CN109182244A - A method of improving Bacillus natto biomass under the high temperature conditions - Google Patents
A method of improving Bacillus natto biomass under the high temperature conditions Download PDFInfo
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- CN109182244A CN109182244A CN201810863786.8A CN201810863786A CN109182244A CN 109182244 A CN109182244 A CN 109182244A CN 201810863786 A CN201810863786 A CN 201810863786A CN 109182244 A CN109182244 A CN 109182244A
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- high temperature
- bacillus natto
- natto
- shaking table
- seed
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a kind of methods of raising Bacillus natto biomass under the high temperature conditions, include the following steps: bafillus natto cultivating 4-8h in seed culture medium, the concentration that seed culture medium volume 0.2-0.6% is added is the corn peptide aqueous solution of 0.1-0.2g/ml, 37 DEG C of culture 4-8h, obtain seed liquor on the shaking table of 150-250r/min；The seed liquor is inoculated in fermentation medium by the inoculum concentration of 1%-3%, 50-55 DEG C is cultivated 24-36h on the shaking table of 150-250r/min.The pretreated seed liquor of different content corn peptide is passed through in access in the fermentation medium provided by the invention, and Bacillus natto has better growth performance under high temperature (50-55 DEG C) stress conditions, than control group (pre-processing without corn peptide).Method of the invention, it is simple and easy, it can effectively and quickly improve biomass of the Bacillus natto under 50 DEG C of -55 DEG C of high temperature.
Description
Technical field
The present invention relates to a kind of methods of raising Bacillus natto biomass under the high temperature conditions, belong to biotechnology neck
Domain.
Background technique
Natto originates from ancient Chinese, bean product is made by fermenting bacillus natto by soya bean, has stickiness, and smell is more smelly,
Taste is slightly sweet.Natto is increasingly becoming the favorite nutraceutical of the people of other countries and health food, with very high nutriture value
Value, contained protein, vitamin B, vitamin E, calcium and iron are all higher than common soybean.Natto also has many physiology
Health-care effect, such as antitumaous effect, antioxidation, blood pressure lowering, anti-curing osteoporosis, hemostasis effect, antibacterial action with
And the effects of thrombus dissolving.
Bacillus natto, nickname bafillus natto (Bacillus natto) belong to bacillus subtilis Bacillus natto subspecies.Industry
Production natto needs Bacillus natto to ferment at 25~40 DEG C to soya bean, and 25~40 DEG C are also the suitable growth temperature of other miscellaneous bacterias
Degree is easy to by living contaminants.In order not to be made the obtained natto deliciousness of fermentation by other living contaminants more preferably, and nutriture value
Value is higher, needs to improve industrial fermentation temperature up to 45~50 DEG C.Data shows that the very strong heat resistance of Bacillus natto reaches as high as 50
DEG C, but since hot environment seriously inhibits the growth performance and metabolic activity of thallus, the physiological activity of cell is affected, so that
The biomass of Bacillus natto reduces.Therefore, the biomass of raising Bacillus natto at high temperature has industrial fermentation production natto very heavy
The meaning wanted.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of raising Bacillus natto biomass under the high temperature conditions
Method.
Technical solution of the present invention is summarized as follows:
A method of Bacillus natto biomass under the high temperature conditions is improved, is included the following steps:
(1) by bafillus natto (Bacillus natto) in seed culture medium, on the shaking table of 150-250r/min
The corn peptide aqueous solution of the 0.1-0.2g/mL of the 0.2%-0.6% of seed culture fluid volume is added in 37 DEG C of culture 4-8h,
37 DEG C of culture 4-8h, obtain seed liquor on the shaking table of 150-250r/min；
(2) seed liquor is inoculated in fermentation medium by the inoculum concentration of 1%-3%, 50-55 DEG C in 150-250r/
24-36h is cultivated on the shaking table of min.
Beneficial effects of the present invention: access in the fermentation medium provided by the invention is located in advance by different content corn peptide
The seed liquor of reason, Bacillus natto have more under high temperature (50-55 DEG C) stress conditions than control group (pre-processing without corn peptide)
Good growth performance.Method of the invention, it is simple and easy, Bacillus natto can effectively and be quickly improved under 50 DEG C of -55 DEG C of high temperature
Biomass.
Specific embodiment
Bafillus natto (Bacillus natto) belongs to bacillus subtilis Pseudomonas, deposit number BNCC185324,
In in June, 2017 structure receive biological (www.bnbio.com) from north, address: the Chaoyang District, Beijing City garden Shuan Hui No. 5 building of first.
