CN102433287A - Direct-vat bacillus subtilis leaven and preparation method thereof - Google Patents
Direct-vat bacillus subtilis leaven and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a direct-vat-set bacillus subtilis leaven and a preparation method thereof, which comprises the steps of bacillus subtilis seed culture, bacillus subtilis high-density amplification culture and thallus leaven preparation by a freeze-drying method, and finally the direct-vat-set bacillus subtilis leaven is obtained, wherein the weight percentages of culture mediums for the bacillus subtilis seed culture and the high-density amplification culture are as follows: sucrose 2, bean pulp 1, monopotassium phosphate 0.1, magnesium sulfate 0.05, water to 100%, pH 7.0-7.2. The concentration conditions for preparing the thallus leavening agent by the freeze drying method are as follows: centrifuging at 5000r/min for 10min at 4 deg.C with lyophilized protectant of 5% maltodextrin, 4% sucrose, 0.8% trehalose, and 0.2% tween. The survival rate of the direct-vat-set bacillus subtilis leaven produced by the method is 95.3 percent, and the viable count is 2.69 multiplied by 1010cfu/g, inIdeal liquid and solid fermentation effects can be achieved according to the inoculation amount of 1 per mill. A preparation method of a novel high-activity direct-vat-set bacillus subtilis starter is developed based on the bacillus subtilis and is applied to liquid fermentation and solid fermentation of bacillus subtilis related products.
Description
Technical field
The invention belongs to the biotechnological formulation field, especially a kind of direct-throwing fermentation of bacillus subtilis agent and preparation method thereof.
Background technology
Throw type leaven is meant that a series of height concentrate and standardized lyophilize starter culture, can directly add heat treated raw material and ferment, and need not to its carry out activation, other pre-treatment work such as spread cultivation.The application of direct-throwing fermentation of bacillus subtilis agent does not at present appear in the newspapers both at home and abroad.
Summary of the invention
The objective of the invention is to overcome the weak point of prior art, provide a kind of simple to operate, viable bacteria content is very high, vigor is high, direct-throwing fermentation of bacillus subtilis agent that inoculum size is little and preparation method thereof.
The present invention realizes that the technical scheme of purpose is following:
The preparation method of a kind of direct-throwing fermentation of bacillus subtilis agent comprises that subtilis seed culture, subtilis high-density amplification culture, thalline concentrate, add protective material, freeze-drying prepares the thalline starter, obtains the agent of direct-throwing fermentation of bacillus subtilis at last; The substratum weight percent of said subtilis seed culture and high-density amplification culture is: sucrose 2; Dregs of beans 1, potassium primary phosphate 0.1, sal epsom 0.05; Add water to 100%; PH7.0-7.2, freeze-drying prepare the concentrated condition of thalline starter: centrifugal rotational speed 5000r/min, centrifugal 10min; Centrifuging temperature is 4 ℃, and the protective material that is adopted is the aqueous solution that contains 5% maltodextrin, 4% sucrose, 0.8% trehalose and 0.2% tween.
And; The name of said subtilis is called TK-1; Specific name Bacillus subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
And said seed culture condition is: cultivate for 37 ± 1 ℃, incubation time is 20h.
And the survival rate of subtilis is 95.3% in the agent of said direct-throwing fermentation of bacillus subtilis, and viable count is 2.69 * 10
10Cfu/g.
And the agent of said direct-throwing fermentation of bacillus subtilis is applied to liquid state fermentation by 1 ‰ inoculum size, and the highest viable count is 1.03 * 10
10Cfu/mL.
And the agent of direct-throwing fermentation of bacillus subtilis is applied to solid state fermentation by 1 ‰ inoculum size, and the highest viable count is 0.985 * 10
10Cfu/g.
The agent of a kind of direct-throwing fermentation of bacillus subtilis is characterized in that: comprise lyophilized vaccine and subtilis, said lyophilized vaccine is 5% maltodextrin, 4% sucrose, 0.8% trehalose, 0.2% tween.
And; The name of said subtilis is called TK-1; Specific name Bacillus subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
Advantage of the present invention and positively effect are:
1, after subtilis TK-1 bacterial strain passes through fed-batch fermentation high-density amplification culture among the present invention; Adopt freeze-drying to prepare the thalline starter; Using the survival rate of the direct-throwing fermentation of bacillus subtilis agent of present method production is 95.3%, and viable count is 2.69 * 10
10Cfu/g, the inoculum size according to 1 ‰ just can reach the liquid and solid state fermentation effect of ideal.
