CN109468259A - A kind of culture medium for promoting gemma to generate - Google Patents
A kind of culture medium for promoting gemma to generate Download PDFInfo
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Abstract
The present invention relates to a kind of culture mediums that promotion gemma generates, the culture medium includes the component of following parts by weight: corn flour: 3-5, peptone: 0.3-0.5, bean cake powder: 2-3, magnesium salts: 0.01-0.03, chloride: 0.001-0.01, sylvite: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.The culture medium is used as liquid fermentation medium.Compared with prior art, culture medium of the present invention can effectively shorten the time of gemma generation, and operation is simple, and significantly improves gemma yield.
Description
Technical field
The present invention relates to a kind of culture mediums, more particularly, to a kind of culture medium that promotion gemma generates.
Background technique
Bacillus subtilis is widely used as probiotics in field of microbial fermentation.Bacillus subtilis is in animal body
It is interior to promote to grow beneficial to anaerobic bacteria, and the organic acids such as lactic acid are generated, gut pH is reduced, inhibits other pathogenic bacteria indirectly
Growth；It can be improved immunoglobulin and antibody level, enhance cellular immunity and humoral immune function, improve herd immunity；
The enzymes such as alpha-amylase, protease, lipase, cellulase are synthesized, in alimentary canal together with the intracorporal digestive enzymes of animal
It plays a role, is capable of the composition of the flora of balance intestinal, improve intestinal health.
In the industrial production, the fermentation of bacillus subtilis mainly has solid fermentation and liquid fermentation, and solid fermentation is opposite
For it is more stable, but bacterium amount is had certain limitations, liquid fermentation can be such that bacterium amount is greatly improved, form high bacterium amount, but
It is that high bacterium amount does not represent high gemma, the thallus that gemma is not formed in liquid fermentation can be most of dead in spray drying,
And if gemma rate is low in bacterium powder, and the thallus for not forming gemma can be dead in a short time, greatly reduces bacterium in bacterium powder
Content and guarantee its stability.Thus it is guaranteed that the gemma proportion in liquid fermentation, is of great significance.
Chinese patent CN201510174012.0 discloses the preparation method of a Bacillus species bacteriocin preparation, described
Preparation method is the following steps are included: series bacillus (Paenibacillus sp.) is inoculated in culture series bacillus by (1)
In culture medium, concussion fermented and cultured obtains fermentation liquid, by gained fermentation liquid centrifuging and taking supernatant；(2) by gained supernatant and second
Alcohol mixing, the volume ratio that ethyl alcohol is mixed with supernatant are 2:1~4:1, and gained mixed liquor is stood, and the temperature of standing is 2~10
DEG C, the time of standing is 12~36 hours, and sediment is collected by centrifugation in mixed liquor, removes solvent to obtain the final product.The culture medium is matched
Side be preferably: nitrogen source 1~3%, carbon source 1~10%, l-cysteine 0.01~0.03%, sodium sulphate 0.01~0.03%,
Na2HPO412H2O 0.1~0.3%, sodium acetate 1~3%, sodium chloride 0.05~0.15%, sodium bicarbonate 0.1~0.3%,
Surplus is water.
Chinese patent CN201810095745.9 discloses a kind of preparation method of bacillus amyloliquefaciens G5 Bacillus powder,
One, preparation produces gemma culture medium, produces gemma nutrient media components are as follows: cornstarch, glucose, dregs of beans, calcium carbonate, ammonium chloride, phosphorus
Sour hydrogen dipotassium, magnesium sulfate and manganese sulfate；Two, gemma fermentation liquid is cultivated；Three, Bacillus powder is prepared, in Xiang Suoshu gemma fermentation liquid
Add ammonium sulfate and diatomite；Then plate-frame filtering is carried out；Obtain the Bacillus powder of bacillus amyloliquefaciens G5.Whereby, this hair
In the preparation method of the bacillus amyloliquefaciens G5 Bacillus powder of bright offer, production gemma culture medium raw material is common, is conveniently easy to get, and drops
Low production cost；Number of spores is more in obtained gemma fermentation liquid, gemma rate >=98%；Using ammonium sulfate and diatomite as wadding
The time of Bacillus powder preparation is shortened in solidifying agent, and living bacteria count is more in obtained Bacillus powder, and the yield of effective viable bacteria >=
95%.Produce gemma culture medium each component are as follows: 10~40g/L of cornstarch, 0.5~3g/L of glucose, 5~25g/L of dregs of beans, carbonic acid
0.1~1.5g/L of calcium, 0.1~1.0g/L of ammonium chloride, 0.1~1.0g/L of dipotassium hydrogen phosphate, 0.01~0.7g/L of magnesium sulfate and sulphur
Sour 0.01~0.7g/L of manganese.
