CN109055270A - A kind of high-density cultivation method and culture medium for bacillus - Google Patents
A kind of high-density cultivation method and culture medium for bacillus Download PDFInfo
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- CN109055270A CN109055270A CN201811012929.0A CN201811012929A CN109055270A CN 109055270 A CN109055270 A CN 109055270A CN 201811012929 A CN201811012929 A CN 201811012929A CN 109055270 A CN109055270 A CN 109055270A
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of high-density cultivation method and culture medium for bacillus.High-density cultivation method of the present invention for bacillus includes inclined-plane culture, shaking flask culture, seed culture and fermented and cultured.Fermentation medium is made of following components and its concentration in culture medium of the present invention: sucrose 8-15g/L, maize cob meal 25-50g/L, peptone 15-30g/L, yeast powder 15-30g/L, cornstarch 30-40g/L, magnesium sulfate 0.5-0.9g/L, zinc chloride 0.1-0.7g/L, potassium dihydrogen phosphate 0.5-1.3g/L, dipotassium hydrogen phosphate 0.8-1.5g/L, ammonium sulfate 0.03-0.1g/L, fish intestinal juice 0.1-1g/L.The spore forming rate and gemma density of the bacillus of culture medium and cultural method culture provided by the invention are significantly increased.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of high-density cultivation methods and culture for bacillus
Base.
Background technique
Bacillus is a section for bacterium, is a kind of bacillus or coccus that can form gemma, including bacillus, gemma
Lactobacillus, fusobacterium, Desulfotomaculum and Sporosarcina etc..They are strong to extraneous injurious factor resistance, together
When have the function of that organic matter decomposition power is strong, generate metabolism product abundant, antibacterial, harmful power of going out by force, deodorization etc..
Studies have shown that bacillus can generate a variety of digestive ferments, digestion and absorption of the animal to nutriment is helped.Gemma bar
Bacterium has stronger protease, amylase and lipase active, while also having the enzyme of complex carbohydrates in degradation feed,
Such as pectase, cellulase, these enzymes can destroy the cell wall of plant feed cell, and the nutriment of cell is promoted to discharge
Out, and the anti-nutritional factors in feed can be eliminated, reduces the obstacle that anti-nutritional factors utilizes animal digestion.Therefore, gemma
Bacillus becomes the first choice of Substitutes For Antibiotic in additive for microbe feedstuff.
But bacteriostasis is concentrated mainly on to the research of bacillus at present, antibacterial substance isolating and purifying and characterizing,
And the producing enzyme feature and the application effect research in dregs of beans etc. of bacillus subtilis, and improving bacillus thallus yield
Fermentation technique in terms of, High Density Cultivation technique then report it is less.
Domestic and foreign literature report bacillus culture technique, fermentation liquid gemma density majority hundred million/milliliter of 30-100 it
Between, production level is lower.The high density that Chinese patent application CN102433283A discloses a kind of feeding bacillus subtilis is raw
Production. art is used as culture medium using glucose, starch, yeast powder, peptone, beancake powder, inorganic salts etc., only in 30 liters of fermentors
On tested, highest viable count be 15,000,000,000/milliliter, but without explanation whether be suitable for other bacillus type cultures,
The specific viable count that bacillus reaches after High Density Cultivation is not pointed out yet.Chinese patent application CN102586153A is disclosed
A kind of fermentation culture method is used as culture medium using peptone, soluble starch, inorganic salts etc., and fermentation liquid spore concentration is most
Height only 4,300,000,000.
Therefore, existing high density fermentation culture technology gemma density in actually culture is not high, and production cost is higher, no
It is suitble to promote the use of.Also, the prior art is not suitable for mainly for the High Density Cultivation of a certain or several bacillus
In all bacillus.
Summary of the invention
For the above technical problem, the purpose of the present invention is to provide a kind of high-density cultivation methods for bacillus
And culture medium.The present invention is directed to obtain a kind of high-density cultivation method and culture medium suitable for various bacillus.
Technical scheme is as follows:
A kind of culture medium for bacillus High Density Cultivation, including slant medium, seed culture medium and fermentation training
Support base；The fermentation medium is made of following components and its concentration: sucrose 8-15g/L, maize cob meal 25-3g/L, peptone
15-30g/L, yeast powder 15-30g/L, cornstarch 30-40g/L, magnesium sulfate 0.5-0.9g/L, zinc chloride 0.1-0.7g/L, phosphorus
Acid dihydride potassium 0.5-1.3g/L, dipotassium hydrogen phosphate 0.8-1.5g/L, ammonium sulfate 0.03-0.1g/L, fish intestinal juice 0.1-1g/L.
