CN102660482B - Corynebacterium pekinense and application thereof - Google Patents
Corynebacterium pekinense and application thereof Download PDFInfo
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- CN102660482B CN102660482B CN 201210152838 CN201210152838A CN102660482B CN 102660482 B CN102660482 B CN 102660482B CN 201210152838 CN201210152838 CN 201210152838 CN 201210152838 A CN201210152838 A CN 201210152838A CN 102660482 B CN102660482 B CN 102660482B
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Abstract
The invention relates to the field of microorganisms and discloses Corynebacterium pekinense. The collection number of the Corynebacterium pekinense is CGMCC No. 5923. The Corynebacterium pekinense MHZ-0111 of which the collection number is CGMCC No. 5923 is obtained by performing ultraviolet radiation on wild type Corynebacterium pekinense AS 1.299 which is taken as a starting strain, and screening by using a neomycin-containing culture medium; and the Corynebacterium pekinense can greatly reduce the yield of lactic acid in the fermentation process, particularly in the fermentation of L-glutamic acid, so that the problem about the inhibition of the lactic acid is solved, and the production of a final metabolite is facilitated. Therefore, the Corynebacterium pekinense of which the collection number is CGMCC No. 5923 can be widely applied to the fermentation to reduce the lactic acid.
Description
Technical field
The present invention relates to microorganism field, relate to a kind of Beijing rod bacillus and application thereof specifically.
Background technology
Microbial fermentation namely refers to utilize microorganism, under suitable condition, raw material is converted into the process of the product that necessary for human wants through specific pathways metabolism.Microbial fermentation is divided into aerobic fermentation and anaerobically fermenting, industrial fermentation at present nearly all is to be raw material with saccharic (mainly being dextrose plus saccharose), utilize the microbial metabolism approach to synthesize the product useful to the mankind, most of in all fermentations is aerobic fermentation.
No matter aerobic fermentation or anaerobically fermenting, glycolysis-is the total pathways metabolism of Institute of Micro-biology, and microorganism all can experience the process of glucose → 6-glucose 1-phosphate1-→ glyceraldehyde 3-phosphate → pyruvic acid, and pyruvic acid is glucolytic inevitable outcome, if further fermentation can form multiple product.For example L-L-glutamic acid (amino acid) fermentation, 2,3-butyleneglycol fermentation and nisin fermentation etc., be exactly to generate acetyl-CoA by pyruvic acid in the L-glutamic acid fermentation, be condensed into citric acid with oxaloacetic acid then, enter the krebs cycle pathway and generate α-Tong Wuersuan, and then generate L-L-glutamic acid; Be to generate the 3-oxobutanol by pyruvic acid in 2, the 3-butyleneglycol fermentation, and then generate 2,3-butyleneglycol; And nisin also is to transform generation by pyruvic acid through multistep after the experience glycolysis-.
Yet, the limited situation of dissolved oxygen very easily occurs in some aerobic fermentation processes, in case the dissolved oxygen deficiency, pyruvic acid will enter the lactic acid metabolism approach, generates by product-lactic acid, and can not change into other materials such as acetyl-CoA, the lactic acid accumulation also surpasses certain limit, will suppress the generation of final product, influences the High-efficient Production of product, therefore can in the aerobic fermentation process, reduce lactic acid production, be conducive to the generation of final product.Simultaneously, in some anaerobic fermentation process, may also need suitable reduction lactic acid to reach some purpose in the production.
Therefore, in the microbial fermentation field, filtering out the novel strain that can reduce lactic acid production during the fermentation has very important meaning to microbial fermentation.But, still there is not the report of this class bacterial strain in the prior art, particularly be applicable to the report that falls lactobacillus strain of amino acid fermentation.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of Beijing rod bacillus and application thereof, make described Beijing rod bacillus can reduce the output of lactic acid in the fermenting process, be applied in the microbial fermentation.
For achieving the above object, the present invention with wild-type Beijing rod bacillus AS 1.299(available from Institute of Microorganism, Academia Sinica) be starting strain, by ultraviolet ray it is carried out physical mutagenesis, to be coated on the substratum that contains Xin Meisu through the suspension bacteria liquid of the rod bacillus AS of the Beijing behind the uv irradiating 1.299 then, filter out the bacterial strain with neomycin resistance from containing the Xin Meisu substratum, called after bacterial strain MHZ-0111.
