in 1000 milliliters of triangular flasks, (liquid amount is 500 millis
Rise) in carry out, shaking table hunting speed be 200 revs/min, cultivation temperature keep 30 DEG C, cultivate 48 hours.
(3) Solid Fermentation of Uncooked Material
The preparation of fermentation medium：Corn stalk powder 1g, wheat bran 5g, glucose 1g, NH4Cl 0.6g, KH2PO40.3g,
FeSO4·7H2O 0.1g, NaCl 10g, deionized water 60ml；After culture medium is mixed evenly, loads tray and keep thickness to exist
5cm or so.
Inoculation：The seed liquor that kind of age is 48 hours is inoculated into fermentation medium, inoculum concentration 30ml,
After seed liquor and culture medium are mixed evenly, sealed using 8 layers of gauze.
Culture：Tray is put into constant incubator, fermented and cultured terminates after 5 days under the conditions of 37 DEG C.
Enzyme activity determination：By culture medium and running water with 1 after fermentation ends：10 ratio mixing, shaking table vibrate 1 hour, from
The heart removes culture medium and thalline spore, obtains the zymotic fluid of cellulase, measure filter paper enzyme activity is 12.32U/g.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (9)
1. a kind of ocean aspergillus niger ZJUBE 2010 (Aspergillus sp.ZJUBE 2010), it is characterised in that the ocean is black
Aspergillus is preserved in China typical culture collection center on June 2nd, 2010, and deposit number is CCTCC NO：M2010132.
2. ocean 2010 applications in terms of fermenting and producing cellulase of aspergillus niger ZJUBE described in claim 1.
3. the answering in terms of charcoal source is degraded by fermentation process as monose of ocean aspergillus niger ZJUBE 2010 described in claim 1
With.
4. application as claimed in claim 3, it is characterised in that the fermentation process comprises the following steps：
(1) strains of ocean aspergillus niger ZJUBE 2010 are seeded on Spore cultivation base and cultivate spore；
(2) spore suspension in step (1) is inoculated into seed culture medium and cultivates seed liquor；
(3) seed liquor in step (2) is inoculated into raw material fermentation culture medium and carries out Solid Fermentation of Uncooked Material；Wherein, raw material solid-state
Fermentation is specially：It is that 20~50% strains are directly seeded in without HTHP by mass percent at a temperature of 35~40 DEG C
Cultivated 3~5 days in treated raw material fermentation culture medium.
5. application as claimed in claim 4, it is characterised in that in step (1), the Spore cultivation base potato glucose
Agar medium, Spore cultivation temperature are 30~35 DEG C, and incubation time is 3~5 days.
6. application as claimed in claim 5, it is characterised in that in step (2), the seed culture medium includes mass fraction
For 1~5% organic nitrogen source for being 0.25~3% using carbon source and mass fraction, the seed culture medium advances in inoculation
The processing of row conventional sterilant, the seed liquor cultivation temperature is 30 DEG C, incubation time is 2 days.
7. application as claimed in claim 6, it is characterised in that described to include slowly utilizing carbon source using carbon source；It is described to have
Machine nitrogen source includes peptone.
8. application as claimed in claim 6, it is characterised in that in step (3), Solid Fermentation of Uncooked Material is carried out in tray,
And the ulking thickness of the raw material fermentation culture medium is 3~5 centimetres；
Raw material fermentation culture medium includes described in 100ml：5~12g sodium chloride, the 1~6g carbon sources quickly utilized, 1 slowly utilized
~25g carbon sources, and 5~25g cellulase induction things；The material-water ratio of the raw material fermentation culture medium is 3：5.
9. application as claimed in claim 7, it is characterised in that the carbon source quickly utilized includes grape sugar, the slowly profit
Carbon source includes corn stalk powder, rice straw powder；The cellulase induction thing is wheat bran.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061537A (en) * | 2021-04-02 | 2021-07-02 | 深高蓝德环保科技集团股份有限公司 | Method for preparing aspergillus niger microbial inoculum |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649309A (en) * | 2009-08-27 | 2010-02-17 | 北京利德曼生化股份有限公司 | Method for producing cellulase through solid-state fermentation of raw materials |
| CN102010855A (en) * | 2010-07-30 | 2011-04-13 | 浙江大学 | Liquid fermentation method for producing salt-tolerant cellulase from sea aspergillus niger |
| CN102010856A (en) * | 2010-07-30 | 2011-04-13 | 浙江大学 | Solid fermentation method for producing salt-tolerant cellulase by using oceanic Aspergillus niger |
| CN105838697A (en) * | 2016-01-18 | 2016-08-10 | 浙江大学舟山海洋研究中心 | Fermentation method for producing salt-tolerant cellulase from marine aspergillus niger |
-
2017
- 2017-08-14 CN CN201710689555.5A patent/CN107488595A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649309A (en) * | 2009-08-27 | 2010-02-17 | 北京利德曼生化股份有限公司 | Method for producing cellulase through solid-state fermentation of raw materials |
| CN102010855A (en) * | 2010-07-30 | 2011-04-13 | 浙江大学 | Liquid fermentation method for producing salt-tolerant cellulase from sea aspergillus niger |
| CN102010856A (en) * | 2010-07-30 | 2011-04-13 | 浙江大学 | Solid fermentation method for producing salt-tolerant cellulase by using oceanic Aspergillus niger |
| CN105838697A (en) * | 2016-01-18 | 2016-08-10 | 浙江大学舟山海洋研究中心 | Fermentation method for producing salt-tolerant cellulase from marine aspergillus niger |
Non-Patent Citations (3)
| Title |
|---|
| DONG-SHENG XUE等: "Optimization of a Natural Medium for Cellulase by a Marine Aspergillus niger Using Response Surface Methodology", 《APPL BIOCHEM BIOTECHNOL》 * |
| 魏丹华: "海洋黑曲霉生料固态发酵产纤维素酶及酶的固定化研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 齐景伟等编著: "《反刍动物生物饲料开发与应用》", 31 March 2014, 内蒙古大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061537A (en) * | 2021-04-02 | 2021-07-02 | 深高蓝德环保科技集团股份有限公司 | Method for preparing aspergillus niger microbial inoculum |
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