The slant medium is made of following components and its concentration: dehydrated potato powder 150-180g/L, glucose 20-25g/
L, agar powder 25g/L.
The seed culture medium is made of following components and its concentration: sucrose 40-60g/L, peptone 15-20g/L, yeast
Powder 15-20g/L, cornstarch 20-25g/L, magnesium sulfate 0.3-0.8g/L, potassium dihydrogen phosphate 2-5g/L.
A method of using the above-mentioned culture medium culture bacillus for bacillus High Density Cultivation, including it is as follows
Step:
S1. bacillus is inoculated into slant medium, is 25-30 DEG C in temperature, under the conditions of pH=6-7, cultivates 4-9
It, is made slant tube strain；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 25-30 DEG C in temperature, pH=5-7 is stirred
Revolving speed is mixed to cultivate 5-6 days, obtaining shake-flask seed culture solution under the conditions of 180-200r/min；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, is 25-35 DEG C in temperature, under the conditions of speed of agitator 200-250r/min, pH=5-7, cultivates 18h, obtain seed culture
Liquid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 5-10%
Middle fermentation controls pH=6-7 in fermentation process, and at 30-38 DEG C, speed of agitator is controlled in 110-130r/ for cultivation temperature control
Min, 30% or more, ferment 10h, adds same amount of fermentation medium for oxyty control, continues to cultivate 10h, repeats to mend
Add culture operation 3-6 times to get.
Bacillus described in the step S1 is selected from bacillus subtilis, bacillus licheniformis, bacillus coagulans, left-handed
Lactic acid bacillus or racemic lactic acid bacillus.
The maize cob meal being added in fermentation medium of the present invention is with the broken processing of corn ear using stringent screening system
At, have many advantages, such as even tissue, hardness be suitable for, good toughness, water imbibition is strong, wear-resisting property is good.Containing sugar in maize cob meal
54.5%, thick protein 2.2%, crude fat 0.4%, crude fibre 29.7%, minerals 1.2%.
Studies have shown that maize cob meal is added in fermentation medium of the present invention, glucose content in culture solution is reduced, and introduce
The additives such as albumen, fat and fiber, meanwhile, in addition the water sorption of maize cob meal, substantially increases the survival of bacillus
Rate.
The magnesium ion and zinc ion for being conducive to culture absorption are added in fermentation medium provided by the invention, is conducive to bud
Absorption of the spore bacillus to metal ion has the function of promoting growth.
Fish intestinal juice is added in fermentation medium provided by the invention, facilitates the quick suction of carbohydrate, albumen and vitamin
It receives, to promote the quick breeding and growth of bacillus.
Acidity and oxygen concentration in fermentation process is effectively guaranteed in such a way that fermentation medium is added in gradation in the present invention
Control, and improve the utilization rate of nutriment, saved the energy.
Compared with prior art, beneficial effects of the present invention:
(1) high-density cultivation method provided in the present invention for bacillus, the cultural method are suitable for withered grass bud
The high density training of spore bacillus, bacillus licheniformis, bacillus coagulans, D-lactic acid bacillus and racemic lactic acid bacillus
It supports.Using the high-density cultivation method provided by the present invention for bacillus, the bud of bacillus in obtained fermentation liquid
Spore formation rate can be 95% or more, gemma density minimum 1.5 × 1010/ milliliter.
(2) cultural method of gradation addition fermentation medium provided by the invention, is effectively guaranteed in fermentation process sour
The control of degree and oxygen concentration, and the utilization rate of nutriment is improved, save the energy.
(3) provided by the present invention for the culture medium of bacillus High Density Cultivation, wherein fermentation medium uses corn
Source of the core powder as carbohydrate, albumen and fiber, reduces the glucose content of fermentation phase, is added to other auxiliary materials, improves
The spore forming rate of bacillus.
(4) provided by the present invention for the culture medium of bacillus High Density Cultivation, metal ion zinc and magnesium is added, simultaneously
Fish intestinal juice is added, promotes absorption and utilization of the bacillus to nutriment, so that tachyauxesis is bred, improves gemma and generates
Rate.