2, the present invention adopts freeze-drying to prepare the agent of direct-throwing fermentation of bacillus subtilis first, compares with former liquid seed inoculation, and the starter inoculum size is little, directly uses, and labor savings shortens the production cycle, can prevent effectively that again bacterial classification from polluting and degeneration; Preserve convenient management, advantages such as product with stable quality.
3, the present invention has confirmed that subtilis adopts centrifugal rotational speed 5000r/min in concentration process, 4 ℃ of centrifugal 10min down, and centrifugal yield is 98.83%.
4, the present invention has confirmed that protective material is 5% maltodextrin, 4% sucrose, 0.8% trehalose, 0.2% tween in the thalline freezing dry process, and final subtilis freeze-drying survival rate is 93%, and viable count is 2.69 * 10
10Cfu/g.
5, the direct-throwing fermentation of bacillus subtilis agent of adopting the inventive method preparation is applied to liquid state fermentation by 1 ‰ inoculum size, and the highest viable count is 1.03 * 10
10Cfu/mL, the direct-throwing fermentation of bacillus subtilis agent of employing the inventive method preparation is applied to solid state fermentation by 1 ‰ inoculum size, and the highest viable count is 0.985 * 10
10Cfu/g.
6, the fermentation of bacillus subtilis agent of the present invention's preparation has the following advantages: viable bacteria content is very high, and vigor is high, thereby inoculum size is little; Can directly use, avoid lengthy and tedious enlarged culturing process, both labor savings shortens the production cycle, can prevent effectively that again bacterial classification from polluting and degeneration; Preserve convenient management; Be easy to control production technique, product with stable quality; , simple rehydration can directly use after handling as the seed bacterial strain.
Description of drawings
Fig. 1 carries out the influence of microorganism collection time to the starter viable count for the present invention;
Fig. 2 is the influence of lyophilized vaccine to the starter viable count, annotates: No. 1 10% maltodextrin; No. 2 10% sucrose; No. 3 10% trehaloses; No. 4 10% tweens; No. 5 5% SANMALT-S+5% sucrose; No. 6 5% maltodextrin+4% sucrose+0.8% trehalose+0.2% tweens; No. 7 5% maltodextrin+4% sucrose+1% tweens; No. 8 5% maltodextrin+4% sucrose+1% trehaloses;
Fig. 3 is seeded in the effect comparison of liquid substratum for fermentation of bacillus subtilis agent and fermented liquid.
Fig. 4 is seeded in the effect comparison of solid medium for fermentation of bacillus subtilis agent and fermented liquid.
Embodiment
Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
1. the seed liquor of thalline preparation
The bacterial classification of glycerine pipe cold storage is received in the test tube that liquid seed culture medium is housed, and is that 37 ℃, rotating speed are to cultivate under the condition of 150r/min in temperature.
Wherein liquid seed culture medium (g/L) is: peptone 10g, and Nacl 10g, yeast soak powder 5g, and cultivate in shaking table pH7.0～7.2, and its temperature is 37 ℃, and rotating speed is 150r/min.
3. the high-density amplification culture of thalline
Inoculum size with 5% is inoculated in the 200L jar with subtilis and carries out fermentation culture, and liquid amount 160L cultivates 20h in 37 ± 1 ℃.The employing peristaltic pump adds the NaOH solution of 2mol/L and regulates pH, makes it to maintain about 6.8, and fermentation is adopted and left standstill fed-batch fermentation, carries out gentle agitation after the feed supplement, and the soya-bean oil of interpolation 0.3% is made skimmer.
4. bacterial classification is collected
It is a step of whole technology key that bacterial classification is collected; Comparatively suitable method is to adopt supercentrifuge to carry out spinning to carry out vacuum lyophilization then at present; It can adapt to the suitability for industrialized production requirement fully, so mostly adopt centrifugal and the cryodesiccated way of vacuum-drying.
5. the protectant selection of thalline
After having collected a large amount of subtilis thalline, must add the bacterial classification protective material so that subtilis avoids the damage of extraneous adverse factor.Adding protective material can improve survival rate, the survival time of subtilis effectively and keep the original characteristic of subtilis.
6. lyophilize
Thalline and the protective material collected are carried out carrying out pre-freeze behind the thorough mixing; The pre-freeze temperature is controlled at-20 ℃～-30 ℃; The pre-freeze time decides with the biomass surface-area, and mostly between-20 ℃～-30 ℃, dried starter moisture content is lower than 3% and is advisable drying temperature.