The present invention carries out physio-biochemical characteristics according to the method for the elegant pearl in east, Cai Miaoying chief editor " common bacteria system identification handbook " to it with the above-mentioned bacterial strain MHZ-0111 that filters out and detects, and detected result is consistent with wild-type Beijing rod bacillus.In addition, analyze through the 16S DNA detection, bacterial strain MHZ-0111 and wild-type Beijing rod bacillus homology in conjunction with physiological and biochemical property, identify that it is Beijing rod bacillus up to 99%.
Beijing rod bacillus MHZ-0111 of the present invention has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 20th, 2012, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.5923.
The L-glutamic acid fermentation simultaneous test of Beijing rod bacillus MHZ-0111 of the present invention and wild-type Beijing rod bacillus AS 1.299 shows, it can significantly reduce lactic acid production, the Lactic Acid from Fermentation Broth content that Beijing of the present invention rod bacillus MHZ-0111 fermentation obtains is 4g/L, and the Lactic Acid from Fermentation Broth content that the rod bacillus AS1.299 fermentation of wild-type Beijing obtains is 15g/L, and lactic acid production has reduced by 73.3%.
Xin Meisu is a kind of aminoglycoside antibiotics, disturb bacterioprotein synthetic on the intracellular rrna by being incorporated into, make it the protein of resulting anomaly, and the bacterial cell membrane permeability is increased, cause in the cell important physiologically substance to leak outside and present germicidal action, stronger to bacterium germicidal action stationary phase, be sterilization stationary phase class microbiotic.After wild strain obtains neomycin resistance, may be because the protein opposing Xin Meisu of Beijing of the present invention rod bacillus MHZ-0111 resulting anomaly, these unusual protein cause the metabolism synthetase series to change the feedback inhibition of some product, perhaps inhibition to a certain degree the activity of serum lactic dehydrogenase, thereby cause the lactic acid production of mutant strain to decline to a great extent.
As well known to those skilled in the art, no matter be that anaerobically fermenting is produced the by product lactic acid of taking out of when producing other products in lactic acid or the aerobic fermentation specially, all need microorganism through the glycolysis-pathways metabolism, generated through the serum lactic dehydrogenase effect by pyruvic acid, above-mentioned test-results shows, Beijing of the present invention rod bacillus has the L-glutamic acid fermentation in the activity, particularly aerobic fermentation that reduces lactic acid production in the fermenting process.
Therefore, Beijing rod bacillus MHZ-0111 of the present invention can be applied to reduce lactic acid production in the fermentation.Wherein said fermentation is preferably aerobic fermentation, and described aerobic fermentation is preferably amino acid fermentation, and described amino acid is preferably L-L-glutamic acid.
In addition, the present invention also provides a kind of L-glutamic acid fermentation method, be that Beijing rod bacillus of CGMCCNo.5923 is inoculated in seed culture medium and expands numerously with deposit number, the culture that will expand then after numerous changes the fermention medium fermentation over to, and keeping the pH value between yeast phase is 6.8-7.4.
Wherein, described seed culture medium is well known in the art, and the present invention is preferably by 25g/L glucose, 5g/L urea, 30g/L corn steep liquor, 1g/L KH
2PO
4, 0.4g/L MgSO
47H
2O, 2mg/L ferrous sulfate, 2mg/L manganous sulfate are formed, and the pH value is 6.8-7.4.
Described fermention medium is well known in the art, and the present invention is preferably by 80g/L glucose, 20g/L urea, 8g/L corn steep liquor, 1.2g/L KH
2PO
4, 0.7g/L MgSO
47H
2O, 2mg/L ferrous sulfate, 2mg/L manganous sulfate, 200ug/L VitB1,2mL/L 1% phenolsulfonphthalein are formed, and the pH value is 6.8-7.4.
Culture after expansion of the present invention is numerous and the volume ratio of fermention medium are preferably 1: 10.
