CN102943057B - Bacillus coagulans and high-density fermentation process thereof - Google Patents
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- 238000000855 fermentation Methods 0.000 title claims abstract description 58
- 230000004151 fermentation Effects 0.000 title claims abstract description 58
- 241000193749 Bacillus coagulans Species 0.000 title claims abstract description 28
- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 28
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 27
- 239000008103 glucose Substances 0.000 claims abstract description 27
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- 239000001888 Peptone Substances 0.000 claims description 10
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Abstract
The invention discloses bacillus coagulans and a high-density fermentation process thereof. The Latin chemical name of the strain is as follows: bacillus coagulans, the referenced strains are: NJYHHWG 877005, wherein the preservation date is 2012, 10 and 18 months, and the registration number of the preservation center is CGMCC No. 6681; the process provided by the invention mainly takes the pH value and the glucose concentration as guidance, and regulates and controls the concentrations of different substrates in stages to realize feedback material supplementing culture so as to greatly improve the fermentation unit and the production yield.
Description
Technical field
The invention belongs to fermentation engineering field, relate to the fermentation of a kind of Bacillus coagulans and feedback supplement thereof and realize the production technique of high-density culture.
Background technology
Bacillus coagulans is extensive in distributed in nature, and its gemma has stronger resistivity to heat and other lethal genes, also high to the resistance of antibiotic and other pharmaceutical chemicalss.The ability of adding useful Bacillus coagulans and have the nutritive substances such as secretion various proteolytic enzyme, amylase, lipase, superoxide-dismutase (SOD), antibiotic and multiple amino acids, VITAMIN just because of this characteristic, therefore can be widely used in each fields such as medicines and health protection, agricultural and food.
Bacillus coagulans is one of bacterial classification having in genus bacillus application potential, and its fungistatic effect is remarkable.This bacterium principal character and ordinary lactic acid bacteria are quite similar, can, in caecum, colonic field planting, in large intestine, ferment after oral, and during the fermentation, can produce short chain fatty acid, as lactic acid and acetic acid etc.; Bacillus coagulans is facultative anaerobe, can adapt to the intestinal environment of hypoxemia, and entering after enteron aisle can consume free oxygen breeding, is conducive to the growth of anaerobion milk-acid bacteria and bifidus bacillus; This bacterium has high patience to acid and bile, can carry out lactic fermentation, and the L～lactic acid of generation can reduce enteron aisle pH value, suppresses harmful bacteria; Owing to can forming gemma, so more benefit and recover micro ecology of gastrointestinal tract balance than the genus bacillus of lactic acid producing not.Thereby promote intestinal microecology balance, improve immunity of organism and resistance against diseases, prevention of intestinal tract disease.Bacillus coagulans has very strong restraining effect to various plants pathogenic bacteria, also can be used as agricultural produce material and uses.Bacillus coagulans CGMCC No.6681 through field antibacterial and biological and ecological methods to prevent plant disease, pests, and erosion test also find its to melon, really, the various crop disease such as the gray molds of the cash crop such as vegetables, banded sclerotial blight, Powdery Mildew, black spot all has significant prevention effect.
In biotechnology industry platform, the middle and lower reaches technology such as cell large scale cultivation are the weakest sport technique segments of China, the present invention is directed to the bottleneck problem running in Bacillus coagulans industrialization process furthers investigate, and the industrial fermentation technique that cell large scale is cultivated is explored, to reduce the production cost of Bacillus coagulans, for scale operation, the popularization of biological pesticide provide and can use for reference template.
Summary of the invention
The object of the invention is to improve Bacillus coagulans fermentation unit and production output, for scale operation, the popularization of probiotic bacterium provide a kind of Bacillus coagulans and high cell density fermentation thereof; Mainly to realize high density fermentation by feedback supplement fermentation process.