7. mensuration viable count
The general colony counting method that adopts; Operation steps is: take by weighing the starter 1g for preparing, be dissolved in the SPSS of 9ml, shake up the bacterium liquid of getting 1ml with liquid-transfering gun in the back and add in the SPSS of another 9ml; Dilute, take turns doing ten times of gradient dilutions.After having diluted, get suitable extent of dilution 1ml respectively with liquid-transfering gun and throw flat board into, pour the substratum mixing into, take turns doing three parallel, put 38 ℃ of incubators into and be inverted and leave standstill cultivation.
8. the packing of starter
The starter viable count needs greater than 10
10Cfu/g, final powder formulation product must carry out the vacuum seal packing, and in cryopreservation, the quality guaranteed period is approximately 1a.
Below be some Determination of Parameters that the direct-throwing subtilis prepared the working method of agent, and the optimization of some culture condition.
The screening of direct-throwing fermentation of bacillus subtilis agent preparation condition
1. the Bacillus subtilis strain collection time confirms
Bacterial growth has experienced lag phase, logarithmic phase, balance period and paracme respectively, and at different times, growing state is different, respectively live bacterial count is carried out in its sampling.
Screening instance one
Liquid state fermentation substratum: sucrose 2g, dregs of beans 1g, potassium hydrogenphosphate 0.1g, sal epsom 0.05g, pH7.0～7.2 inoculations and culture condition: inoculum size 5% (v/w), 37 ± 1 ℃ of culture temperature, incubation time 18h.The viable count that records is 3.01 * 10
10Cfu/ml.
Screening instance two
Liquid state fermentation substratum: sucrose 2g, dregs of beans 1g, potassium primary phosphate 0.1g, sal epsom 0.05g, pH7.0～7.2 inoculations and culture condition: inoculum size 5% (v/w), 37 ± 1 ℃ of culture temperature, incubation time 20h.The viable count that records is 3.43 * 10
10Cfu/ml.
Screening instance three
Liquid state fermentation substratum: sucrose 2g, dregs of beans 1g, potassium primary phosphate 0.1g, sal epsom 0.05g, pH7.0～7.2
Inoculation and culture condition: inoculum size 5% (v/w), 37 ± 1 ℃ of culture temperature, incubation time 22h.The viable count that records is 3.36 * 10
10Cfu/ml.
The result
The righttest incubation time of liquid seed fermentation liquid is 20h (seeing accompanying drawing 1).
2. confirming of centrifugal rotational speed when liquid bacterial agent is collected and time
To increase the liquid bacterial agent that the subtilis after bacterium is cultivated is collected through whizzer, revolution when centrifugal and time are bigger to the influence of thalline, and under the equal conditions, along with the increase of centrifugal revolution, the survival rate of centrifugal back bacterium reduces,
Screening instance 1
With the thalline liquid that ferments, under the condition of 4000r/min, centrifugal 10min, centrifugal preceding viable count 3.43 * 10
10Cfu/ml, viable count is 3.25 * 10 in the sedimentation liquid of centrifugal back
10Cfu/ml, centrifugal back sedimentation liquid thalline survival rate is 94.75%.
With the thalline liquid that ferments, under the condition of 4000r/min, centrifugal 15min, centrifugal preceding viable count 3.43 * 10
10Cfu/ml, viable count is 3.34 * 10 in the sedimentation liquid of centrifugal back
10Cfu/ml, centrifugal back sedimentation liquid thalline survival rate is 97.38%.
With the thalline liquid that ferments, under the condition of 5000r/min, centrifugal 10min, centrifugal preceding viable count 3.43 * 10
10Cfu/ml, viable count is 3.39 * 10 in the sedimentation liquid of centrifugal back
10Cfu/ml, centrifugal back sedimentation liquid thalline survival rate is 98.83%.
The result: the centrifugal optimum condition is 5000r/min, and centrifugation time is 10min (seeing table 1).
3. lyophilized vaccine confirms
Protectant concentration of below mentioning is the weight percent concentration of the aqueous solution of tie substance.
Behind the thalline and the good lyophilized vaccine mixing that goes out, it is freezed fully, carry out vacuum lyophilization then.Must carry out pre-freeze to sample, the temperature of this research pre-freeze-25 ℃～-30 ℃, pre-freeze time 4hr, freezing thickness is 0.5mm, carries out lyophilize again, condition is: cold well temperature-30 ℃, 50 ℃ of plate temperature, freeze-drying time 20h.