Fish intestinal juice is the intestinal juice of carp, mainly contains pancreatic juice and bile, wherein the organic matter in pancreatic juice is a variety of digestion
Enzyme may act on sugar, fat and three kinds of food ingredients of protein, and bile is mutually coordinated with pancreatic juice, promotion albumen, carbohydrate and other
The decomposition of nutriment is conducive to the quick absorption to these nutriments.
Specific embodiment
It is further detailed With reference to embodiment, as described below is several embodiments of the invention, but
The range that this should not be interpreted as to the above-mentioned theme of the present invention is limited to example below.The spirit and principles in the present invention are not being departed from
Within any modification for making, and the equivalent replacement or improvement made according to ordinary skill knowledge and customary means,
It should all include within the scope of the present invention.
Embodiment 1, bacillus subtilis High Density Cultivation
It chooses bacillus subtilis and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Slant medium is made of following components and its concentration:
Potato 180g/L, glucose 25g/L, agar powder 25g/L add water 1000mL to be configured to slant medium, sterilizing
It is spare.
Seed culture medium is made of following components and its concentration:
Sucrose 60g/L, peptone 15g/L, yeast powder 15g/L, cornstarch 25g/L, magnesium sulfate 0.3g/L, biphosphate
Potassium 5g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration: sucrose 12g/L, maize cob meal 25g/L, peptone 15g/L,
Yeast powder 15g/L, cornstarch 30g/L, magnesium sulfate 0.5g/L, zinc chloride 0.7%, potassium dihydrogen phosphate 0.57g/L, phosphoric acid hydrogen two
Potassium 1.3g/L, ammonium sulfate 0.03g/L, fish intestinal juice 0.1g/L sterilize spare.
The high-density cultivation method of bacillus subtilis, steps are as follows:
S1. bacillus subtilis is inoculated into slant medium, is 25 DEG C, under the conditions of pH=6 in temperature, cultivates 4 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 25 DEG C, pH=5 in temperature, speed of agitator
Under the conditions of 180rpm, cultivates 5 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, is 25 DEG C in temperature, under the conditions of speed of agitator 200rpm, pH=5, cultivates 18h, obtain seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 5%
It ferments, pH=6 is controlled in fermentation process, at 30 DEG C, speed of agitator is controlled in 110r/min, oxyty control for cultivation temperature control
30% or more, ferment system 10h, adds same amount of fermentation medium, continues to cultivate 10h, and culture operation 3 times is added in repetition,
To obtain the final product.
By the culture of above-mentioned condition, the spore forming rate of bacillus subtilis is 92% in fermentation liquid, and gemma density is
1.5×1010/ milliliter.
Embodiment 2, bacillus licheniformis High Density Cultivation
It chooses bacillus licheniformis and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Potato 180g/L, glucose 25g/L, agar powder 25g/L add water 1000mL to be configured to slant medium, sterilizing
It is spare.
Seed culture medium is made of following components and its concentration:
Sucrose 60g/L, peptone 15g/L, yeast powder 15g/L, cornstarch 25g/L, magnesium sulfate 0.3g/L, biphosphate
Potassium 5g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration: sucrose 15g/L, maize cob meal 25g/L, peptone 30g/L,
Yeast powder 15g/L, cornstarch 20g/L, magnesium sulfate 0.9g/L, zinc chloride 0.1%, potassium dihydrogen phosphate 1.3g/L, phosphoric acid hydrogen two
Potassium 0.8g/L, ammonium sulfate 0.1g/L, fish intestinal juice 1g/L sterilize spare.
The high-density cultivation method of bacillus licheniformis, steps are as follows:
S1. bacillus licheniformis is inoculated into slant medium, at 35 DEG C, under the conditions of pH=7, cultivates 9 days, is made oblique
Face test tube strains；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, at 30 DEG C, pH=7, speed of agitator is
Under the conditions of 200rpm, cultivates 6 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, is 35 DEG C in temperature, under the conditions of speed of agitator 250rpm, pH=7, cultivates 18h, obtain seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 10%
It ferments, pH=7 is controlled in fermentation process, at 38 DEG C, speed of agitator is controlled in 130r/min, oxyty control for cultivation temperature control
30% or more, ferment system 10h, adds same amount of fermentation medium, continues to cultivate 10h, and culture operation 6 times is added in repetition,
To obtain the final product.
By the culture of above-mentioned condition, the spore forming rate of bacillus licheniformis is 93% in fermentation liquid, and gemma density is
1.5×1010/ milliliter.