The culture medium that above-mentioned two patent is related to mainly prepares bacteria preparation or bacterium powder, and there is no special promotion gemma bars
The function of bacterium production gemma rate.
Chinese patent CN102181389A discloses a kind of method of breeding Bacillaceae with high spore yield, and this method is root
According to the thermal stability characteristics of gemma, the means cultivated after being handled using stepped heating select the bacillus of high yield gemma rate.
Its step includes: bacillus activation, level-one heat treatment, level-one culture, second level heat treatment, second level culture, three-level heating
Processing, third stage culture and collection, obtain the high yield gemma rate bacillus.Although the above method improves the production of bacillus
Gemma rate, but the method needs longer incubation time, by activating and by third stage culture, at least 3-5 days every time, cultivates one
It is secondary at least to need 2 weeks time, the time of culture domestication is elongated, and after culture, it is also necessary to carry out different degrees of
Heat treatment, needs careful setting temperature and time, increases control cost.
Chinese patent CN201810052769.6 discloses a kind of strain domestication method of raising bacillus production gemma rate,
The following steps are included: bacillus is activated；By the strain after activation successively according to from normal incubation medium to barren culture medium, so
It is further added by the sequence that nutrient concentrations are cultivated into normal incubation medium afterwards and carries out secondary culture, culture medium is according to concentration setting 3
More than a, continuous passage 3 is more than generation.Above-mentioned patent using change culture medium nutriment number come change strain existence
Environment, and then the production gemma rate of bacillus is improved, but above-mentioned cultural method is still excessively complicated, there are many required program, training
Feeding time and efficiency have to be hoisted.
Therefore, a kind of culture medium that promotion gemma generates is established, can either effectively shorten time of sporulation, and can be big
The formation rate of gemma and the total amount of gemma are improved greatly, is of great significance for industrial production.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of promotion gemma to generate
Culture medium.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of culture medium for promoting gemma to generate, the culture medium includes the component of following parts by weight:
Corn flour: 3-5, peptone: 0.3-0.5, bean cake powder: 2-3, magnesium salts: 0.01-0.03, chloride: 0.001-
0.01, sylvite: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.
Preferably, which includes the component of following parts by weight:
Corn flour: 3-5, peptone: 0.3-0.5, bean cake powder: 2-3, glucose: 1-2, magnesium salts: 0.01-0.03, chlorination
Object: 0.001-0.01, sylvite: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.
It is further preferred that the culture medium includes the component of following parts by weight:
Corn flour: 4.5, peptone: 0.3, bean cake powder: 2, glucose: 1, magnesium salts: 0.015, chloride: 0.001, sylvite:
0.02, calcium salt: 0.03, manganese salt: 0.004.
In culture medium of the present invention, it is preferable that the magnesium salts is epsom salt or magnesium sulfate.
In culture medium of the present invention, it is preferable that the manganese salt is manganese sulfate or manganese sulfate monohydrate.
In culture medium of the present invention, it is preferable that the chloride is potassium chloride or sodium chloride.
In culture medium of the present invention, it is preferable that the calcium salt is calcium nitrate or calcium carbonate.
In culture medium of the present invention, it is preferable that the sylvite is potassium dihydrogen phosphate or dipotassium hydrogen phosphate.
