CN104152429A - Screening and liquid fermentation method of high-yield bacillus subtilis strain of nattokinase - Google Patents

Screening and liquid fermentation method of high-yield bacillus subtilis strain of nattokinase Download PDF

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CN104152429A
CN104152429A CN201310178561.6A CN201310178561A CN104152429A CN 104152429 A CN104152429 A CN 104152429A CN 201310178561 A CN201310178561 A CN 201310178561A CN 104152429 A CN104152429 A CN 104152429A
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nattokinase
fermentation
screening
medium
fermention medium
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戴东升
王星星
杨仁佳
安俊
郑慧
周鹏
姚振芳
王斐
陈婧
禹玉洪
任武贤
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Yabao Pharmaceutical Group Corp
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Shanxi Yabao Pharmaceutical Group Corp
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

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Abstract

The invention relates to a screening and liquid fermentation method of a high-yield bacillus subtilis strain of nattokinase in the technical field of biology. By using the screening and liquid fermentation method, a high-yield strain YBNK-1 of nattokinase is screened and is proven to be bacillus subtilis after being detected by Guangdong detection center of microbiology, and the detection report number is Guangdong microbiological detection 2012ZD0175. By virtue of a liquid fermentation formula and fermentation process optimization, the average activity of the obtained nattokinase is more than 2000 urokinases in every milliliter. The screening and liquid fermentation method can be used for laying a basis for industrial production of nattokinase.