Embodiment 3, bacillus coagulans High Density Cultivation
It chooses bacillus coagulans and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Potato 180g/L, glucose 25g/L, water 1000ml, agar powder 25g/L are configured to slant medium, sterilize standby
With.
Seed culture medium is made of following components and its concentration:
Sucrose 60g/L, peptone 15g/L, yeast powder 15g/L, cornstarch 25g/L, magnesium sulfate 0.3g/L, biphosphate
Potassium 5g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration: sucrose 11g/L, maize cob meal 29g/L, peptone 21g/L,
Yeast powder 27g/L, cornstarch 34g/L, magnesium sulfate 0.7g/L, zinc chloride 0.4%, potassium dihydrogen phosphate 0.9g/L, phosphoric acid hydrogen two
Potassium 1.1g/L, ammonium sulfate 0.08g/L, fish intestinal juice 0.5g/L sterilize spare.
The high-density cultivation method of bacillus coagulans, steps are as follows:
S1. bacillus coagulans are inoculated into slant medium, are 32 DEG C, under the conditions of pH=6 in temperature, cultivate 5 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 30 DEG C, pH=7 in temperature, speed of agitator
Under the conditions of 190rpm, cultivates 5 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, is 32 DEG C in temperature, under the conditions of speed of agitator 220rpm, pH=6, cultivates 18h, obtain seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 8%
It ferments, pH=6.5 is controlled in fermentation process, at 35 DEG C, speed of agitator is controlled in 120r/min, oxyty for cultivation temperature control
30% or more, ferment 10h, adds same amount of fermentation medium for control, continues to cultivate 10h, culture operation 4 is added in repetition
It is secondary to get.
By the culture of above-mentioned condition, the spore forming rate of bacillus coagulans is 95% in fermentation liquid, and gemma density is
1.6×1010/ milliliter.
Comparative example 1, bacillus coagulans High Density Cultivation
It chooses bacillus coagulans and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Slant medium is made of following components and its concentration:
Potato 180g/L, glucose 25g/L, water 1000ml, agar powder 25g/L are configured to slant medium, sterilize standby
With.
Seed culture medium is made of following components and its concentration:
Sucrose 60g/L, peptone 15g/L, yeast powder 15g/L, cornstarch 25g/L, magnesium sulfate 0.3g/L, biphosphate
Potassium 5g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration: sucrose 11g/L, glucose 29g/L, peptone 21g/L, ferment
Female powder 27g/L, cornstarch 34g/L, magnesium sulfate 0.7g/L, zinc chloride 0.4%, potassium dihydrogen phosphate 0.9g/L, dipotassium hydrogen phosphate
1.1g/L, ammonium sulfate 0.08g/L, fish intestinal juice 0.5g/L sterilize spare.
The high-density cultivation method of bacillus coagulans, steps are as follows:
S1. bacillus coagulans are inoculated into slant medium, are 32 DEG C, under the conditions of pH=6 in temperature, cultivate 5 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 30 DEG C, pH=7 in temperature, speed of agitator
Under the conditions of 190rpm, cultivates 5 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, temperature is 32 DEG C, under the conditions of speed of agitator 220rpm, pH=6, cultivates 18h, obtains seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 8%
It ferments, pH=6.5 is controlled in fermentation process, at 35 DEG C, speed of agitator is controlled in 120r/min, oxyty for cultivation temperature control
30% or more, ferment 10h, adds same amount of fermentation medium for control, continues to cultivate 10h, culture operation 4 is added in repetition
It is secondary to get.
By the culture of above-mentioned condition, the spore forming rate of bacillus coagulans is 80% in fermentation liquid, and gemma density is
1.2×1010/ milliliter.
Comparative example 1 and the difference of embodiment 3 are, maize cob meal is replaced with grape in the fermentation medium of comparative example 1
Sugar.As a result the spore forming rate of bacillus coagulans is substantially reduced with gemma density in fermentation liquid.
Comparative example 2, bacillus coagulans High Density Cultivation
It chooses bacillus coagulans and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Slant medium is made of following components and its concentration:
Potato 180g/L, glucose 25g/L, water 1000ml, agar powder 25g/L are configured to slant medium, sterilize standby
With.
