CN105062947B - A kind of production method of high gemma rate bacillus licheniformis - Google Patents
A kind of production method of high gemma rate bacillus licheniformis Download PDFInfo
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- CN105062947B CN105062947B CN201510525282.1A CN201510525282A CN105062947B CN 105062947 B CN105062947 B CN 105062947B CN 201510525282 A CN201510525282 A CN 201510525282A CN 105062947 B CN105062947 B CN 105062947B
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- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 241000726221 Gemma Species 0.000 title claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 43
- 230000004151 fermentation Effects 0.000 claims abstract description 43
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims abstract description 11
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims abstract description 11
- 239000011565 manganese chloride Substances 0.000 claims abstract description 11
- 235000002867 manganese chloride Nutrition 0.000 claims abstract description 11
- 229940099607 manganese chloride Drugs 0.000 claims abstract description 11
- 230000001954 sterilising effect Effects 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 239000007640 basal medium Substances 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229920002261 Corn starch Polymers 0.000 claims description 4
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 4
- 239000002518 antifoaming agent Substances 0.000 claims description 4
- 239000008120 corn starch Substances 0.000 claims description 4
- 229940099112 cornstarch Drugs 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical group [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 239000006260 foam Substances 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 239000003921 oil Substances 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 230000003519 ventilatory effect Effects 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000000034 method Methods 0.000 description 5
- 229940099596 manganese sulfate Drugs 0.000 description 4
- 239000011702 manganese sulphate Substances 0.000 description 4
- 235000007079 manganese sulphate Nutrition 0.000 description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 2
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229910052748 manganese Inorganic materials 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- 229910001437 manganese ion Inorganic materials 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000003918 fraction a Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A kind of production method of high gemma rate bacillus licheniformis, manganese chloride is not added in ferment tank culture medium, and fill into 0.03 ~ 0.04% sterilizing manganese chloride solution of fermentation volume in the 23 ~ 25h that ferments, the present invention adds manganese chloride by the middle and later periods of fermenting, neither influence bacillus licheniformis early growth, and improve bacillus licheniformis at spore rate, shorten fermentation period, to reduce fermenting and producing cost, better product quality is obtained.
Description
Technical field
The invention belongs to technical field of biological fermentation, and in particular to a kind of producer of high gemma rate bacillus licheniformis
Method.
Background technology
Bacillus licheniformis has the activity of stronger protease, lipase, amylase, and nutrient in feed can be promoted to drop
Solution stimulates the development of animal immune organ, and enhancing immunity of organisms can generate resistant activity material enzyme, and take by force with unique biology
Oxygen mechanism of action can inhibit the growth and breeding of pathogenic bacteria, be a kind of green feed additive, bacillus licheniformis formed gemma with
Have resistance strong afterwards, it is heat-resisting, it is not easy death, is easily stored.So improve the lichen bacillus ferments during at spore method
It is an emphasis of fermentation production process.
Existing general production process is the manganese sulfate that 0.02% is added in the basal medium that ferments, manganese ion tool
Play the role of promoting sporulation, but manganese ion can also inhibit the growth and breeding of Bacillus licheniformis body, so the dosage of manganese
Limitation is received, it is low at spore rate, only 85% or so, to improve into spore rate will extend fermentation time, increase production cost.
Invention content
The purpose of the present invention is that solve the deficiencies in the prior art and provides a kind of high gemma rate bacillus licheniformis
Production method.