By above technical scheme as can be known, deposit number of the present invention is that Beijing rod bacillus MHZ-0111 of CGMCC No.5923 is starting strain with wild-type Beijing rod bacillus AS 1.299, behind uv irradiating, obtain by containing the screening of Xin Meisu substratum, it can significantly reduce the output of lactic acid in the fermenting process, particularly in the L-glutamic acid fermentation, thereby improve the problem that lactic acid suppresses, be beneficial to the production of final meta-bolites.Therefore, deposit number of the present invention is the generation that reduces lactic acid during Beijing rod bacillus of CGMCC No.5923 can be widely used in fermenting.
The explanation of biological preservation information
Classification name: Beijing rod bacillus, Corynebacterium pekinense. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 20th, 2012, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.5923.
Embodiment
The embodiment of the invention discloses a kind of Beijing rod bacillus and application thereof.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Bacterial strain of the present invention is described by preferred embodiment, and the related personnel obviously can change or suitably change and combination product as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
In order further to understand the present invention, the present invention is described in detail below in conjunction with embodiment.
Embodiment 1: the screening of Beijing rod bacillus MHZ-0111 of the present invention
Starting strain-wild-type Beijing rod bacillus AS 1.299 is seeded in the liquid nutrient medium 30 ℃ cultivates the bacteria suspension that obtained to be in logarithmic phase in 11 hours, then this bacteria suspension concentration is adjusted to every milliliter and contains and have an appointment 10
8Individual cell is placed on ultraviolet ray and shone 35 seconds down, to be coated on the perfect medium flat board that contains the 0.5mg/L Xin Meisu through the bacteria suspension of uviolizing at last, 30 ℃ are continued to cultivate 2 days, according to feature picking list bacterium colonies such as colonial morphology, colors, and called after MHZ-0111.
Wherein, the liquid culture based formulas is as follows: glucose 25g/L, urea 5g/L, corn steep liquor 30g/L, KH
2PO
41g/L, MgSO
47H
2O 0.4g/L, ferrous sulfate 2mg/L, manganous sulfate 2mg/L are settled to 1000ml with dissolved in distilled water and after regulating pH to 6.8-7.4.
Perfect medium is composed as follows: Xin Meisu 0.5mg/L, extractum carnis 10g, peptone 10g, NaCl5g, glucose 5g, agar 18g are settled to 1000ml with dissolved in distilled water and after regulating pH to 6.8-7.4.
The above-mentioned bacterial strain MHZ-0111 that filters out is carried out physio-biochemical characteristics according to the method for the elegant pearl in east, Cai Miaoying chief editor " common bacteria system identification handbook " to it detect, detected result is consistent with wild-type Beijing rod bacillus.In addition, analyze through the 16S DNA detection, bacterial strain MHZ-0111 and wild-type Beijing rod bacillus homology in conjunction with physiological and biochemical property, identify that it is Beijing rod bacillus up to 99%.
Embodiment 2:L-glutamic acid fermentation lactic acid production simultaneous test
1, fermentation process
Beijing of the present invention rod bacillus MHZ-0111 is seeded in expand in the seed culture medium of the following stated numerous, in 30 ℃ of shaking culture 11 hours, the gained culture is inoculated in the fermention medium of the following stated, the volume ratio of culture and fermention medium is 1: 10(those skilled in the art can suitably regulate according to the amount of the fermention medium in the actual production, this ratio is preferred proportion), 30 ℃ of shaking culture are to no longer producing (being generally about 40 hours) till the L-L-glutamic acid.In fermention medium during shaking culture, add an amount of urea according to phenolsulfonphthalein as the medium pH changing conditions of indicator indication, keep pH value in the fermention medium between 6.8-7.4.
Seed culture medium:
25g/L glucose, 5g/L urea, 30g/L corn steep liquor, 1g/L KH
2PO
4, 0.4g/LMgSO
47H
2O, 2mg/L ferrous sulfate, 2mg/L manganous sulfate are with dissolved in distilled water and regulate pH to 6.8-7.4.
Fermention medium: 80g/L glucose, 20g/L urea, 8g/L corn steep liquor, 1.2g/L KH
2PO
4, 0.7g/L MgSO
47H
2O, 2mg/L ferrous sulfate, 2mg/L manganous sulfate, 200ug/L VitB1,2mL/L 1% phenolsulfonphthalein are with dissolved in distilled water and regulate pH to 6.8-7.4.