Seed culture medium is made of following components and its concentration:
Sucrose 60g/L, peptone 15g/L, yeast powder 15g/L, cornstarch 25g/L, magnesium sulfate 0.3g/L, biphosphate
Potassium 5g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration: sucrose 11g/L, glucose 29g/L, peptone 21g/L, ferment
Female powder 27g/L, cornstarch 34g/L, magnesium sulfate 0.7g/L, potassium dihydrogen phosphate 0.9g/L, dipotassium hydrogen phosphate 1.1g/L, ammonium sulfate
0.08g/L, fish intestinal juice 0.5g/L sterilize spare.
The high-density cultivation method of bacillus coagulans, steps are as follows:
S1. bacillus coagulans are inoculated into slant medium, are 32 DEG C, under the conditions of pH=6 in temperature, cultivate 5 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 30 DEG C, pH=7 in temperature, speed of agitator
Under the conditions of 190rpm, cultivates 5 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, temperature is 32 DEG C, under the conditions of speed of agitator 220rpm, pH=6, cultivates 18h, obtains seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 8%
It ferments, pH=6.5 is controlled in fermentation process, at 35 DEG C, speed of agitator is controlled in 120r/min, oxyty for cultivation temperature control
30% or more, ferment 10h, adds same amount of fermentation medium for control, continues to cultivate 10h, culture operation 4 is added in repetition
It is secondary to get.
By the culture of above-mentioned condition, the spore forming rate of bacillus coagulans is 85% in fermentation liquid, and gemma density is
1.35×1010/ milliliter.
Comparative example 2 and the difference of embodiment 3 are, are not added with zinc chloride in the fermentation medium of comparative example 2.As a result it ferments
The spore forming rate of bacillus coagulans and gemma density reduce in liquid.
Comparative example 3, bacillus coagulans High Density Cultivation
It chooses bacillus coagulans and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Slant medium is made of following components and its concentration:
Potato 180g/L, glucose 25g/L, water 1000ml, agar powder 25g/L are configured to slant medium, sterilize standby
With.
Seed culture medium is made of following components and its concentration:
Sucrose 60g/L, peptone 15g/L, yeast powder 15g/L, cornstarch 25g/L, magnesium sulfate 0.3g/L, biphosphate
Potassium 5g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration: sucrose 11g/L, glucose 29g/L, peptone 21g/L, ferment
Female powder 27g/L, cornstarch 34g/L, magnesium sulfate 0.7g/L, zinc chloride 0.4%, potassium dihydrogen phosphate 0.9g/L, dipotassium hydrogen phosphate
1.1g/L, ammonium sulfate 0.08g/L sterilize spare.
The high-density cultivation method of bacillus coagulans, steps are as follows:
S1. bacillus coagulans are inoculated into slant medium, are 32 DEG C, under the conditions of pH=6 in temperature, cultivate 5 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 30 DEG C, pH=7 in temperature, speed of agitator
Under the conditions of 190rpm, cultivates 5 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, temperature is 32 DEG C, under the conditions of speed of agitator 220rpm, pH=6, cultivates 18h, obtains seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 8%
It ferments, pH=6.5 is controlled in fermentation process, at 35 DEG C, speed of agitator is controlled in 120r/min, oxyty for cultivation temperature control
30% or more, ferment 10h, adds same amount of fermentation medium for control, continues to cultivate 10h, culture operation 4 is added in repetition
It is secondary to get.
By the culture of above-mentioned condition, the spore forming rate of bacillus coagulans is 83% in fermentation liquid, and gemma density is
1.1×1010/ milliliter.
Comparative example 3 and the difference of embodiment 3 are, fish intestinal juice is not added in the fermentation medium of comparative example 3.As a result it sends out
The spore forming rate of bacillus coagulans is substantially reduced with gemma density in zymotic fluid.
Comparative example 4, bacillus coagulans High Density Cultivation
It chooses bacillus coagulans and carries out High Density Cultivation.
Configure culture culture medium, including slant medium, seed culture medium and fermentation medium.
Slant medium is made of following components and its concentration:
Potato 160g/L, glucose 22g/L, water 1000ml, agar powder 20g/L are configured to slant medium, sterilize standby
With.
Seed culture medium is made of following components and its concentration:
Sucrose 45g/L, peptone 8g/L, yeast powder 7g/L, cornstarch 20g/L, magnesium sulfate 0.6g/L, potassium dihydrogen phosphate
3g/L is configured to seed culture medium, sterilizes spare.