Corn seed: the day tower 619 bought from Tianjin ZTDD-Geneseed Technology Co., Ltd..
Above-mentioned raw materials source is in order to enable those skilled in the art to more fully understand the present invention, but not to this hair
It is bright to limit.
The preparation of corn peptide aqueous solution: it weighs a certain amount of full niblet and is ground into corn flour, be packed into the triangular flask of 1L
In, it is made into the corn flour suspension that mass fraction is 5% with phosphate buffer (concentration 0.1moi/L, PH=6.8), stirring is equal
It is even, it is put into 55 DEG C in water bath with thermostatic control shaker, pH value is adjusted to 8.5；By enzyme and corn flour suspension quality than for 4% it is enzyme
Alkali protease is added in amount, hydrolyzes 1.5h；Maintain pH value 8.5 with the NaOH aqueous solution of 4mol/L.Triangular flask is put 90 DEG C of people
In water-bath, 15min is kept to inactivate enzyme；Triangular flask is taken out, cools down room temperature rapidly, 4000r/min centrifugation 10min takes supernatant
Filtrate is freeze-dried to obtain corn peptide powder by liquid through 0.22 μm of membrane filtration.
Corn peptide powder is taken to be dissolved in the corn peptide aqueous solution for being made into 0.1-0.2g/ml in sterile water.
Seed culture medium: peptone 1wt%, beef extract 0.3wt%, NaCl 0.5wt%, glucose 0.5wt%, yeast
Cream 0.5wt%, PH 7.0.
Fermentation medium: glucose 2wt%, peptone 0.5wt%, NaCl 0.5wt%, K2HPO40.4wt%,
KH2PO40.2wt%, pH 7.0.
Embodiment 1 (G1)
A method of Bacillus natto biomass under the high temperature conditions is improved, is included the following steps:
(1) the bafillus natto glycerin storage liquid 10ul of -20 DEG C of preservations is taken to be inoculated in 50ml seed culture medium,
37 DEG C of culture 6h on the shaking table of 200r/min；Aseptically, the concentration that seed culture medium volume 0.6% is added is 0.15g/
The corn peptide aqueous solution of ml, 37 DEG C of culture 6h, obtain seed liquor on the shaking table of 200r/min；
(2) seed liquor is inoculated in fermentation medium by 2% inoculum concentration, 53 DEG C on the shaking table of 200r/min
Cultivate 30h.
Embodiment 1 is 3 parallel.
Reference examples 1 (CK1)
(1) the bafillus natto glycerin storage liquid 10ul of -20 DEG C of preservations is taken to be inoculated in 50ml seed culture medium,
37 DEG C of culture 6h on the shaking table of 200r/min；Aseptically, the sterile water of seed culture medium volume 0.6% is added,
37 DEG C of culture 6h, obtain seed liquor on the shaking table of 200r/min；
(2) seed liquor is inoculated in fermentation medium by 2% inoculum concentration, 53 DEG C on the shaking table of 200r/min
Cultivate 30h.
Reference examples 1 are 3 parallel.
Thalline were collected by centrifugation for the bacteria suspension for taking after fermentation, after sterile water washing 3 times, 10ml sterile water is added, shakes
It is even, CK1, the OD600 value (such as table 1) of G1 are measured with ultraviolet-uisible spectrophotometer
It is 1.34 that experiment, which shows that control group 1 measures OD600 value,；
It is 1.51 that example 1 group, which measures OD600, improves 12.69% than control group.
Table 1
Embodiment 2 (G2)
A method of Bacillus natto biomass under the high temperature conditions is improved, is included the following steps:
(1) the bafillus natto glycerin storage liquid 10ul of -20 DEG C of preservations is taken to be inoculated in 50ml seed culture medium,
37 DEG C of culture 4h on the shaking table of 150r/min；Aseptically, the concentration that seed culture medium volume 0.2% is added is 0.2g/
The corn peptide aqueous solution of ml, 37 DEG C of culture 4h, obtain seed liquor on the shaking table of 150r/min；
(2) seed liquor is inoculated in fermentation medium by 1% inoculum concentration, 50 DEG C on the shaking table of 150r/min
Cultivate 36h.
Embodiment 2 is 3 parallel.
Reference examples 2 (CK2)
(1) the bafillus natto glycerin storage liquid 10ul of -20 DEG C of preservations is taken to be inoculated in 50ml seed culture medium,
37 DEG C of culture 4h on the shaking table of 150r/min；Aseptically, the sterile water of seed culture medium volume 0.2% is added,
37 DEG C of culture 4h, obtain seed liquor on the shaking table of 150r/min；
(2) seed liquor is inoculated in fermentation medium by 1% inoculum concentration, 50 DEG C on the shaking table of 150r/min
Cultivate 36h.
