CN109722408A - Promote the method for producing bacillus subtilis gemma - Google Patents
Promote the method for producing bacillus subtilis gemma Download PDFInfo
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- CN109722408A CN109722408A CN201711033059.0A CN201711033059A CN109722408A CN 109722408 A CN109722408 A CN 109722408A CN 201711033059 A CN201711033059 A CN 201711033059A CN 109722408 A CN109722408 A CN 109722408A
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Abstract
The present invention relates to field of fermentation engineering, the method for promoting producing bacillus subtilis gemma is disclosed, this method includes that bacillus subtilis is seeded in fermentation medium to cultivate, the conditions for the training include: 28-32 DEG C, 95-120rpm；The inoculum concentration of bacillus subtilis is 2 × 107‑8×107CFU/ml fermentation medium；The fermentation medium contains the yeast extract of 0.2-0.5 weight %, the molasses of 0.5-1 weight % and the glucose of 0.3-0.8 weight %, and pH 7-7.5.The present invention can shorten the budding time of the gemma during fermentation of bacillus subtilis and improve Number of spores and bud ratio, to advantageously reduce production cost.
Description
Technical field
The present invention relates to field of fermentation engineering, and in particular to a method of promote producing bacillus subtilis gemma.
Background technique
Bacillus subtilis is a kind of aerobic production bacillus being widely present in nature.It has very strong fat
Enzyme, protease, amylase isoreactivity, metabolism is vigorous, free from environmental pollution to person poultry harmless, wide in livestock feed industrial application
It is general.The dry bacterium of withered grass gemma is one of common strain of feeding micro-ecological preparation, and bacillus exists in the maturity period with spore state,
Has many advantages, such as natural acidproof and bile tolerance.Bacillus subtilis, into target animal enteron aisle, is taken by force oxygen through biology and made by field planting
With, pathogenic microorganisms of flying up and down, and a variety of digestive ferments and nutriment are generated, generate beneficial metabolic product.So bacillus subtilis
Bacterium can not only improve raw material flavor, it is often more important that its adjustable alimentary canal health enhances the immune function of animal body, finally
The generation for preventing disease achievees the purpose that promote target animal growth and improve food conversion ratio, thus bacillus subtilis quilt
It is developed into feeding micro-ecological preparation more and more.
One of an important factor for fermentative medium formula is influence microbial fermentation.It is generally believed that gemma is bacteriotrophy
Body must feed exhaustion when, especially in carbon source, nitrogen source or phosphatic insufficient situation, the water content that is formed in the cell
It is low, the strong hypopus of resistance.Studies have shown that gemma caused by bacillus subtilis have heat-resisting, drought-enduring, uvioresistant and
A variety of resistance such as organic solvent.Due to the extremely strong resistance of gemma, during interchangeable manufacturing production and high temperature drying,
It can guarantee the activity of probiotics, improve product quality.
Currently, being primarily present the problems such as sporulation time is long, and bud ratio is low in industrial processes.Therefore, promote withered
It is more beneficial research direction that careless bacillus, which produces gemma,.
Summary of the invention
The purpose of the invention is to overcome the problems, such as that sporulation time length of the existing technology is low with bud ratio, mention
For a kind of method for promoting producing bacillus subtilis gemma.
To achieve the goals above, one aspect of the present invention provides a kind of method for promoting producing bacillus subtilis gemma,
This method includes that bacillus subtilis is seeded in fermentation medium to cultivate, the conditions for the training include: 28-32
DEG C, 95-120rpm；The inoculum concentration of bacillus subtilis is 2 × 107-8×107CFU/ml fermentation medium；The fermented and cultured
Base contains the yeast extract of 0.2-0.5 weight %, the molasses of 0.5-1 weight % and the glucose of 0.3-0.8 weight %, and pH
7-7.5。
Through the above technical solutions, the present invention can shorten the budding time of the gemma during fermentation of bacillus subtilis and
Number of spores and bud ratio are improved, to advantageously reduce production cost.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
In the present invention, in the absence of explanation to the contrary, before the pH of culture medium refers both to initial pH value, namely sterilizing
PH value, other ingredients, dosage when being preparation (before sterilizing)；" CFU " used is Colony Forming Unit, can be passed through
The method of plate culture count measures.
The method provided by the invention for promoting producing bacillus subtilis gemma includes that bacillus subtilis is seeded to fermentation
It is cultivated in culture medium, the conditions for the training include: 28-32 DEG C, and 95-120rpm (it preferably includes: 30 ± 0.5 DEG C, 100
±5rpm)；The inoculum concentration of bacillus subtilis is 2 × 107-8×107CFU/ml fermentation medium；The fermentation medium contains
The yeast extract of 0.2-0.5 weight %, the molasses of 0.5-1 weight % and the glucose of 0.3-0.8 weight %, and pH 7-7.5.
According to the preferred embodiment of the present invention, the fermentation medium contains the yeast extract of 0.3-0.35 weight %,
The molasses of 0.7-0.9 weight % and the glucose of 0.5-0.7 weight %, and pH 7.2-7.4.In preferred embodiment,
The fermentation medium contains the yeast extract of 0.32 ± 0.01 weight %, the molasses and 0.6 ± 0.01 of 0.8 ± 0.01 weight %
The glucose of weight %, and pH 7.2-7.4.