Description

The screening of Nattokinase high yield bacillus subtilis bacterial strain and the method for liquid state fermentation
Technical field
The present invention relates to the method for the screening of a kind of Nattokinase high yield bacillus subtilis bacterial strain and liquid state fermentation in biological technical field.
Background technology
30 years from now on, China's elderly population will get more and more, and the elderly's healthy problem is more and more subject to people's concern and attention.Cardiovascular and cerebrovascular embolism class diseases is one of serious and the most common disease of harm senior health and fitness.At present, thrombosis becomes one of disease that mortality ratio is the highest gradually, and thrombus is a kind of important means of this class disease for the treatment of.Therefore, the research of thrombolytic drug is widely paid attention to.Current as the widely application clinically of first-generation thrombolytic drug streptokinase (SK), urokinase (UK), but they are fibrin proenzyme in human activin inner blood, thereby fibrin degradation, easily causes general activation of fibrinolysis and with bleeding tendency.SK also has antigenicity and has anaphylaxis simultaneously.Although and scleroproein is had to very high selectivity as the t-PA of s-generation fibrinolytic medicine, but in order to obtain good thrombolytic effect, apply heavy dose (100mg) clinically thus part lost fibrinous selectivity, therefore still can cause the hemorrhage side effect of Denging, and there is the shortcomings such as fibrin-specific is poor, Half-life in vivo is short, production cost is high, the heavy dose of continuous use of need.Given this, development of new thrombolytic drug has become one of important study hotspot.At present, Nattokinase (NK), as coming from food, highly effective and safe and can directly being absorbed, be played the plasmin of solution fibrin effect by digestive tube, has security good, long action time in body, the advantages such as effective drug duration length.Becoming the focus of current Thrombolytic Drugs research.
The main reason of nowadays influence Nattokinase industrialization is, production cost is high, and the production cycle is long.Therefore filter out the bacterial strain of a strain high-yield nattokinase, and set up a kind of suitable zymotechnique, significant to the industrialization of Nattokinase.
Summary of the invention
The invention provides a kind of fermention medium for Nattokinase liquid state fermentation, it comprises: soy peptone, Zulkovsky starch, Na 2hPO 412H 2o, KH 2pO 4, CaCl 2, MgSO 47H 2o.
According to the present invention, described fermention medium comprises: soy peptone 1.0-2.5%, Zulkovsky starch 2.0-4%, Na 2hPO 412H 2o0.4-0.5%, KH 2pO 40.15-0.25%, CaCl 20.01-0.03%, MgSO 47H 2o0.05-007%.
The present invention also provides a kind of method of Nattokinase liquid state fermentation, it is characterized in that, described method comprises that following step is poly-:
(1) go bail for and be stored in the bacterial classification (preferably semi-ring) on solid medium, be seeded in (pH is preferably 6.8-7.2) in seed culture medium,
(2) constant temperature culture (preferably 10-14 hour), is inoculated into afterwards in described fermention medium and ferments.
According to the present invention, step (1) is carried out on the Bechtop of having sterilized.
According to the present invention, the inoculum size being wherein inoculated in fermention medium is 2-10%, preferably 2-4%.
According to the present invention, be seeded in the operational condition in seed culture medium described in step (1): preferred temperature is 34-37 ℃, preferred rotating speed is 160-300rpm.
According to the present invention, the fermentation in step (2) is liquid state fermentation, and preferred above-mentioned liquid state fermentation can be fermented in shaking flask, also can be in fermentation cylinder for fermentation, and the fermentor tank of 5L for example.
According to the present invention, in shaking flask, ferment, medium pH is preferably 6.9-7.1, the preferred 33-18 ℃ of temperature, 160rpm constant temperature culture 48-54 hour.
According to the present invention, in fermentation cylinder for fermentation, take the variation of the dissolved oxygen (DO) along with fermentor tank and improve the rotating speed of fermentor tank stirrer, along with the prolongation of fermentation time, tune up stirring velocity, preferably stirring velocity is brought up to 700rpm from 300rpm, more preferably from 320rpm, brings up to 700rpm.
According to the present invention, in fermentation cylinder for fermentation, fermentor tank liquid amount is 50-70%, inoculum size is 2-4%, and air flow is 0.5-0.8vvm, and cultivation pH is 6.9-7.1 (with ammoniacal liquor and second acid for adjusting pH), temperature is 35-37 ℃, and whole fermentation period is 28-32 hour.
According to the present invention, described seed culture medium is nutrient broth medium, comprises peptone 1.0%, extractum carnis 0.5%, and NaCl0.5%, pH value 7.0, or add therein some damping fluid compositions.
The present invention also provides a kind of gemma Bacillus subtilus strain YBNK-1 of high-yield nattokinase, through Guangdong Province microbiological analysis inspection center, is accredited as subtilis, and examining report is numbered the micro-inspection in Guangdong 2012ZD0175.
Above-mentioned bacterial strains is through China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date: on 04 12nd, 2013, preserving number is CGMCC NO.7467, be defined as subtilis, Bacillus subtilis.
The present invention also provides a kind of screening method of described superior strain: by purifying and the ultraviolet mutagenesis of ruling repeatedly, be purified into 8 strain Bacillus nattos from commercially available natto, select through the strongest bacterial strain YBNK-1 of the dull and stereotyped detection of active of fiber.