Technical scheme of the present invention is: a kind of genus bacillus, its Classification And Nomenclature is coagulating bacillus strain, the Latin formal name used at school of bacterial classification is: Bacillus coagulans, the bacterial strain of ginseng certificate is: NJYHHWG 877005, preservation date is on October 18th, 2012, and registering on the books and number in preservation center is CGMCC No.6681.
The present invention also provides a kind of technique of utilizing above-mentioned Bacillus coagulans to carry out high density fermentation, and its concrete steps are as follows: from nutrient agar picking Bacillus coagulans thalline, access seed liquor culture media shaking vase is cultivated to obtain fermentation seed liquid; Get in the fermentor tank that fermention medium is housed after 4～20% the fermentation seed liquid access sterilizing of fermention medium volume and carry out fermentation culture; Concentration by feedback supplement method control nutritive substance is carried out high density fermentation, controls temperature, air flow and rotating speed, and fermentation culture 48～78h obtains fermented liquid.
Above-mentioned cultivation and fermentation seed liquor is picking thalline from 35～42 DEG C of nutrient agars of cultivating 50～72h, access is equipped with in the aseptic Erlenmeyer flask of seed liquor substratum, under 35～42 DEG C of conditions, shaking table is cultivated, and controls rotating speed 140～200rpm, obtains fermentation seed liquid after 32～48h; It is 5～25% of Erlenmeyer flask volume that the seed liquor wherein filling in Erlenmeyer flask is cultivated base unit weight.
Preferably described seed liquor nutrient media components is: peptone 8～15g/L, and yeast extract paste 3～8g/L, NaCl4～8g/L, glucose 35～70g/L, pH is controlled at 6.5～7.8.
Preferably fermention medium component: peptone 8～15g/L, yeast extract paste 3～8g/L, NaCl 4～8g/L, glucose 35～70g/L, CaCO
310～15g/L, K
2hPO
40.8～1.5g/L, MgSO
47H
2o 0.1～0.6g/L, ZnSO
47H
2o 0.005～0.015g/L, MnCl
24H
2o 0.001～0.005g/L, pH remains on 6.5～7.8; Fermentor tank liquid amount is 20～45% of fermentor tank volume.
Preferably described feedback supplement adds for carrying out stage by stage feed supplement stream, and fermentation is carried out 16～26h and started current adding substrate A, and instructs bottoms stream acceleration with pH value in fermented liquid, makes pH value remain on 6.5～7.8; Carry out 12～24h in fermentation and start, current adding substrate B, and instruct bottoms stream acceleration with the concentration of glucose, make the interior concentration of glucose in fermentor tank be controlled at 50～70g/L, fermentation 32～44h; Wherein substrate A composition is: CaCO
355～85g/L, (NH
4)
2hPO
450～80g/L; Substrate B composition is: glucose 200～400g/L, yeast extract paste 50～80g/L, K
2hPO
44.5～8.5g/L, MgSO
47H
2o 0.57～3.5g/L.
Preferably in fermentation culture process, control temperature at 36～42 DEG C; Pass into sterile air, keep air flow at 3～6L/ (Lmin) to control dissolved oxygen 30～45%; Control rotating speed at 140～200rpm.
CGMCC No.6681 bacterial strain has following character:
1, form and cultural characteristic:
This bacterium is G+ bacteria, and thalline is rod, (0.8～1) μ m × (3～4) μ m, single or one-tenth short chain.Produce oval gemma, mostly inclined to one side thalline one end bacterium, some sporocysts expand, and have mobility.Environment resistibility is very strong to external world, processes 10min for 90 DEG C, and processing 5min for 100 DEG C can inactivation, and pH value is also can grow for 4.0～9.0 o'clock.Irregular at nutrient agar upper limb, be flattened round white colony, 2～3mm size.Facultative anaerobe, growth temperature is 20～55 DEG C, optimum growth temperature is 35～45 DEG C.