Method of the invention can shorten the gemma budding time of bacillus subtilis, and therefore, the time of the culture can
To be no longer than 15 hours, preferably 8-15 hours, more preferably 9-10 hours.
According to the present invention, in order to make bacillus subtilis adapt to culture environment, before the method can also be included in culture
Bacillus subtilis is seeded in seed culture medium and is activated.
A preferred embodiment of the invention, the seed culture medium contain the yeast powder of 0.4-0.8 weight %,
The peptone of 0.5-1.2 weight %, the glucose of 0.4-0.6 weight % and the sodium chloride of 0.8-1.2 weight %, and pH 7.2-
7.4.In preferred embodiment, the seed culture medium contains the yeast powder of 0.5 ± 0.01 weight %, 1 ± 0.01 weight
Measure the peptone of %, the sodium chloride of the glucose of 0.5 ± 0.01 weight % and 1 ± 0.01 weight %, and pH 7.2-7.4.
A preferred embodiment of the invention, the condition of the activation include: 28-32 DEG C, 110-130rpm, eventually
Point OD600Value 0.1-0.2 (more preferably includes 30 ± 0.5 DEG C, 120 ± 2rpm, terminal OD600Value 0.1 ± 0.05).In addition, activation
When, the inoculum concentration of bacillus subtilis is preferably 1 × 106-4×106CFU/ml seed culture medium.
A kind of particularly preferred embodiment according to the present invention, which comprises
(1) bacillus subtilis is seeded in seed culture medium and is activated, the condition of the activation includes 30 ± 0.5
DEG C, 120 ± 2rpm, terminal OD600Value 0.1 ± 0.05；The inoculum concentration of bacillus subtilis is 1 × 106-4×106CFU/ml seed
Culture medium；The seed culture medium contains the yeast powder of 0.5 ± 0.01 weight %, the peptone of 1 ± 0.01 weight %, 0.5 ±
The sodium chloride of the glucose of 0.01 weight % and 1 ± 0.01 weight %, and pH 7.2-7.4；
(2) bacillus subtilis after activation is seeded in fermentation medium again and is cultivated, the condition of the culture
It include: 30 ± 0.5 DEG C, 100 ± 5rpm；The inoculum concentration of bacillus subtilis is 2 × 107-8×107CFU/ml fermentation medium；
The fermentation medium contains the yeast extract of 0.32 ± 0.01 weight %, the molasses and 0.6 ± 0.01 of 0.8 ± 0.01 weight %
The glucose of weight %, and pH 7.2-7.4.
In the preferred embodiment, the time of the culture can further be foreshortened to no longer than 9.5 hours (such as 9
Or 9.5 hours).
Culture medium used in the present invention typically contains water, and content is selected as needed.Culture medium of the invention contains
It is formed by mentioned component or by the above ingredient and water.Wherein, yeast extract, yeast powder, peptone and molasses are conventional use
Carry out the substance of the culture medium of preparing microorganism, can be commercially available.
In the present invention, the bacillus subtilis (Bacillus subtilis) can be commonly used in the art various withered
Careless bacillus can be commercially available.
The present invention will be described in detail by way of examples below.
In following embodiment, yeast powder, peptone and yeast extract are purchased from Angel Yeast company；Molasses and corn pulp are
The commercially available product of COFCO Biochemical (Anhui) Co., Ltd.；Bacillus subtilis is purchased from ATCC,6633TM。
Embodiment 1-2
(1) bacillus subtilis being stored in ultra low temperature freezer is shifted to an earlier date into 0.5h natural thaw, is then seeded to seed
It is activated in culture medium, the condition of the activation includes 30 DEG C, 120rpm, terminal OD600Value 0.1；Bacillus subtilis connects
Kind amount is 2 × 106CFU/ml seed culture medium；The seed culture medium contains the yeast powder of 0.5 weight %, the egg of 1 weight %
White peptone, the sodium chloride of the glucose of 0.5 weight % and 1 weight %, surplus are water, and pH 7.2-7.4；
(2) bacillus subtilis after activation is seeded in fermentation medium again and is cultivated, the condition of culture and connect
Kind amount is shown in Table 1；The fermentation medium contains the yeast extract of 0.32 weight %, the molasses of 0.8 weight % and 0.6 weight %'s
Glucose, surplus are water, and pH 7.2-7.4；
During the cultivation process, 0h, start starting when after every 3h sample microscopy, bacterium colony count, until dyeing microscopic examination obtains
Bud ratio stops culture 95% or more.
Wherein, the test method of bud ratio are as follows: each sample takes 3ml, and 0.1ml dilution spread plate therein is taken to calculate viable bacteria
Sum.Produce gemma after, stoste carry out 80 DEG C of water-bath 10min, spread plate calculate gemma number, and according to formula " bud ratio=
Gemma number/total viable count × 100% " calculates bud ratio, and the results are shown in Table 1.
