CN106190885A - A kind of fermentation of bacillus subtilis culture medium and application thereof - Google Patents
A kind of fermentation of bacillus subtilis culture medium and application thereof Download PDFInfo
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- CN106190885A CN106190885A CN201510225498.6A CN201510225498A CN106190885A CN 106190885 A CN106190885 A CN 106190885A CN 201510225498 A CN201510225498 A CN 201510225498A CN 106190885 A CN106190885 A CN 106190885A
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Abstract
The present invention relates to a kind of fermentation of bacillus subtilis culture medium and application thereof, fermentation medium includes following components: bean cake 30g/L, Semen Maydis powder 10g/L, yeast powder 0.2g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, calcium carbonate 0.1g/L, manganese sulfate 0.2g/L, sodium chloride 5g/L.Compared with prior art, the fermentation medium of the present invention makes viable count 200-400cfu/ml in producing bacillus subtilis product, spore forming rate 87～92%.
Description
Technical field
The present invention relates to a kind of culture medium, especially relate to a kind of fermentation of bacillus subtilis culture medium and answer
With, belong to microbial fermentation engineering field.
Background technology
Bacillus subtilis is that the important probiotic bacteria of a class produces bacterial strain, relative to other types probiotic bacteria (as
Lactic acid bacteria), bacillus cereus is typically simple to nutritional requirement, and accretion rate is fast, and is easily isolated, cultivates
And preservation, the harshest to Industrialized processing technique condition, additionally, the spore that bacillus cereus produces is to hot, purple
The poor environments such as outside line, ionizing radiation and low pH (2-3) have extremely strong resistivity.Therefore, spore bar
Bacterium is prone to preserve as probiotics, easy to use.Active component is high, has the biggest promotion and application to be worth,
Bacillus cereus is often using the form of spore as feed additive.
Bacillus subtilis in incubation, due to condition of culture grasp bad, often occur number of viable low,
The problems such as spore forming rate is low.Improve viable count and especially improve spore forming rate, the form with spore is made
Positive effect is had for feed additive production.Document report fermentation of bacillus subtilis technology is prepared at present
Rate is about 80-120cfu/ml, and wherein spore yield is at 40%-90%.Research emphasis focus mostly on culture medium,
The aspect such as cultivation temperature and initial pH, ignores sweat pH and regulates and controls the impact that may bring.
Chinese patent CN102168055A discloses a kind of fermentation of bacillus subtilis method of high spore rate,
The method is through activating bacillus subtilis, two-step fermentation cultivation productive target product.First inoculate the withered of activation
Grass bacillus cereus, cultivation temperature is 35～38 DEG C, tank pressure 0.02～0.06Mpa, ventilation ratio are 1: 0.5～1:
1, agitation revolution is 150rpm～200rpm, fermentation culture 8～14 hours, obtains seed bacterium solution, then will plant
Daughter bacteria liquid inoculated and cultured 20～26 hours, obtain high spore rate bacillus subtilis fluid product.In this patent,
Fermentation medium consists of: sucrose 0.20～0.70%, carbamide 0.10～0.40%, dipotassium hydrogen phosphate 0.40～0.80
%, potassium dihydrogen phosphate 0.20～0.40%, yeast extract 0.02～0.06%, iron chloride 0.01～0.04%, carbonic acid
Calcium 0.01～0.04%, magnesium sulfate 0.05～0.20%, manganese sulfate 0.01～0.04%, starch 0.30～0.80%,
Bean cake 1.00～1.50%, Semen Glycines 0.80～1.20%, bubble enemy 0.01～0.02%, surplus is water, pH6.0～7.0.
This medium component is complicated, especially carbon source and nitrogen source.Existing bean cake has again Semen Glycines；Carbamide at high temperature may be used
Carry out condensation reaction, generate biuret, contracting triuret and cyanuric acid.Cyanuric acid is readily generated melamine
Amine.
Summary of the invention
Defect that the purpose of the present invention is contemplated to overcome above-mentioned prior art to exist and a kind of high viable bacteria is provided
The fermentation of bacillus subtilis culture medium of spore forming rate several, high and application thereof.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of fermentation of bacillus subtilis culture medium, including following components: bean cake 25-35g/L, Semen Maydis powder
5-15g/L, yeast powder 0.1-1g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate 3g/L, magnesium sulfate
0.1-1g/L, ferrous sulfate 0.1-1g/L, calcium carbonate 0.1-1g/L manganese sulfate 0.1-1g/L, sodium chloride 1-10g/L,
PH is 7.0-7.5.