Screening instance 1
Thalline is mixed with lyophilized vaccine by a certain percentage, condition such as above-mentioned, the lyophilized vaccine composition is 10% maltodextrin, and recording freezing survival rate is 51.3%, and the freeze-drying survival rate is 46.1%, and the lyophilized powder viable count is 0.83 * 10
10Cfu/g.
Thalline is mixed with lyophilized vaccine by a certain percentage, condition such as above-mentioned, freeze-dried protection composition is: 5% maltodextrin, 4% sucrose, 1% trehalose, recording freezing survival rate is 90.1%, freeze-drying survival rate 88.7%, the lyophilized powder viable count is 1.22 * 10
10Cfu/g.
Thalline is mixed with lyophilized vaccine by a certain percentage; Condition such as above-mentioned, freeze-dried protection composition is: 5% maltodextrin, 4% sucrose, 0.8% trehalose, 0.2% tween, recording freezing survival rate is 89.6%; The freeze-drying survival rate is 88.4%, and the lyophilized powder viable count is 2.69 * 10
10Cfu/g.
The result
The freeze-dried protection composition that should adopt is: 5% maltodextrin, 4% sucrose, 0.8% trehalose, 0.2% tween (seeing accompanying drawing 2).
Conclusion: this experiment adopts this method to produce the agent of direct-throwing fermentation of bacillus subtilis, and this starter bacteria containing amount is high, and energetic, viable count reaches 2.69 * 10
10Cfu/g.
4. the application of fermentation of bacillus subtilis agent in liquid state fermentation
Screening instance 1
The fermentation of bacillus subtilis agent is added in sterilized 0.85% saline water, and normal temperature rehydration 20min inserts the liquid state fermentation substratum of the bacterium of going out then by 0.1% inoculum size; Form (%): sucrose 2, dregs of beans 1, potassium hydrogenphosphate 0.1; Sal epsom 0.05, fermentation culture 18h is carried out in pH7.0～7.2 under 37 ℃ of conditions; Survey viable count, record viable count and be: 9.96 * 10
9Cfu/mL.
Subtilis that will be after liquid activation is inserted the liquid state fermentation substratum of the bacterium of going out by 5% inoculum size, forms (%): sucrose 2, dregs of beans 1; Potassium hydrogenphosphate 0.1, sal epsom 0.05, pH7.0～7.2; Under 37 ℃ of conditions, carry out fermentation culture 18h, survey viable count.
Recording viable count is: 8.53 * 10
9Cfu/mL.
The result
Use direct-throwing bacillus licheniformis starter to contrast in the liquid state fermentation substratum by inoculum size 0.1% and inoculum size 5% liquid inoculation, the highest viable count is respectively 1.03 * 10
10Cfu/mL and 0.903 * 10
9Cfu/mL (accompanying drawing 3).Obviously reduce inoculum size, and can reach better ferment effect.
Five, the application of direct-throwing fermentation of bacillus subtilis agent in solid state fermentation
Screening instance 1
The fermentation of bacillus subtilis agent is added in sterilized 0.85% saline water; Normal temperature rehydration 20min; Inserting what went out bacterium by 0.1% inoculation then is main solid-state fermentation culture medium with the dregs of beans; Form: dregs of beans: wheat bran: Semen Maydis powder=3: 1: 1, leave standstill cultivation 18h at 37 ℃ of constant incubators, survey viable count.
Recording viable count is: 8.21 * 10
9Cfu/g.
It is main solid-state fermentation culture medium with the dregs of beans that subtilis that will be after liquid activation is inserted what went out bacterium by the inoculum size of 5% (v/w), forms: dregs of beans: wheat bran: Semen Maydis powder=3: 1: 1, and leave standstill at 37 ℃ of constant incubators and to cultivate 18h, survey viable count.
Recording viable count is: 7.4 * 10
9Cfu/g.
The result
Use the agent of direct-throwing fermentation of bacillus subtilis to contrast in solid-state fermentation culture medium by inoculum size 0.1% and inoculum size 5% liquid inoculation, the highest viable count is respectively 9.85 * 10
9Cfu/g and 8.43 * 10
9Cfu/g (accompanying drawing 4).There is the lag phase of about 12h in starter inoculation back thalline, and growth then is quick, and final ferment effect is desirable, can obviously reduce inoculum size.