It is further preferred that the magnesium salts is epsom salt, the manganese salt is manganese sulfate, and the chloride is chlorination
Potassium, the calcium salt are calcium nitrate, and the sylvite is potassium dihydrogen phosphate.
Culture medium of the present invention is used as liquid fermentation medium.
When culture medium of the present invention is used as liquid fermentation medium, in every 1L liquid fermentation medium, containing with the following group
Point:
Corn flour: 3-5g, peptone: 0.3-0.5g, bean cake powder: 2-3g, epsom salt: 0.01-0.03g, potassium chloride:
0.001-0.01g, potassium dihydrogen phosphate: 0.01-0.03g, calcium nitrate: 0.01-0.05g, manganese sulfate: 0.003-0.005g.
Culture medium of the present invention is in use, technological condition for fermentation are as follows: temperature: 36-38 DEG C, ventilation: 3-5m3/ h, pH:
7.0-7.4, revolving speed: 200-300rpm, preferably temperature: 37 DEG C, ventilation: 3-5m3/ h, pH:7.2, revolving speed: 200rpm.
Culture medium of the present invention is in use, using following methods:
A. prepared by bacillus seed liquor
It is followed by the bacillus for being stored in inclined-plane is activated in Shake flask medium, in 36-38 DEG C, 200-300rpm is shaken
Bed culture, obtains bacillus seed liquor；
B. strain fermentation
Using culture described in claim 1 as fluid nutrient medium, using fermentor to bacillus seed liquor obtained by step A
It ferments, the gemma amount of bacillus reaches 94.74-97.86%, and gemma amount reaches 1.73-1.95 × 1010CFU/mL。
In the present invention, the bacillus is the bacillus or coccus that can form gemma, including bacillus, gemma cream bar
Pseudomonas and Sporosarcina, it is preferred that be bacillus subtilis, bacillus licheniformis.
Medium component in the present invention is adjusted mainly for salt component, and the present invention utilizes potassium chloride, adds nitric acid
Calcium, potassium dihydrogen phosphate, epsom salt and manganese sulfate etc. can make thallus be in suitable growing environment, effectively facilitate simultaneously
The generation of gemma shortens the time, and improves spore production.
Wherein MgSO4.7H2The Mg of O2+It is that EMP, TCA approach and lysine generate important zymoexciter, Mg2+With MnSO4
In Mn2+The generation of gemma can be effectively facilitated；KCl equilibrium osmotic pressure serves adjust osmotic pressure in the medium；
KH2PO4In K+It is the co-factor of certain enzymes (fructokinase, phosphopyruvic acid transphosphorylase etc.), maintains potential difference and infiltration
Pressure, but also be to play important pH value buffer function；Ca(NO3)2In Ca2+The activator of a variety of enzymes, more with cell membrane
Permeability it is related；And NO3-Can be for inorganic nitrogen-sourced, the time that the bacillus subtilis spore that can effectively shorten is formed, simultaneously
Substantially increase the formation rate of gemma and the total amount of gemma.The collaboration effect of these components make its in industrial processes significantly
The electric power and manpower saved by fermentation time reduction are saved, so that fermentation efficiency is obviously improved.
Compared with prior art, culture medium of the present invention can effectively shorten the time of gemma generation, and can substantially increase
The formation rate of gemma and the total amount of gemma, operation is simple, and significantly improves gemma yield.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.
Comparative example
1. prepared by bacillus subtilis seed liquor
It is followed by the bacillus subtilis for being stored in inclined-plane is activated in 100mL Shake flask medium, in 37 DEG C, 200rpm
Shaking table culture is for 24 hours.
The formula of the fermentation medium used in the comparative example are as follows: peptone: 10g/L, yeast powder 5g/L, sodium chloride 5g/
L, glucose 2g/L.
2. strain fermentation
It is fermented using 50L fermentor, is fermented using conventional zymotechnique to bacterial strain.Technological condition for fermentation
Are as follows: temperature: 37 DEG C, ventilation: 3-5m3/ h, pH:7.2, revolving speed: 200rpm.