2, physio-biochemical characteristics
The major physiological biochemical character of Bacillus coagulans CGMCC No.6681 bacterial strain is in table 1:
The physiological and biochemical property of table 1 bacterial strain
Note :+: positive or growth;-: negative or do not grow
Beneficial effect:
The present invention is directed to the bottleneck problem running in course of industrialization and study, a kind of technique of utilizing Bacillus coagulans to carry out high density fermentation is provided.This technique is mainly taking pH value and glucose concn as guidance, and the concentration that regulates and controls stage by stage different substrates is cultivated to realize feedback supplement, makes thalline biomass improve 30% left and right, and viable count reaches 5.8 × 10
9this invention of cfu/mL is easy to operate succinct, is conducive to suitability for industrialized production, for scale operation, the popularization of probiotic bacterium provide and can use for reference template.
Preservation information
Its Classification And Nomenclature of above-mentioned Bacillus coagulans is Bacillus coagulans, the microorganism (strain) of ginseng certificate is: NJYHHWG 877005, China Committee for Culture Collection of Microorganisms's common micro-organisms center (No. 3, Chaoyang District Beijing great Tun road first, Institute of Microorganism, Academia Sinica) is independently screened and be preserved in this bacterial strain Shi Ben seminar.It is referred to as CGMCC, and preservation date is on October 18th, 2012, and the numbering of registering on the books is CGMCC No.6681.
Embodiment
Below case study on implementation of the present invention is elaborated, the implementation case is implemented under taking technical solution of the present invention as prerequisite, provided detailed embodiment and the concrete process of serving as, but protection scope of the present invention is not limited to following case study on implementation.
Example 1:
Picking Bacillus coagulans thalline from 35 DEG C of nutrient agars of cultivating 50h, access is equipped with in the aseptic Erlenmeyer flask of seed liquor substratum, and in Erlenmeyer flask, fermention medium volume accounts for 5% of Erlenmeyer flask volume.Seed liquor nutrient media components is: peptone 8g/L, and yeast extract paste 3g/L, NaCl 4g/L, glucose 35g/L, pH is controlled at 6.5.Under 35 DEG C of conditions, shaking table is cultivated, and controls rotating speed 140rpm, obtains fermentation seed liquid after 32h.Get in the fermentor tank that fermention medium is housed after the fermentation seed liquid access sterilizing of fermention medium volume 4% and carry out fermentation culture.Fermention medium component: peptone 8g/L, yeast extract paste 3g/L, NaCl 4g/L, glucose 35g/L, CaCO
310g/L, K
2hPO
40.8g/L, MgSO
47H
2o 0.1g/L, ZnSO
47H
2o 0.005g/L, MnCl
24H
2o 0.001g/L, pH remains on 6.5, and fermentor tank liquid amount is 20% of fermentor tank volume.Fermentation is carried out 16h and is started current adding substrate A, and instructs bottoms stream acceleration with pH value in fermented liquid, makes pH value remain on 6.5; Carry out 12h in fermentation and start, current adding substrate B, and instruct bottoms stream acceleration with the concentration of glucose, make the interior concentration of glucose in fermentor tank be controlled at 50g/L, fermentation 32h; Wherein substrate A composition is: CaCO
355g/L, (NH
4)
2hPO
450g/L; Substrate B composition is: glucose 200g/L, yeast extract paste 50g/L, K
2hPO
44.5g/L, MgSO
47H
2o 0.57g/L.In fermentation culture process, control temperature at 36 DEG C; Pass into sterile air, keep air flow at 3L/ (Lmin) to control dissolved oxygen 30%; Control rotating speed at 140rpm.In fermented liquid, viable count is 8.5 × 10
8cfu/mL.