As preferably, described fermentation medium includes following components: bean cake 30g/L, Semen Maydis powder 10g/L,
Yeast powder 0.2g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, sulphuric acid is sub-
Ferrum 0.1g/L, calcium carbonate 0.1g/L manganese sulfate 0.2g/L, sodium chloride 5g/L.
The application of a kind of described fermentation of bacillus subtilis culture medium, it is characterised in that this culture medium is used for
Preparation bacillus subtilis.
The preparation of bacillus subtilis comprises the following steps:
(1) bacillus subtilis is seeded on slant medium, controls slant medium pH7.3-7.5,
Controlling temperature is 36～38 DEG C, cultivates 24～48h, until growing spore.Described slant medium include with
Lower component: peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L, agar
20g/L。
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36～38 DEG C, cultivates 16～24h.Described seed culture medium includes following group
Point: peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L.
(3) seed liquor through seed culture is inoculated on the fermentation medium of the present invention, carries out fermentation training
Supporting, the temperature controlling fermentation culture is 35～38 DEG C, starts by 5～6 hours not control ph from fermentation,
Between 5～6 hours to 8 hours, interpolation ammonia control ph is between 6.0-6.5, starts to mend after 8 hours
Adding phosphoric acid solution makes pH control between 7.0-7.5, to fermentation ends, the producing bacillus subtilis product obtained
Middle viable count 200-400cfu/ml, spore forming rate 87～92%.Fermentation culture total time is 48-56h.
Compared with prior art, the present invention has the following advantages and beneficial effect:
1, use new fermentative medium formula, especially carbon source and nitrogen source are optimized, carbon source and nitrogen
The optimization in source is by numerous carbon sources, nitrogen source are carried out monofactorial screening, choose on this basis some because of
Son, redesign variable concentrations and the orthogonal test of factor；Preferably obtained by repeatedly orthogonal test and checking.
The present invention is by choosing the serial enzymes of metabolic process index, and monitoring enzyme delta data alive show that the present invention is fermented
The formula of culture medium.Such as: limit CaCO30.1-1g/L is due to CaCO3Be conducive to the formation of spore,
Spore is rich in calcium ion, and chemical composition dipicolinic acid knot specific to most of calcium ion and spore
Close, it is therefore desirable to judged the excellent effect of this culture medium by the amount measuring Ca-dipicolinic acid.
2, during the fermentation, not control ph is started by 5～6 hours from fermentation, at 5～6 hours to 8
It is not added with between hour or interpolation ammonia control ph, between 6.0-6.5, starts to add phosphorus after 8 hours on a small quantity
Acid solution makes pH control between 7.0-7.5, and to fermentation ends, viable count is up to 200-400cfu/ml, bud
Spore formation rate about 90%.The present invention uses the fermentating controling condition being not used in other culture medium, this culture medium
Starting by 5～6 hours in fermentation, this stage is that amylase, protease etc. decompose the conversion of culture medium substrate in a large number
For available nitrogen source and the stage of carbon source, add ammonia and can live with the substrate reactions decomposed and inhibitory enzyme；8 hours
Growth later is substantially accelerated pH value and is quickly risen, it is to avoid pH value is too high needs acid adding control.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Comparative example
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 36～38 DEG C, cultivates 24～48h, until
Grow spore；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36～38 DEG C, cultivates 16～24h, and seed culture medium includes following components: egg
White peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 30g/L, glucose 5g/L, Semen Maydis powder 5g/L, yeast powder 0.2g/L, phosphoric acid
Potassium dihydrogen 1.5g/L, dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, calcium carbonate
0.1g/L manganese sulfate 0.2g/L, sodium chloride 5g/L, PH are 7.0, and the fermentation medium sterilizing being configured to is followed by
Enter seed liquor, 35～38 DEG C, rotating speed be 200rpm, pH value control the fermentation cylinder for fermentation at 6.8-7.2
Cultivating 51 hours its fermentation levels is 110cfu/ml wherein spore accounting 80%.