Conclusion
Through the research to direct-throwing fermentation of bacillus subtilis agent producing process, subtilis concentrates adopts final subtilis survival rate to reach 93.2%, and the viable count of starter is 2.69 * 10
10Cfu/g, the inoculum size by 1 ‰ just can reach ideal liquid state or solid state fermentation effect, and the highest viable count is respectively 1.03 * 10
10Cfu/mL, 9.85 * 10
9Cfu/g.
Table 1 centrifugation time of the present invention and rotating speed are to the influence of starter viable count
Claims (8)
1. the preparation method of direct-throwing fermentation of bacillus subtilis agent is characterized in that: comprise that subtilis seed culture, subtilis high-density amplification culture, thalline concentrate, add protective material, freeze-drying prepares the thalline starter, obtains the agent of direct-throwing fermentation of bacillus subtilis at last; The substratum weight percent of said subtilis seed culture and high-density amplification culture is: sucrose 2; Dregs of beans 1, potassium primary phosphate 0.1, sal epsom 0.05; Add water to 100%; PH 7.0-7.2, freeze-drying prepare the concentrated condition of thalline starter: centrifugal rotational speed 5000r/min, centrifugal 10min; Centrifuging temperature is 4 ℃, and the protective material that is adopted is the aqueous solution that contains 5% maltodextrin, 4% sucrose, 0.8% trehalose and 0.2% tween.
2. the preparation method of direct-throwing fermentation of bacillus subtilis according to claim 1 agent; It is characterized in that: the name of said subtilis is called TK-1; Specific name: subtilis Bacillus subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
3. the preparation method of direct-throwing fermentation of bacillus subtilis according to claim 1 agent is characterized in that: said seed culture condition is: cultivate for 37 ± 1 ℃, incubation time is 20h.
4. the preparation method of direct-throwing fermentation of bacillus subtilis according to claim 1 agent is characterized in that: the survival rate of subtilis is 95.3% in the agent of said direct-throwing fermentation of bacillus subtilis, and viable count is 2.69 * 10
10Cfu/g.
5. the preparation method of direct-throwing fermentation of bacillus subtilis according to claim 5 agent is characterized in that: the agent of said direct-throwing fermentation of bacillus subtilis is applied to liquid state fermentation by 1 ‰ inoculum size, and the highest viable count is 1.03 * 10
10Cfu/mL.
6. the preparation method of direct-throwing fermentation of bacillus subtilis according to claim 5 agent is characterized in that: the agent of direct-throwing fermentation of bacillus subtilis is applied to solid state fermentation by 1 ‰ inoculum size, and the highest viable count is 0.985 * 10
10Cfu/g.
7. direct-throwing fermentation of bacillus subtilis agent is characterized in that: comprise lyophilized vaccine and subtilis, said lyophilized vaccine is 5% maltodextrin, 4% sucrose, 0.8% trehalose, 0.2% tween.
8. direct-throwing fermentation of bacillus subtilis according to claim 7 agent; It is characterized in that: the name of said subtilis is called TK-1; Specific name Bacillus subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
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| CN109868238A (en) * | 2019-02-28 | 2019-06-11 | 浙江宝录检测技术有限公司 | Bacillus subtilis employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN112175834A (en) * | 2019-07-05 | 2021-01-05 | 中粮生物化学（安徽）股份有限公司 | Application of lactobacillus plantarum in preservation of bacillus subtilis solid microbial inoculum and method for prolonging preservation period of bacillus subtilis |
| CN112175834B (en) * | 2019-07-05 | 2022-08-05 | 中粮生物科技股份有限公司 | Application of lactobacillus plantarum in preservation of bacillus subtilis solid microbial inoculum and method for prolonging preservation period of bacillus subtilis |
| CN112920961A (en) * | 2019-12-05 | 2021-06-08 | 中粮生物科技股份有限公司 | Method for culturing bacillus subtilis |
| CN110819579A (en) * | 2019-12-24 | 2020-02-21 | 中国海洋大学 | A kind of preparation method of solid Bacillus subtilis inoculum |
| CN112980717B (en) * | 2020-12-24 | 2023-07-07 | 云南省微生物发酵工程研究中心有限公司 | Solid bacillus microbial agent and preparation method thereof |
| CN112980717A (en) * | 2020-12-24 | 2021-06-18 | 云南省微生物发酵工程研究中心有限公司 | Solid bacillus microbial agent and preparation method thereof |
| CN113005040A (en) * | 2021-01-22 | 2021-06-22 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and using method thereof |
| CN113462573A (en) * | 2021-07-12 | 2021-10-01 | 河北科技大学 | Preservation method of agricultural bacillus liquid microbial inoculum |
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