Comparative example 1
According to the method culture bacillus subtilis of embodiment 3, the difference is that, " the 0.32 weight % in fermentation medium
Yeast extract " replace with " corn pulps of 1.5 weight % ", the results are shown in Table 1.
Comparative example 2-3
According to the method culture bacillus subtilis of embodiment 3, the difference is that, in step (2), according to connecing shown in table 1
Kind amount inoculation, the results are shown in Table 1.
Comparative example 4
According to the method culture bacillus subtilis of embodiment 3, the difference is that, the fermentation medium contains 0.6 weight
The yeast extract of % is measured, the glucose of the molasses of 0.32 weight % and 0.8 weight %, the results are shown in Table 1.
Table 1
Can be seen that by the result of table 1 is had using the embodiment 1 of currently preferred cultivation temperature than embodiment 2
Obvious better effect, although the budding time of embodiment 1 is slightly longer than embodiment 2, total Number of spores and bud ratio are all excellent
In embodiment 2.In addition, comparing embodiment 1 and comparative example 1-4, which can be seen that mode only according to the invention, controls fermentation training
The type and inoculum concentration for supporting base can effectively shorten the budding time, and improve Number of spores and bud ratio.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
Claims (7)
1. a kind of method for promoting producing bacillus subtilis gemma, which is characterized in that this method includes connecing bacillus subtilis
Kind is cultivated into fermentation medium, the conditions for the training include: 28-32 DEG C, 95-120rpm；Bacillus subtilis
Inoculum concentration is 2 × 107-8×107CFU/ml fermentation medium；The fermentation medium contains the yeast leaching of 0.2-0.5 weight %
Powder, the molasses of 0.5-1 weight % and the glucose of 0.3-0.8 weight %, and pH 7-7.5.
2. according to the method described in claim 1, wherein, the fermentation medium contains the yeast leaching of 0.3-0.35 weight %
Powder, the molasses of 0.7-0.9 weight % and the glucose of 0.5-0.7 weight %, and pH7.2-7.4.
3. method according to claim 1 or 2, wherein the time of the culture is 8-15 hours.
4. according to the method described in claim 1, wherein, the method also includes being seeded to bacillus subtilis before culture
It is activated in seed culture medium.
5. according to the method described in claim 4, wherein, the seed culture medium contains the yeast powder of 0.4-0.8 weight %,
The peptone of 0.5-1.2 weight %, the glucose of 0.4-0.6 weight % and the sodium chloride of 0.8-1.2 weight %, and pH 7.2-
7.4。
6. method according to claim 4 or 5, wherein the condition of the activation includes: 28-32 DEG C, 110-130rpm,
Terminal OD600Value 0.1-0.2；The inoculum concentration of bacillus subtilis is 1 × 106-4×106CFU/ml seed culture medium.
7. according to the method described in claim 1, wherein, which comprises
(1) bacillus subtilis being seeded in seed culture medium and is activated, the condition of the activation includes 30 ± 0.5 DEG C,
120 ± 2rpm, terminal OD600Value 0.1 ± 0.05；The inoculum concentration of bacillus subtilis is 1 × 106-4×106The training of CFU/ml seed
Support base；The seed culture medium contains the yeast powder of 0.5 ± 0.01 weight %, the peptone of 1 ± 0.01 weight %, 0.5 ±
The sodium chloride of the glucose of 0.01 weight % and 1 ± 0.01 weight %, and pH 7.2-7.4；
(2) bacillus subtilis after activation is seeded in fermentation medium again and is cultivated, the conditions for the training include:
30 ± 0.5 DEG C, 100 ± 5rpm；The inoculum concentration of bacillus subtilis is 2 × 107-8×107CFU/ml fermentation medium；The hair
Ferment culture medium contains the yeast extract of 0.32 ± 0.01 weight %, the molasses of 0.8 ± 0.01 weight % and 0.6 ± 0.01 weight %
Glucose, and pH7.2-7.4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175859A (en) * | 2019-07-05 | 2021-01-05 | 中粮生物化学（安徽）股份有限公司 | High-density fermentation method of bacillus subtilis |
| CN114410516A (en) * | 2022-01-05 | 2022-04-29 | 中盐工程技术研究院有限公司 | Fermentation method of bacillus subtilis with high spore rate |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103937717A (en) * | 2014-04-22 | 2014-07-23 | 华南农业大学 | Method for efficiently improving germination rate of Bacillus subtilis |
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| CN103937717A (en) * | 2014-04-22 | 2014-07-23 | 华南农业大学 | Method for efficiently improving germination rate of Bacillus subtilis |
Non-Patent Citations (3)
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| 刘春红 等: "枯草芽胞杆菌B201产芽孢培养基优化", 《中国生物防治学报》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175859A (en) * | 2019-07-05 | 2021-01-05 | 中粮生物化学（安徽）股份有限公司 | High-density fermentation method of bacillus subtilis |
| CN112175859B (en) * | 2019-07-05 | 2022-11-18 | 中粮生物科技股份有限公司 | High-density fermentation method of bacillus subtilis |
| CN114410516A (en) * | 2022-01-05 | 2022-04-29 | 中盐工程技术研究院有限公司 | Fermentation method of bacillus subtilis with high spore rate |
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