Embodiment 1-5
1. prepared by bacillus subtilis seed liquor
The bacillus subtilis for being stored in inclined-plane is inoculated into Shake flask medium, in 37 DEG C, 200rpm shaking table culture 22-
For 24 hours, it then crosses to 37 DEG C of LB plate cultures, isolates single bacterium colony, activate 2-3 times repeatedly.By the bacillus subtilis after activation
Bacterium is connected in 100mL Shake flask medium, and in 37 DEG C, for 24 hours, seed liquor is made in 200rpm shaking table culture.
The formula of fermentation medium used in embodiment 1-5 is as shown in table 1.
Culture medium prescription in 1 embodiment 1-5 of table
2. strain fermentation
It is fermented using 50L fermentor, is fermented using conventional zymotechnique to bacterial strain.Technological condition for fermentation
Are as follows: temperature: 37 DEG C, ventilation: 3-5m3/ h, pH:7.2, revolving speed: 200rpm.
3. the calculating of gemma rate: after fermentation, being coated with the dilution method of counting using plate to carry out detecting total bacterium amount；Into mistake
After 80 DEG C of water-baths after 20min, plate coating dilution count detection gemma production quantity is carried out.The wherein calculation of gemma rate are as follows:
Gemma productivity=(gemma production quantity/total viable bacteria amount) * 100%.
Comparative example and the fermentation medium of embodiment 1-5 cultivate bacillus subtilis, and the results are shown in Table 2.
2 bacillus subtilis spore of table generates situation
From result it follows that fermentation medium of the invention can make withered grass bud in the case where not influencing viable bacteria total amount
The gemma rate of spore bacillus reaches 94.74-97.86%, and gemma amount reaches 1.79-1.85 × 1010CFU/mL, the contracting of sporulation time
Short 5-7h, i.e., do not influence total bacterium amount, in contrast, improves gemma amount, and shorten the sporulation time, gemma is greatly facilitated
Formation efficiency.
Embodiment 6-8
1. prepared by bacillus seed liquor
The bacillus for being stored in inclined-plane is inoculated into Shake flask medium, in 37 DEG C, 200rpm shaking table culture for 24 hours, then is drawn
Line is isolated single bacterium colony, is activated 2 times repeatedly to 37 DEG C of LB plate cultures.Bacillus after activation is connected to 100mL shaking flask
In culture medium, in 37 DEG C, for 24 hours, seed liquor is made in 200rpm shaking table culture.
The bacillus cultivated in embodiment 6-8 is respectively synanthrin lactobacillus, urea sporosarcina, lichens gemma
Bacillus.
2. strain fermentation
It is fermented using 50L fermentor, the formula (unit is g/L) of fermentation medium: corn flour: 4.5, peptone:
0.3, bean cake powder: 2, glucose: 1, magnesium salts: 0.015, chloride: 0.001, sylvite: 0.02, calcium salt: 0.03, manganese salt: 0.004,
It is fermented using conventional zymotechnique to bacterial strain.Technological condition for fermentation are as follows: temperature: 37 DEG C, ventilation: 4m3/ h, pH:7.2,
Revolving speed: 250rpm.
3. the calculating of gemma rate: after fermentation, being coated with the dilution method of counting using plate to carry out detecting total bacterium amount；Into mistake
After 80 DEG C of water-baths after 20min, plate coating dilution count detection gemma production quantity is carried out.The wherein calculation of gemma rate are as follows:
Gemma productivity=(gemma production quantity/total viable bacteria amount) * 100%.
The fermentation medium of embodiment 6-8 cultivates bacillus, and the results are shown in Table 2.
3 bacillus spore of table generates situation
From result it follows that fermentation medium of the invention can make gemma bar in the case where not influencing viable bacteria total amount
The gemma amount of bacterium reaches 1.73-1.95 × 1010CFU/mL, gemma rate reach 95.53-97.14%, and the sporulation time shortens
The formation efficiency of gemma is greatly facilitated in 6-7h.