2, L-glutamic acid fermentation simultaneous test
Beijing of the present invention rod bacillus MHZ-0111 and wild-type Beijing rod bacillus AS 1.299 are fermented simultaneously according to aforementioned production method, the yeasting of two kinds of bacterial strains, inoculum size are all identical, and two kinds of lactic acid contents that bacterial strain synthesized are measured by the bio-sensing instrument in the back that stops to ferment.
The result shows, wild-type Beijing synthetic lactic acid content of rod bacillus AS 1.299 fermentations is 15g/L, the synthetic lactic acid content of Beijing rod bacillus MHZ-0111 fermentation of the present invention only is 4g/L, and the mutant strain lactic acid production has reduced by 73.3% than starting strain AS 1.299.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (6)
1. Beijing rod bacillus is characterized in that deposit number is CGMCC No.5923.
2. deposit number is Beijing rod bacillus of CGMCC No.5923 reduces lactic acid production in the L-glutamic acid fermentation application.
3. L-glutamic acid fermentation method, it is characterized in that, be that Beijing rod bacillus of CGMCC No.5923 is inoculated in seed culture medium and expands numerously with deposit number, the culture that will expand then after numerous changes the fermention medium fermentation over to, and keeping the pH value between yeast phase is 6.8-7.4.
4. according to the described fermentation process of claim 3, it is characterized in that described seed culture medium is by 25g/L glucose, 5g/L urea, 30g/L corn steep liquor, 1g/L KH
2PO
4, 0.4g/L MgSO
47H
2O, 2mg/L ferrous sulfate, 2mg/L manganous sulfate are formed, and the pH value is 6.8-7.4.
5. according to the described fermentation process of claim 3, it is characterized in that described fermention medium is by 80g/L glucose, 20g/L urea, 8g/L corn steep liquor, 1.2g/L KH
2PO
4, 0.7g/L MgSO
47H
2O, 2mg/L ferrous sulfate, 2mg/L manganous sulfate, 200ug/L VitB1,2mL/L1% phenolsulfonphthalein are formed, and the pH value is 6.8-7.4.
6. according to the described fermentation process of claim 3, it is characterized in that the culture after described expansion is numerous and the volume ratio of fermention medium are 1:10.
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| CN 201210152838 CN102660482B (en) | 2012-05-17 | 2012-05-17 | Corynebacterium pekinense and application thereof |
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| CN103642766B (en) * | 2013-07-02 | 2016-12-28 | 廊坊梅花生物技术开发有限公司 | Albumen, DNA molecular, convert host and this conversion host for the method producing L valine containing this DNA |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1225946A (en) * | 1998-12-28 | 1999-08-18 | 山东大学 | Glutamine fermentation production process |
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2012
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1225946A (en) * | 1998-12-28 | 1999-08-18 | 山东大学 | Glutamine fermentation production process |
Non-Patent Citations (4)
| Title |
|---|
| 北京棒杆菌AS1.299发酵生产谷氨酸的研究;王冠蕾等;《承德石油高等专科学校学报》;20071231;第9卷(第4期);第45－47页 * |
| 浅谈棒杆菌的谷氨酸分泌机理;高德富等;《发酵科技通讯》;20040430;第33卷(第2期);第15－16页 * |
| 王冠蕾等.北京棒杆菌AS1.299发酵生产谷氨酸的研究.《承德石油高等专科学校学报》.2007,第9卷(第4期),第45－47页. |
| 高德富等.浅谈棒杆菌的谷氨酸分泌机理.《发酵科技通讯》.2004,第33卷(第2期),第15－16页. |
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Owner name: XINJIANG PLUM FLOWER AMINO ACID CO., LTD. Free format text: FORMER OWNER: TONGLIAO MEIHUA BIOTECHNOLOGY CO., LTD. Effective date: 20131204 |
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Effective date of registration: 20131204 Address after: 831300 the Xinjiang Uygur Autonomous Region Wujiaqu City People's Road No. 3092 Patentee after: Xinjiang Meihua Amino Acid Co., Ltd. Address before: Figure 028000 the Inner Mongolia Autonomous Region Muli town Horqin district Tongliao City Patentee before: Tongliao Meihua Biotechnology Co., Ltd. |