The advantage of this technical scheme
The technical program natto bacterial classification used is after fermentor tank liquid state fermentation, and gained natto kinase activity reaches every milliliter of 1800-2100 urokinase unit.Through multiple batches of fermentation test, the technical program has simple to operate, and raw material is easy to get, and fermentation period is short, and enzyme is lived high, meets the requirement that industrialization is produced.
Accompanying drawing explanation
Fig. 1 is that different time points Nattokinase is two kinds of carbon source SDS-PAGE detected results.
Fig. 2 is that Nattokinase solusphere method detects bacterial classification active detected result (DF: Zulkovsky starch in two kinds of carbon source substratum; PT: glucose).
Fig. 3 is for expressing in the fermention medium of 4% glucose and 4% Zulkovsky starch for (9/10/11) Nattokinase kind of an age
SDS-PAGE detected result (DF: Zulkovsky starch; PTT: glucose).
Fig. 4 is for being the active detected result (DF: Zulkovsky starch of (9/10/11) Nattokinase solusphere method in the fermention medium of 4% glucose kind of an age; PTT: glucose).
Fig. 5 is for being the active detected result (DF: Zulkovsky starch of (9/10/11) Nattokinase solusphere method in the fermention medium of 4% Zulkovsky starch kind of an age; PTT: glucose).
Embodiment
The formula of slant medium: peptone 1.0%, extractum carnis 0.5%, NaCl0.5%, agar 2%, pH value 7.0.
Fermention medium: peptone 1.0%, Zulkovsky starch 2.0%, Na 2hPO 412H 2o0.4%, KH 2pO40.15%, CaCl 20.01%, MgSO 47H 2o0.05%.
Seed culture medium: peptone 1.0%, extractum carnis 1.0%, NaCl1.0%, sterilizing 20min at 7.0,121 ℃ of pH values.
Embodiment 1: the screening of superior strain
1) in the Bechtop of having sterilized, get high purity natto cellulose capsule, be dissolved in sterilized 10ml seed culture medium, after it fully dissolves, get 200 μ l and be inoculated into 30ml/250ml (in triangular flask);
2) 160rpm, 35 ℃ of constant-temperature shaking culture;
3) cultivate after 10h, in sterilized Bechtop, get 100 μ l bacterium liquid, be coated with dull and stereotyped;
Be inverted flat board, 35 ℃ of constant temperature culture;
4) 35 ℃ of constant temperature culture 24h, single bacterium colony that picking grows fine, is inoculated in sterilizing 30ml/250ml (triangular flask) seed culture medium;
5) with 3% (V/V) inoculum size, be inoculated in sterilized 30ml/250ml (triangular flask) fermention medium;
6) 35 ℃, 160rpm constant-temperature shaking culture, at different point in time sampling, detects Nattokinase expression.
Embodiment 2: the situation that Nattokinase superior strain is expressed in different carbon sources
1) in the Bechtop of having sterilized, get the Bacillus natto superior strain of above-mentioned screening, be inoculated into sterilized 30ml/250ml (triangular flask) seed culture and concentrate;
2) 35 ℃, 160rpm, cultivates after 8h, and 3% inoculum size of take is inoculated into respectively sterilized carbon source in the fermention medium of glucose, Zulkovsky starch;
3) 35 ℃, 160rpm constant-temperature shaking culture;
4) sampling at set intervals, stand-by.
Result is as follows:
Nattokinase take in the fermention medium that starch is carbon source as seen from Figure 1, Figure 2, and typical curve is y=0.2724x+1.4852 (R2=0.983), and maximum activity is 693U/ml, and carbon source is now Zulkovsky starch.
Embodiment 3: Nattokinase superior strain bacterial classification in age not of the same race is expression in 4% Zulkovsky starch basis fermention medium
1) in the Bechtop of having sterilized, get the Bacillus natto superior strain of above-mentioned screening, be inoculated in sterilized 30ml/250ml (triangular flask) seed culture medium;
2) 35 ℃, 160rpm, cultivates 9h, 10h, and after 11h, 3% inoculum size of take is inoculated into respectively sterilized carbon source in the fermention medium of 4% Zulkovsky starch and 4% glucose;
3) 35 ℃, 160rpm constant-temperature shaking culture;
4) sampling at set intervals, stand-by.
From Fig. 3, Fig. 4, Fig. 5, kind age is 10h, and after expression, 48h activity can approach 2000U/ml, and further illustrates and using Zulkovsky starch and than usining glucose, be more conducive to the expression of Nattokinase as carbon source as carbon source.
Embodiment 4: Nattokinase 500ml shake flask test
Go bail for and be stored in the YBNK-1 bacterial classification on slant medium, be inoculated in 30 milliliters of seed culture mediums.37 ℃ of constant temperature 160-200rpm cultivate 12 hours, and 160rpm constant temperature culture 48-54 hour is housed in the 500ml triangular flask of 60ml fermention medium with 3% inoculum size (v/v) access.Average enzyme work reaches every milliliter of 2000 urokinase units.
Embodiment 5: the expression of Nattokinase in 5L fermentor tank
Go bail for and be stored in the YBNK-1 bacterial classification on slant medium, be inoculated in 30 milliliters of seed culture mediums.37 ℃ of constant temperature 160-200rpm cultivate 12 hours.In 5L fermentor tank, pack 3L fermention medium into, 121 ℃ of sterilizings 20 minutes, mixing speed is 300rpm, and air flow is 0.5vvm, when fermentation jar temperature is down to 37 ℃, with ammoniacal liquor, adjusting pH is 7.0, then with 3% (v/v) inoculum size, bacterial classification is inoculated in fermention medium, fermentation period is 28-32 hour, wherein stirring velocity increases along with the prolongation of fermentation time, from initial stirring velocity, is that 300rpm is increased to 700rpm.The average activity of gained Nattokinase is every milliliter of 2000 urokinase units.