Reference examples 2 are 3 parallel.
Thalline were collected by centrifugation for the bacteria suspension for taking after fermentation, after sterile water washing 3 times, 10ml sterile water is added, shakes
It is even, CK2, the OD600 value (such as table 2) of G2 are measured with ultraviolet-uisible spectrophotometer
It is 1.43 that experiment, which shows that control group 2 measures OD600 value,；
It is 1.59 that 2 groups of embodiment, which measure OD600, improves 11.19% than control group.
Table 2
Embodiment 3 (G3)
A method of Bacillus natto biomass under the high temperature conditions is improved, is included the following steps:
(1) the bafillus natto glycerin storage liquid 10ul of -20 DEG C of preservations is taken to be inoculated in 50ml seed culture medium,
37 DEG C of culture 8h on the shaking table of 250r/min；Aseptically, the concentration that seed culture medium volume 0.4% is added is 0.1g/
The corn peptide aqueous solution of ml, 37 DEG C of culture 8h, obtain seed liquor on the shaking table of 250r/min；
(2) seed liquor is inoculated in fermentation medium by 3% inoculum concentration, 55 DEG C on the shaking table of 250r/min
Culture is for 24 hours.
Embodiment 3 is 3 parallel.
Reference examples 3 (CK3)
(1) the bafillus natto glycerin storage liquid 10ul of -20 DEG C of preservations is taken to be inoculated in 50ml seed culture medium,
37 DEG C of culture 8h on the shaking table of 250r/min；Aseptically, the sterile water of seed culture medium volume 0.4% is added,
37 DEG C of culture 8h, obtain seed liquor on the shaking table of 250r/min；
(2) seed liquor is inoculated in fermentation medium by 3% inoculum concentration, 55 DEG C on the shaking table of 250r/min
Culture is for 24 hours.
Reference examples 3 are 3 parallel.
Thalline were collected by centrifugation for the bacteria suspension for taking after fermentation, after sterile water washing 3 times, 10ml sterile water is added, shakes
It is even, CK3, the OD600 value (such as table 3) of G3 are measured with ultraviolet-uisible spectrophotometer
It is 1.21 that experiment, which shows that control group 3 measures OD600 value,；
It is 1.42 that 3 groups of embodiment, which measure OD600, improves 17.36% than control group.
Table 3
Claims (1)
1. a kind of method for improving Bacillus natto biomass under the high temperature conditions, it is characterized in that including the following steps:
(1) bafillus natto is cultivated into seed culture medium 4-8h, the concentration of seed culture medium volume 0.2-0.6% is added
For the corn peptide aqueous solution of 0.1-0.2g/ml, 37 DEG C of culture 4-8h, obtain seed liquor on the shaking table of 150-250r/min；
(2) seed liquor is inoculated in fermentation medium by the inoculum concentration of 1%-3%, 50-55 DEG C in 150-250r/min
Shaking table on cultivate 24-36h.
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| CN201810863786.8A CN109182244B (en) | 2018-08-01 | 2018-08-01 | Method for improving biomass of bacillus natto under high-temperature condition |
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| CN201810863786.8A CN109182244B (en) | 2018-08-01 | 2018-08-01 | Method for improving biomass of bacillus natto under high-temperature condition |
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| CN109182244B CN109182244B (en) | 2021-11-02 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001066773A2 (en) * | 2000-03-09 | 2001-09-13 | E.I. Dupont De Nemours And Company | Nucleic acids that code for oxidosqualene cyclases |
| CN105838636A (en) * | 2015-12-02 | 2016-08-10 | 四川大学 | Method for improving biomass of lactic acid bacteria under high salt condition |
| CN107574213A (en) * | 2016-11-21 | 2018-01-12 | 广西大学 | A kind of method that corn peptide is prepared using fermenting bacillus natto |
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2018
- 2018-08-01 CN CN201810863786.8A patent/CN109182244B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001066773A2 (en) * | 2000-03-09 | 2001-09-13 | E.I. Dupont De Nemours And Company | Nucleic acids that code for oxidosqualene cyclases |
| CN105838636A (en) * | 2015-12-02 | 2016-08-10 | 四川大学 | Method for improving biomass of lactic acid bacteria under high salt condition |
| CN107574213A (en) * | 2016-11-21 | 2018-01-12 | 广西大学 | A kind of method that corn peptide is prepared using fermenting bacillus natto |
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