Embodiment 1
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 36～38 DEG C, cultivates 24～48h, until
Grow spore；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36～38 DEG C, cultivates 16～24h, and seed culture medium includes following components: egg
White peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 30g/L, Semen Maydis powder 10g/L, yeast powder 0.2g/L, potassium dihydrogen phosphate 1.5g/L,
Dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, calcium carbonate 0.1g/L manganese sulfate 0.2g/L,
Sodium chloride 5g/L, PH are 7.4, access seed liquor, 35～38 after the fermentation medium sterilizing being configured to
DEG C, rotating speed be 200rpm, pH value control 7.0--7.4 fermentation cylinder for fermentation cultivate 54 hours its fermentation
Level is 240cfu/ml wherein spore accounting 88%.
Embodiment 2
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 36～38 DEG C, cultivates 24～48h, until
Grow spore；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36～38 DEG C, cultivates 16～24h, and seed culture medium includes following components: egg
White peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 30g/L, Semen Maydis powder 10g/L, yeast powder 0.2g/L, potassium dihydrogen phosphate 1.5g/L,
Dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, calcium carbonate 0.1g/L manganese sulfate 0.2g/L,
Sodium chloride 5g/L, PH are 7.4, access seed liquor, 35～38 after the fermentation medium sterilizing being configured to
DEG C, rotating speed be 200rpm, pH value within 6-8 hour, control between 6.0-6.5, control thereafter at 7.0--7.4
It is 430cfu/ml wherein spore accounting 90% that fermentation cylinder for fermentation cultivates 58 hours its fermentation levels.
Embodiment 3
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 36～38 DEG C, cultivates 24～48h, until
Grow spore；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36～38 DEG C, cultivates 16～24h, and seed culture medium includes following components: egg
White peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 30g/L, Semen Maydis powder 10g/L, yeast powder 0.2g/L, potassium dihydrogen phosphate 1.5g/L,
Dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, calcium carbonate 0.1g/L manganese sulfate 0.2g/L,
Sodium chloride 5g/L, PH are 7.4, access seed liquor, 35～38 after the fermentation medium sterilizing being configured to
DEG C, rotating speed be 200rpm, pH value within 6-8 hour, control between 6.0-6.5, control thereafter at 7.0--7.4
It is 370cfu/ml wherein spore accounting 87% that fermentation cylinder for fermentation cultivates 53 hours its fermentation levels.
Embodiment 4
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 36～38 DEG C, cultivates 24～48h, until
Grow spore；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36～38 DEG C, cultivates 16～24h, and seed culture medium includes following components: egg
White peptone 10g/L, yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 30g/L, Semen Maydis powder 10g/L, yeast powder 0.2g/L, potassium dihydrogen phosphate 1.5g/L,
Dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, calcium carbonate 0.1g/L manganese sulfate 0.2g/L,
Sodium chloride 5g/L, PH are 7.4, access seed liquor, 35～38 after the fermentation medium sterilizing being configured to
DEG C, rotating speed be 200rpm, pH value within 6-8 hour, control between 6.0-6.5, control thereafter at 7.0--7.4
It is 460cfu/ml wherein spore accounting 92% that fermentation cylinder for fermentation cultivates 55 hours its fermentation levels.
Embodiment 5
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 38 DEG C, cultivates 48h, until growing spore
Son；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 38 DEG C, cultivates 24h, and seed culture medium includes following components: peptone 10g/L,
Yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 25g/L, Semen Maydis powder 5g/L, yeast powder 0.1g/L, potassium dihydrogen phosphate 1.5g/L,
Dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L, calcium carbonate 0.1g/L manganese sulfate 0.1g/L,
Sodium chloride 1g/L, PH are 7.0, access seed liquor, 35～38 after the fermentation medium sterilizing being configured to
DEG C, rotating speed be 200rpm, pH value within 6-8 hour, control between 6.0-6.5, control thereafter at 7.0--7.4
It is 480cfu/ml wherein spore accounting 90% that fermentation cylinder for fermentation cultivates 55 hours its fermentation levels.