Claims (9)

1. for a fermention medium for Nattokinase liquid state fermentation, it comprises: soy peptone, Zulkovsky starch, Na 2hPO 412H 2o, KH 2pO 4, CaCl 2, MgSO 47H 2o.
2. fermention medium according to claim 1, it comprises: soy peptone 1.0-2.5%, Zulkovsky starch 2.0-4%, Na 2hPO 412H 2o0.4-0.5%, KH 2pO 40.15-0.25%, CaCl 20.01-0.03%, MgSO 47H 2o0.05-0.07%.
3. the method for a Nattokinase liquid state fermentation, comprise that following step is poly-: go bail for and be stored in the bacterial classification on solid medium, be seeded in (pH is preferably 6.8-7.2) in seed culture medium, constant temperature culture (preferably 10-14 hour), is inoculated into afterwards as fermented in the fermention medium in claim 1 or 2.
4. method according to claim 3, wherein inoculum size is 2-10%, preferably 2-4%.
5. method according to claim 3, wherein said bacterial classification Shi Jing China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, preserving number is 7467.
6. method according to claim 3, wherein said fermentation is to carry out in fermentor tank.
7. method according to claim 6, wherein stirring velocity increases along with the prolongation of fermentation time, and preferably stirring velocity is brought up to 700rpm from 300rpm, more preferably from 320rpm, brings up to 700rpm.
8. according to the method described in claim 6 or 7, fermentor tank liquid amount is 50-70%, and inoculum size is 2-4%, air flow is 0.5-0.8vvm, and cultivation pH is 6.9-7.1 (with ammoniacal liquor and second acid for adjusting pH), and temperature is 35-37 ℃,, whole fermentation period is 28-32 hour.
9. a subtilis, this bacterial strain is through China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and preserving number is 7467.
CN201310178561.6A 2013-05-15 2013-05-15 Screening and liquid fermentation method of high-yield bacillus subtilis strain of nattokinase Pending CN104152429A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087447A (en) * 2015-09-16 2015-11-25 河南省科学院生物研究所有限责任公司 Microwave-resisting bacillus subtilis and application thereof in preparing nattokinase
CN108142832A (en) * 2017-11-08 2018-06-12 珠海市微豆生物技术有限公司 A kind of preparation method for improving black Nattokinase content and activity
CN109706135A (en) * 2019-03-06 2019-05-03 武汉轻工大学 A kind of Nattokinase liquid state fermentation culture medium and the fermentation process for producing Nattokinase

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JPH06261744A (en) * 1993-03-17 1994-09-20 Nippon Opereetaa Kk Bacillus natto strain and natto produced using the same
CN101979531A (en) * 2010-09-26 2011-02-23 湖北国力生物技术开发有限公司 Liquid fermentation method for producing natto kinase in high yield
CN102618522A (en) * 2011-01-28 2012-08-01 湖北国力生物技术开发有限公司 Method for industrially producing nattokinase by using chickpea

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JPH06261744A (en) * 1993-03-17 1994-09-20 Nippon Opereetaa Kk Bacillus natto strain and natto produced using the same
CN101979531A (en) * 2010-09-26 2011-02-23 湖北国力生物技术开发有限公司 Liquid fermentation method for producing natto kinase in high yield
CN102618522A (en) * 2011-01-28 2012-08-01 湖北国力生物技术开发有限公司 Method for industrially producing nattokinase by using chickpea

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087447A (en) * 2015-09-16 2015-11-25 河南省科学院生物研究所有限责任公司 Microwave-resisting bacillus subtilis and application thereof in preparing nattokinase
CN105087447B (en) * 2015-09-16 2018-08-21 河南省科学院生物研究所有限责任公司 One plant of resistance to microwave bacillus subtilis and its application in Nattokinase preparation
CN108142832A (en) * 2017-11-08 2018-06-12 珠海市微豆生物技术有限公司 A kind of preparation method for improving black Nattokinase content and activity
CN109706135A (en) * 2019-03-06 2019-05-03 武汉轻工大学 A kind of Nattokinase liquid state fermentation culture medium and the fermentation process for producing Nattokinase

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