Example 2:
Picking Bacillus coagulans thalline from 38 DEG C of nutrient agars of cultivating 61h, access is equipped with in the aseptic Erlenmeyer flask of seed liquor substratum, and in Erlenmeyer flask, fermention medium volume accounts for 15% of Erlenmeyer flask volume.Seed liquor nutrient media components is: peptone 12g/L, and yeast extract paste 6g/L, NaCl6g/L, glucose 52g/L, pH is controlled at 7.2.Under 38 DEG C of conditions, shaking table is cultivated, and controls rotating speed 170rpm, obtains fermentation seed liquid after 40h.Get in the fermentor tank that fermention medium is housed after the fermentation seed liquid access sterilizing of fermention medium volume 12% and carry out fermentation culture.Fermention medium component: peptone 12g/L, yeast extract paste 6g/L, NaCl 6g/L, glucose 52g/L, CaCO
312g/L, K
2hPO
41.2g/L, MgSO
47H
2o 0.4g/L, ZnSO
47H
2o 0.01g/L, MnCl
24H
2o 0.003g/L, pH remains on 7.2, and fermentor tank liquid amount is 32% of fermentor tank volume.Fermentation is carried out 21h and is started current adding substrate A, and instructs bottoms stream acceleration with pH value in fermented liquid, makes pH value remain on 7.2; Carry out 18h in fermentation and start, current adding substrate B, and instruct bottoms stream acceleration with the concentration of glucose, make the interior concentration of glucose in fermentor tank be controlled at 60g/L, fermentation 38h; Wherein substrate A composition is: CaCO
370g/L, (NH
4)
2hPO
465g/L; Substrate B composition is: glucose 300g/L, yeast extract paste 65g/L, K
2hPO
46.5g/L, MgSO
47H
2o 2.0g/L.In fermentation culture process, control temperature at 39 DEG C; Pass into sterile air, keep air flow at 4.5L/ (Lmin) to control dissolved oxygen 37%; Control rotating speed at 170rpm.In fermented liquid, viable count is 5.8 × 10
9cfu/mL.
Example 3:
Picking Bacillus coagulans thalline from 42 DEG C of nutrient agars of cultivating 72h, access is equipped with in the aseptic Erlenmeyer flask of seed liquor substratum, and in Erlenmeyer flask, fermention medium volume accounts for 25% of Erlenmeyer flask volume.Seed liquor nutrient media components is: peptone 15g/L, and yeast extract paste 8g/L, NaCl 8g/L, glucose 70g/L, pH is controlled at 7.8.Under 42 DEG C of conditions, shaking table is cultivated, and controls rotating speed 200rpm, obtains fermentation seed liquid after 48h.Get in the fermentor tank that fermention medium is housed after the fermentation seed liquid access sterilizing of fermention medium volume 20% and carry out fermentation culture.Fermention medium component: peptone 15g/L, yeast extract paste 8g/L, NaCl 8g/L, glucose 70g/L, CaCO
315g/L, K
2hPO
41.5g/L, MgSO
47H
2o 0.6g/L, ZnSO
47H
2o0.015g/L, MnCl
24H
2o 0.005g/L, pH remains on 7.8, and fermentor tank liquid amount is 45% of fermentor tank volume.Fermentation is carried out 26h and is started current adding substrate A, and instructs bottoms stream acceleration with pH value in fermented liquid, makes pH value remain on 7.8; Carry out 24h in fermentation and start, current adding substrate B, and instruct bottoms stream acceleration with the concentration of glucose, make the interior concentration of glucose in fermentor tank be controlled at 70g/L, fermentation 44h; Wherein substrate A composition is: CaCO
385g/L, (NH
4)
2hPO
480gL; Substrate B composition is: glucose 400g/L, yeast extract paste 80g/L, K
2hPO
48.5g/L, MgSO
47H
2o 3.5g/L.In fermentation culture process, control temperature at 42 DEG C; Pass into sterile air, keep air flow at 6L/ (Lmin) to control dissolved oxygen 45%; Control rotating speed at 200rpm.In fermented liquid, viable count is 3.1 × 10
9cfu/mL.