Fermentation medium is made of following components and its concentration:
Sucrose 14g/L, glucose 34g/L, peptone 16g/L, yeast powder 15g/L, cornstarch 26g/L, magnesium sulfate
0.5g/L, potassium dihydrogen phosphate 0.4g/L, dipotassium hydrogen phosphate 1.1g/L, ammonium sulfate 0.07g/L are configured to fermentation medium, sterilizing
It is spare.
The high-density cultivation method of bacillus coagulans, steps are as follows:
S1. bacillus coagulans are inoculated into slant medium, are 32 DEG C, under the conditions of pH=6 in temperature, cultivate 5 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 30 DEG C, pH=7 in temperature, speed of agitator
Under the conditions of 190rpm, cultivates 5 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies training
It supports, is 32 DEG C in temperature, under the conditions of speed of agitator 220rpm, pH=6, cultivates 18h, obtain seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 8%
It ferments, pH=6.5 is controlled in fermentation process, at 35 DEG C, speed of agitator is controlled in 120r/min, oxyty for cultivation temperature control
Control 30% or more, fermented and cultured 40h to get.
By the culture of above-mentioned condition, the spore forming rate of bacillus coagulans is 70% in fermentation liquid, and gemma density is
1.0×1010/ milliliter.
Comparative example 2 and the difference of embodiment 3 are, do not add culture using gradation during the fermented and cultured of comparative example 2
The feed-type of base.As a result the spore forming rate of bacillus coagulans is substantially reduced with gemma density in fermentation liquid.
Claims (5)
1. a kind of culture medium for bacillus High Density Cultivation, which is characterized in that including slant medium, seed culture medium
And fermentation medium；The fermentation medium is made of following components and its concentration: sucrose 8-15g/L, maize cob meal 25-50g/
L, peptone 15-30g/L, yeast powder 15-30g/L, cornstarch 30-40g/L, magnesium sulfate 0.5-0.9g/L, zinc chloride 0.1-
0.7g/L, potassium dihydrogen phosphate 0.5-1.3g/L, dipotassium hydrogen phosphate 0.8-1.5g/L, ammonium sulfate 0.03-0.1g/L, fish intestinal juice
0.1-1g/L。
2. being used for the culture medium of bacillus High Density Cultivation as described in claim 1, which is characterized in that the inclined-plane culture
Base is made of following components and its concentration: dehydrated potato powder 150-180g/L, glucose 20-25g/L, agar powder 25g/L.
3. being used for the culture medium of bacillus High Density Cultivation as described in claim 1, which is characterized in that the seed culture
Base is made of following components and its concentration: sucrose 40-60g/L, peptone 15-20g/L, yeast powder 15-20g/L, cornstarch
20-25g/L, magnesium sulfate 0.3-0.8g/L, potassium dihydrogen phosphate 2-5g/L.
4. a kind of side using the culture medium culture bacillus described in claim 1 for bacillus High Density Cultivation
Method, which comprises the steps of:
S1. bacillus is inoculated into slant medium, is 25-30 DEG C in temperature, under the conditions of pH=6-7, cultivates 4-9 days,
Slant tube strain is made；
S2. slant tube strain made from step S1 is subjected to shaking flask culture, is 25-30 DEG C, pH=5-7 in temperature, stirring turns
Under the conditions of speed is 180-200r/min, cultivates 5-6 days, obtain shake-flask seed culture solution；
S3. shake-flask seed culture solution made from step S2 is moved in the seeding tank containing seed culture medium and amplifies culture,
It is 25-35 DEG C in temperature, under the conditions of speed of agitator 200-250r/min, pH=5-7, cultivates 18h, obtain seed culture fluid；
S4. seed culture fluid made from step S3 is inoculated into the fermentor equipped with fermentation medium by inoculum concentration 5-10% and is sent out
Ferment, controls pH=6-7 in fermentation process, and at 30-38 DEG C, speed of agitator control is molten in 110-130r/min for cultivation temperature control
30% or more, ferment 10h, adds same amount of fermentation medium for oxygen concentration control, continues to cultivate 10h, culture is added in repetition
Operation 3-6 times to get.
5. being used for the high-density cultivation method of bacillus as claimed in claim 4, which is characterized in that described in the step S1
Bacillus is selected from bacillus subtilis, bacillus licheniformis, bacillus coagulans, D-lactic acid bacillus or racemic lactic acid
Bacillus.
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