To achieve the above object, the present invention uses following technical proposals：
A kind of production method of high gemma rate bacillus licheniformis, includes the following steps：
1) shaking flask culture：Bacillus licheniformis is inoculated on Shake flask medium, inoculum concentration is volume ratio 1:(80 ~ 120),
Carry out shaking table culture；
2) seed tank culture：By step 1）Obtained Bacillus licheniformis liquid is inoculated on seed culture medium, inoculum concentration
Volume ratio 1:(80 ~ 120), are cultivated；
3) fermentation tank culture：By step 2）Obtained Bacillus licheniformis liquid is inoculated on fermentation basal medium, is connect
Kind amount volume ratio 1:(80 ~ 120), carry out fermented and cultured, 35 ~ 37 DEG C of fermentation temperature, and rotating speed is 180 ~ 200rpm/min, ventilatory capacity
It is 1:（0.7~1.0）, fermentation pH is 6.0 ~ 7.8；Whole fermentation time is 30 ~ 36h；
Fermentation basal medium formulation be：Cornstarch 2 ~ 4%；Dregs of beans 1 ~ 3%；Peptone 0.5 ~ 1.5%；Magnesium sulfate 0.04 ~
0.06%；Calcium carbonate 0.5 ~ 0.7%；Potassium dihydrogen phosphate 0.2 ~ 0.4%；Antifoaming agent 0.015 ~ 0.025%；
4) 23 ~ 25h of fermentation fills into 0.03 ~ 0.04% sterilizing manganese chloride solution of fermentation volume.
8 ~ 12g of peptone in every liter of Shake flask medium, 3 ~ 7g of yeast powder, 3 ~ 7g of salt in step 1), remaining is water.
Shaking speed is 180 ~ 220rpm/min, 35 ~ 37 DEG C of temperature, incubation time 10 ~ 12 hours in step 1).
Seed culture based formulas is in step 2)：Peptone 1.5 ~ 2.5%, yeast powder 0.5 ~ 1.5%, sodium chloride 0.4 ~
0.6%, remaining is water, and material content described above is mass fraction.
Condition of culture is in step 2)：180 ~ 220rpm/min of rotating speed, 35 ~ 37 DEG C of temperature, incubation time 10 ~ 12 hours.
Antifoaming agent is silicon oil foam killer in step 3).
The mass fraction a concentration of 5% of step 4) manganese chloride solution.
The present invention adds manganese chloride by the middle and later periods of fermenting, and neither influences the early growth of bacillus licheniformis, and improve
Bacillus licheniformis at spore rate, shorten fermentation period, to reduce fermenting and producing cost, obtain better product product
Matter.
Specific implementation mode
Embodiment 1
A kind of production method of high gemma rate bacillus licheniformis, includes the following steps：
1) shaking flask culture：Bacillus licheniformis is inoculated on Shake flask medium, inoculum concentration is 1 by volume:(80~
120) shaking table culture, is carried out；Shaking speed is 180 ~ 220rpm/min, 35 ~ 37 DEG C of temperature, incubation time 10 ~ 12 hours；Every liter
8 ~ 12g of peptone in Shake flask medium, 3 ~ 7g of yeast powder, 3 ~ 7g of salt, remaining is water；
2) seed tank culture：By step 1）Obtained Bacillus licheniformis liquid is inoculated on seed culture medium, inoculum concentration
By volume 1:(80 ~ 120), are cultivated, 180 ~ 220rpm/min of rotating speed, 35 ~ 37 DEG C of temperature, incubation time 10 ~ 12 hours；
Seed culture based formulas is：Peptone 1.5 ~ 2.5%, yeast powder 0.5 ~ 1.5%, sodium chloride 0.4 ~ 0.6%, remaining is water, the above institute
It is mass fraction to state material content；
3) fermentation tank culture：By step 2）Obtained Bacillus licheniformis liquid is inoculated on fermentation basal medium, is connect
Kind of amount by volume 1:(80 ~ 120), carry out fermented and cultured, 35 ~ 37 DEG C of fermentation temperature, and rotating speed is 180 ~ 200rpm/min, ventilation
Amount is 1:（0.7~1.0）, fermentation pH 6.0-7.8；Whole fermentation time is 30 to 36h；Fermentation basal medium is matched
Fang Wei：Cornstarch 2 ~ 4%；Dregs of beans 1 ~ 3%；Peptone 0.5 ~ 1.5%；Magnesium sulfate 0.04 ~ 0.06%；Calcium carbonate 0.5 ~ 0.7%；Phosphorus
Acid dihydride potassium 0.2 ~ 0.4%；Antifoaming agent 0.015 ~ 0.025%；
4) step 3）0.03 ~ 0.04% sterilization quality score that 23 ~ 25h of middle fermentation fills into fermentation volume is that 5% manganese chloride is molten
Liquid.