Claims (7)
1. a bacillus, its Classification And Nomenclature is coagulating bacillus strain, the Latin formal name used at school of bacterial classification is: Bacillus coagulans, preservation date is on October 18th, 2012, registering on the books and number in preservation center is CGMCC No.6681.
2. utilize Bacillus coagulans as claimed in claim 1 to carry out a technique for high density fermentation, its concrete steps are as follows: from nutrient agar picking Bacillus coagulans thalline, access seed liquor culture media shaking vase is cultivated to obtain fermentation seed liquid; Get in the fermentor tank that fermention medium is housed after 4～20% the fermentation seed liquid access sterilizing of fermention medium volume and carry out fermentation culture; Concentration by feedback supplement method control nutritive substance is carried out high density fermentation, controls temperature, air flow and rotating speed, and fermentation culture 48～78h obtains fermented liquid.
3. technique according to claim 2, it is characterized in that describedly cultivating to such an extent that the step of fermentation seed liquid is: picking thalline from 35～42 DEG C of nutrient agars of cultivating 50～72h, access is equipped with in the aseptic Erlenmeyer flask of seed liquor substratum, under 35～42 DEG C of conditions, shaking table is cultivated, control rotating speed 140～200rpm, after 32～48h, obtain fermentation seed liquid; It is 5～25% of Erlenmeyer flask volume that the seed liquor wherein filling in Erlenmeyer flask is cultivated base unit weight.
4. technique according to claim 2, is characterized in that described seed liquor nutrient media components is: peptone 8～15g/L, and yeast extract paste 3～8g/L, NaCl4～8g/L, glucose 35～70g/L, pH is controlled at 6.5～7.8.
5. technique according to claim 2, is characterized in that fermention medium component is: peptone 8～15g/L, yeast extract paste 3～8g/L, NaCl4～8g/L, glucose 35～70g/L, CaCO
310～15g/L, K
2hPO
40.8～1.5g/L, MgSO
47H
2o0.1～0.6g/L, ZnSO
47H
2o0.005～0.015g/L, MnCl
24H
2o0.001～0.005g/L, pH remains on 6.5～7.8; Fermentor tank liquid amount is 20～45% of fermentor tank volume.
6. technique according to claim 2, is characterized in that described feedback supplement adds for carrying out stage by stage feed supplement stream, and fermentation is carried out 16～26h and started current adding substrate A, and instructs bottoms stream acceleration with pH value in fermented liquid, makes pH value remain on 6.5～7.8; Carry out 12～24h in fermentation and start, current adding substrate B, and instruct bottoms stream acceleration with the concentration of glucose, make glucose concn in fermentor tank be controlled at 50～70g/L, fermentation 32～44h; Wherein substrate A composition is: CaCO
355～85g/L, (NH
4)
2hPO
450～80g/L; Substrate B composition is: glucose 200～400g/L, yeast extract paste 50～80g/L, K
2hPO
44.5～8.5g/L, MgSO
47H
2o0.57～3.5g/L.
7. technique according to claim 2, is characterized in that controlling temperature at 36～42 DEG C in fermentation culture process; Pass into sterile air, keep air flow at 3～6L/ (Lmin) to control dissolved oxygen 30～45%; Control rotating speed at 140～200rpm.
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| CN104232525A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus coagulans preparation |
| CN104738093A (en) * | 2015-03-25 | 2015-07-01 | 南京工业大学 | Preparation method of bacillus coagulans bacterial suspension |
| CN104974966B (en) * | 2015-07-22 | 2017-12-12 | 江南大学 | A kind of bacillus coagulans and its fermentation process in high density and dry bacterium powder preparation method |
| CN105112326B (en) * | 2015-08-19 | 2019-08-20 | 华南理工大学 | A kind of bacillus and its high-density cultivation method |
| CN105695304B (en) * | 2016-03-31 | 2018-02-09 | 昆明三正生物科技（集团）有限公司 | A kind of bacillus coagulans fast culture tank |
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