Embodiment 6
The preparation method of a kind of bacillus subtilis, comprises the following steps:
(1) slant medium includes following components: peptone 10g/L, yeast extract powder 5g/L, glucose
5g/L, sodium chloride 10g/L, agar 20g/L.Bacillus subtilis is seeded on slant medium and carries out tiltedly
Face is cultivated, and controls slant medium pH7.3-7.5, and controlling temperature is 36 DEG C, cultivates 24h, until growing spore
Son；
(2) bacillus subtilis after slant culture is inoculated on seed culture medium, controls seed culture medium
PH7.3-7.5, controlling temperature is 36 DEG C, cultivates 24h, and seed culture medium includes following components: peptone 10g/L,
Yeast extract powder 5g/L, glucose 5g/L, sodium chloride 10g/L；
(3) seed liquor through seed culture is inoculated on fermentation medium, carries out fermentation culture, fermentation
Culture medium consists of: bean cake 35g/L, Semen Maydis powder 15g/L, yeast powder 1g/L, potassium dihydrogen phosphate 1.5g/L,
Dipotassium hydrogen phosphate 3g/L, magnesium sulfate 1g/L, ferrous sulfate 1g/L, calcium carbonate 1g/L manganese sulfate 1g/L,
Sodium chloride 10g/L, PH are 7.5, after the fermentation medium sterilizing being configured to access seed liquor, 38 DEG C,
Rotating speed is 200rpm, pH value controls between 6.0-6.5 for 6-8 hour, controls thereafter at 7.0-7.4 fermentation tank
55 hours its fermentation levels of middle fermentation culture are 470cfu/ml wherein spore accounting 91%.
The above-mentioned description to embodiment is to be understood that for ease of those skilled in the art and make
With invention.These embodiments obviously easily can be made various amendment by person skilled in the art,
And General Principle described herein is applied in other embodiments without through performing creative labour.Cause
This, the invention is not restricted to above-described embodiment, and those skilled in the art are according to the announcement of the present invention, without departing from this
Improvement and amendment that invention category is made all should be within protection scope of the present invention.
Claims (5)
1. a fermentation of bacillus subtilis culture medium, it is characterised in that include following components: bean cake
25-35g/L, Semen Maydis powder 5-15g/L, yeast powder 0.1-1g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate
3g/L, magnesium sulfate 0.1-1g/L, ferrous sulfate 0.1-1g/L, calcium carbonate 0.1-1g/L manganese sulfate 0.1-1g/L,
Sodium chloride 1-10g/L, PH are 7.0-7.5.
A kind of fermentation of bacillus subtilis culture medium the most according to claim 1, it is characterised in that
Described fermentation medium includes following components: bean cake 30g/L, Semen Maydis powder 10g/L, yeast powder 0.2g/L,
Potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate 3g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.1g/L, carbonic acid
Calcium 0.1g/L manganese sulfate 0.2g/L, sodium chloride 5g/L.
3. an application for fermentation of bacillus subtilis culture medium as claimed in claim 1 or 2, it is special
Levying and be, this culture medium, when fermentation of bacillus subtilis is cultivated, starts by 5～6 hours not control from fermentation
PH value processed, between 5～6 hours to 8 hours, pH value controls between 6.0-6.5, pH control after 8 hours
System to fermentation ends, obtains producing bacillus subtilis product between 7.0-7.5.
The application of a kind of fermentation of bacillus subtilis culture medium the most according to claim 3, its feature
It is, viable count 200-400cfu/ml in the producing bacillus subtilis product obtained after fermentation ends, sporulation
Rate 87～92%.
The application of a kind of fermentation of bacillus subtilis culture medium the most according to claim 3, its feature
Being, the condition carrying out fermentation culture is: the temperature controlling fermentation culture is 35～38 DEG C, from the beginning of fermentation
The not control ph by 5～6 hours, added ammonia control ph at 6.0-6.5 between 5～6 hours to 8 hours
Between, start to add phosphoric acid solution after 8 hours and make pH control between 7.0-7.5, to fermentation ends, send out
Ferment cultivation total time is 48-56h.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN111269866A (en) * | 2020-04-14 | 2020-06-12 | 上海邦成生物工程有限公司 | Composite nutrient source for bacillus subtilis fermentation medium and preparation method thereof |
| 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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| CN102168055A (en) * | 2011-03-03 | 2011-08-31 | 大连吉翔农业科技有限公司 | Bacillus subtilis fermenting method with high rate of maturing gemma |
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| CN101619298A (en) * | 2009-07-03 | 2010-01-06 | 武汉科诺生物科技股份有限公司 | Preparation method of bacillus subtilis raw powder of 5 billion live spores per gram |
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| 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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