Bacillus is the bacillus or coccus that can form gemma, including bacillus, Sporolactobacillus and gemma eight
Folded Coccus, it is preferred that be bacillus subtilis, bacillus licheniformis.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of culture medium for promoting gemma to generate, which is characterized in that the culture medium includes the component of following parts by weight:
Corn flour: 3-5, peptone: 0.3-0.5, bean cake powder: 2-3, magnesium salts: 0.01-0.03, chloride: 0.001-0.01, potassium
Salt: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.
2. a kind of culture medium for promoting gemma to generate according to claim 1, which is characterized in that the culture medium includes such as
The component of lower parts by weight:
Corn flour: 3-5, peptone: 0.3-0.5, bean cake powder: 2-3, glucose: 1-2, magnesium salts: 0.01-0.03, chloride:
0.001-0.01, sylvite: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.
3. a kind of culture medium for promoting gemma to generate according to claim 2, which is characterized in that the culture medium includes such as
The component of lower parts by weight:
Corn flour: 4.5, peptone: 0.3, bean cake powder: 2, glucose: 1, magnesium salts: 0.015, chloride: 0.001, sylvite:
0.02, calcium salt: 0.03, manganese salt: 0.004.
4. a kind of culture medium for promoting gemma to generate according to claim 1 or 2 or 3, which is characterized in that the magnesium salts is
Epsom salt or magnesium sulfate,
The manganese salt be manganese sulfate or manganese sulfate monohydrate,
The chloride be potassium chloride or sodium chloride,
The calcium salt be calcium nitrate or calcium carbonate,
The sylvite is potassium dihydrogen phosphate or dipotassium hydrogen phosphate.
5. a kind of culture medium for promoting gemma to generate according to claim 1 or 2, which is characterized in that the culture medium is used
Make liquid fermentation medium.
6. a kind of culture medium for promoting gemma to generate according to claim 5, which is characterized in that the culture medium is used as liquid
When body fermentation medium, in every 1L liquid fermentation medium, contain following components:
Corn flour: 3-5g, peptone: 0.3-0.5g, bean cake powder: 2-3g, epsom salt: 0.01-0.03g, potassium chloride:
0.001-0.01g, potassium dihydrogen phosphate: 0.01-0.03g, calcium nitrate: 0.01-0.05g, manganese sulfate: 0.003-0.005g.
7. a kind of culture medium for promoting gemma to generate according to claim 1, which is characterized in that the culture medium used
Technological condition for fermentation are as follows: temperature: 36-38 DEG C, ventilation: 3-5m3/ h, pH:7.0-7.4, revolving speed: 200-300rpm.
8. a kind of culture medium for promoting gemma to generate according to claim 7, which is characterized in that the culture medium used
Technological condition for fermentation are as follows: temperature: 37 DEG C, ventilation: 3-5m3/ h, pH:7.2, revolving speed: 200rpm.
9. a kind of culture medium for promoting gemma to generate according to claim 1, which is characterized in that the culture medium uses
When, using following methods:
A. prepared by bacillus seed liquor
It is followed by the bacillus for being stored in inclined-plane is activated in Shake flask medium, in 36-38 DEG C, the training of 200-300rpm shaking table
It supports, obtains bacillus seed liquor；
B. strain fermentation
Using component described in claim 1 as fluid nutrient medium, bacillus seed liquor obtained by step A is carried out using fermentor
Fermentation, the gemma amount of bacillus reach 94.74-97.86%, and gemma amount reaches 1.73-1.95 × 1010CFU/mL。
10. a kind of culture medium for promoting gemma to generate according to claim 1, which is characterized in that the bacillus is
The bacillus or coccus of gemma, including bacillus, Sporolactobacillus and Sporosarcina can be formed, it is preferred that be
Bacillus subtilis, bacillus licheniformis.
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| CN111349592A (en) * | 2020-05-11 | 2020-06-30 | 河南大学 | Spore production culture medium and preparation method of bacterial spores |
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