Embodiment 2
A kind of production method of high gemma rate bacillus licheniformis, includes the following steps：
1) shaking flask culture：1L culture mediums are prepared according to formula, in average packing and 10 1000ml triangular flasks, high-temperature sterilization
It after cooling, is inoculated with bacillus licheniformis, inoculum concentration 100, shaking table culture, rotating speed 220rpm/min, 37 DEG C of temperature, when culture
Between 11 hours；
Culture medium prescription：Peptone 10g；3~7g of yeast powder；3~7g of salt is settled to 1L, high steam with distilled water
121 DEG C of sterilizings；
2) seed tank culture：Culture medium prescription：Peptone 2%, yeast powder 1%, sodium chloride 0.5%. drinking water are prepared, volume
300 liters, 121 DEG C of high steam sterilizes 30 minutes, inoculum concentration 1:100, rotating speed 200rpm/min, 36 DEG C of temperature, incubation time
10 hours；
3) fermentation tank culture：10 tons of fermentation basal mediums, 121 DEG C of 30 points of sterilizing sterilizings of high steam are prepared according to formula
Clock is cooled to 35-37 DEG C, by inoculum concentration 1:120 access steps 2）Obtained Bacillus licheniformis strain ferments, temperature 36
DEG C, rotating speed 180rpm/min, ventilatory capacity 1:1；Fermentation pH 6.5；Whole fermentation time is 36 hours；
Fermentation basal medium formulation be：Cornstarch 3%；Dregs of beans 2%；Peptone 1.5%；Magnesium sulfate：0.05%；Carbonic acid
Calcium：0.6%；Potassium dihydrogen phosphate：0.3%；Silicon oil foam killer 0.02%；
4) it ferments 24 hours and fills into 0.04% 5% manganese chloride solution of sterilizing of fermentation volume；
5) it is while with the fermentation basal medium that 0.02% manganese sulfate is added that control carries out fermented and cultured, takes fermentation 24 small
When sample, 30 hours samples, 36 hours samples detect lichens gemma viable count and at spore rate；
6) manganese sulfate is not added for fermentation basal medium, ferments 24 hours, zymotic fluid lichens gemma viable count reaches every milli
12,000,000,000 are risen, the basal medium than manganese sulfate is added improves 2,000,000,000 viable counts；Fermentation fills into the 0.04% of fermentation volume in 24 hours
Sterilize manganese chloride solution, and fermentation reached 85% to 30 hours at spore rate, and fermentation reached 95% or more to 36 hours at spore rate；Sulphur is added
The basal medium of sour manganese ferments was also only 87% to 36 hours at spore rate.
Each process parameter of the production method of bacillus licheniformis is shown in Table 1 ~ 2 in embodiment 3 ~ 16, other same embodiments
2。
Each process parameter table of production method of 1 embodiment of table, 3 ~ 9 bacillus licheniformis
Each process parameter table of production method of 2 embodiment of table, 10 ~ 16 bacillus licheniformis
Claims (4)
1. a kind of production method of high gemma rate bacillus licheniformis, it is characterised in that include the following steps：
1）Shaking flask culture：Bacillus licheniformis is inoculated on Shake flask medium, inoculum concentration is volume ratio 1:(80 ~ 120) carry out
Shaking table culture；
2）Seed tank culture：By step 1）Obtained Bacillus licheniformis liquid is inoculated on seed culture medium, inoculum concentration volume
Than 1:(80 ~ 120), are cultivated；
3）Fermentation tank culture：By step 2）Obtained Bacillus licheniformis liquid is inoculated on fermentation basal medium, inoculum concentration
Volume ratio 1:(80 ~ 120), carry out fermented and cultured, 35 ~ 37 DEG C of fermentation temperature, and rotating speed is 180 ~ 200rpm/min, ventilatory capacity 1:
（0.7~1.0）, fermentation pH is 6.0 ~ 7.8；Whole fermentation time is 30 ~ 36h；
Fermentation basal medium formulation be：Cornstarch 2 ~ 4%；Dregs of beans 1 ~ 3%；Peptone 0.5 ~ 1.5%；Magnesium sulfate 0.04 ~
0.06%；Calcium carbonate 0.5 ~ 0.7%；Potassium dihydrogen phosphate 0.2 ~ 0.4%；Antifoaming agent 0.015 ~ 0.025%；
4）23 ~ 25h of fermentation fills into 0.03 ~ 0.04% sterilizing manganese chloride solution of fermentation volume, and the mass fraction of manganese chloride solution is dense
Degree is 5%；
8 ~ 12g of peptone in every liter of Shake flask medium, 3 ~ 7g of yeast powder, 3 ~ 7g of salt in step 1), remaining is water；
Seed culture based formulas is in step 2)：Peptone 1.5 ~ 2.5%, yeast powder 0.5 ~ 1.5%, sodium chloride 0.4 ~ 0.6%,
Yu Weishui, material content described above are mass fraction.
2. the production method of high gemma rate bacillus licheniformis as described in claim 1, it is characterised in that shaking table in step 1)
Rotating speed is 180 ~ 220rpm/min, 35 ~ 37 DEG C of temperature, incubation time 10 ~ 12 hours.
3. the production method of high gemma rate bacillus licheniformis as described in claim 1, it is characterised in that culture in step 2)
Condition is：180 ~ 220rpm/min of rotating speed, 35 ~ 37 DEG C of temperature, incubation time 10 ~ 12 hours.
4. the production method of high gemma rate bacillus licheniformis as described in claim 1, it is characterised in that defoaming in step 3)
Agent is silicon oil foam killer.
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| CN107523512B (en) * | 2016-06-22 | 2020-07-17 | 浙江京新药业股份有限公司 | High-spore-rate bacillus licheniformis fermentation method |
| CN108795792A (en) * | 2017-05-02 | 2018-11-13 | 浙江京新药业股份有限公司 | A kind of culture medium and fermentation process of bacillus licheniformis |
| CN108633843B (en) * | 2018-04-18 | 2020-10-27 | 中国科学院水生生物研究所 | In-vitro culture method of grass carp intestinal bagworms and preparation method of used culture medium |
| CN110305812B (en) * | 2019-07-05 | 2023-06-13 | 山东苏柯汉生物工程股份有限公司 | Bacillus licheniformis fermentation culture process |
| CN110283752B (en) * | 2019-07-11 | 2021-08-17 | 中国科学院青岛生物能源与过程研究所 | Culture medium and production method of bacillus licheniformis, production method of bacillus licheniformis original powder and product |
| CN113234631A (en) * | 2021-05-20 | 2021-08-10 | 广东海纳川生物科技股份有限公司 | Bacillus licheniformis and fermentation method and application thereof |
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| CN101869182A (en) * | 2010-04-09 | 2010-10-27 | 武汉科诺生物科技股份有限公司 | Preparation method of bacillus licheniformis raw powder with 100 billion live spores per gram |
| CN103865826A (en) * | 2012-12-15 | 2014-06-18 | 重庆江合生物科技有限责任公司 | Method for high density culturing of Bacillus subtilis with high spore formation rate |
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| CN1032570C (en) * | 1993-01-20 | 1996-08-21 | 河南华能科技发展公司 | Method for producing biologic pesticide by lichen gemma |
| CN101497907A (en) * | 2009-03-06 | 2009-08-05 | 河南天方药业股份有限公司 | Fermentation process of spiramycin |
| CN101659936A (en) * | 2009-09-15 | 2010-03-03 | 北京航天恒丰科技发展有限公司 | Method for utilizing metallic ion to promote formation of spore of bacillus subtilis |
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| CN101869182A (en) * | 2010-04-09 | 2010-10-27 | 武汉科诺生物科技股份有限公司 | Preparation method of bacillus licheniformis raw powder with 100 billion live spores per gram |
| CN103865826A (en) * | 2012-12-15 | 2014-06-18 | 重庆江合生物科技有限责任公司 | Method for high density culturing of Bacillus subtilis with high spore formation rate |
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