CN103361288B - Methylotrophic bacillus and application thereof - Google Patents
Methylotrophic bacillus and application thereof Download PDFInfo
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- CN103361288B CN103361288B CN201310265937.7A CN201310265937A CN103361288B CN 103361288 B CN103361288 B CN 103361288B CN 201310265937 A CN201310265937 A CN 201310265937A CN 103361288 B CN103361288 B CN 103361288B
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Abstract
The invention provides methylotrophic bacillus which can produce acetoin at high yield. The methylotrophic bacillus is collected in the China General Microbiological Culture Collection Center (CGMCC) and has the collection number of CGMCCC No.7485. When the methylotrophic bacillus is fermented under a certain conditions, acetoin with the concentration of 15.71g/L can be obtained. The production capacity of most of the reported acetoin producing bacteria is lower than or equivalent to that of the methylotrophic bacillus CGMCCC No.7485; however, according to the strain, except the glucose concentration is preliminarily optimized, other culture conditions and medium components are not subjected to breeding transformation. Thus, after the methylotrophic bacillus is subjected to further breeding transformation and condition optimization, the acetoin yield is further improved. Therefore, the methylotrophic bacillus has wide application prospects.
Description
Technical field
The invention belongs to microorganism and fermentation engineering field, particularly, relate to a kind of Methylotrophic genus bacillus.
Background technology
Acetoin have another name called 3-Hydroxybutanone, vinegar drone, methyl vinyl methyl alcohol.Being present in dairy products and some fruit, is a kind of widely used flavouring agent, and it is edible that China GB 2760-86 specifies that it allows.Acetoin is extremely extensive as fragrance applications scope, and consumption is also comparatively large, and its topmost effect is the production for spices such as cream, dairy products, white wine blending, Yoghourt and strawberry types.In addition, 3-Hydroxybutanone as a kind of platform chemicals, can also be widely used in other numerous industries, and within 2004, USDOE is classified as one of platform chemicals of 30 kinds of preferential developments utilizations.
Domestic and international production acetoin is mainly through chemical synthesis at present, but it exists quality safety worry as flavouring agent.And fermentable production acetoin, there is the advantages such as bioprocess technology environmental friendliness, product safety and raw material can regenerate, the dominant direction that following acetoin is produced will be become.Domesticly at present prepared by acetoin to biology mainly concentrate on the laboratory study development phases such as the optimization of the seed selection of acetoin producing strains and conditions of flask fermentation, not yet enter commercial scale and produce.The producing strains reported is subtilis etc. mainly, yet there are no the relevant report that Methylotrophic genus bacillus prepares acetoin, and the present invention is that microbe fermentation method is prepared acetoin and provided new resources.
Summary of the invention
The present invention screens from high temperature Daqu can the bacterial strain of high yield acetoin, and it has been carried out to the qualification of morphology and genetics aspect, by this bacterium called after Methylotrophic genus bacillus (Bacillus methylotrophicus), and preservation has been carried out to it, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on April 18th, 2013, and deposit number is: CGMCC No.7485.
The invention provides a kind of Methylotrophic genus bacillus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is: CGMCC No.7485.
The present invention also provides a kind of acetoin producing strains, and described acetoin producing strains is Methylotrophic genus bacillus according to claim 1.
The present invention also provides the above-mentioned application of Methylotrophic genus bacillus in preparation acetoin.
Further, the concentration of the acetoin prepared described in is 15.71g/L.
Further, the step of described application is: be that fermenting substrate Methylotrophic genus bacillus is to produce acetoin with glucose.
Further, the concentration of described glucose is 20-60g/L.
Further, the step of described application is: Methylotrophic genus bacillus is cultured to logarithmic phase with containing in the seed culture medium of 20-60g/L glucose, is seeded in the fermention medium containing 20-60g/L glucose, carries out shaking culture.
Further, the concentration of described glucose is 40g/L.
Apply Methylotrophic genus bacillus of the present invention to ferment under certain condition, the acetoin that concentration is 15.71g/L can be obtained.Through contrast, major part reported acetoin producing strains (bacterial strain had be through condition optimizing or breeding transformation) throughput lower than or be equivalent to Methylotrophic genus bacillus CGMCC No.7485 of the present invention.Methylotrophic genus bacillus CGMCC No.7485 in the present invention is except having carried out except initial optimization glucose concn, and other culture condition and substratum composition are all the conventional selections of genus bacillus, also not yet transform through breeding.Can infer thus, Methylotrophic genus bacillus of the present invention is after further breeding transformation and condition optimizing, and acetoin output will be further enhanced, and therefore, Methylotrophic genus bacillus of the present invention has broad application prospects.
Accompanying drawing explanation
Fig. 1 is the flat-plate bacterial colony photo of Methylotrophic genus bacillus CGMCC No.7485 bacterial strain (Bacillus methylotrophicus strain) of the present invention;
Fig. 2 is the gramstaining photo of Methylotrophic genus bacillus CGMCC No.7485 bacterial strain (Bacillus methylotrophicus strain) of the present invention;
Fig. 3 is the spore staining photo of Methylotrophic genus bacillus CGMCC No.7485 bacterial strain (Bacillus methylotrophicus strain) of the present invention;
Fig. 4 is Methylotrophic genus bacillus CGMCC No.7485 bacterial strain (Bacillus methylotrophicus strain) 16Sr DNA cloning bands of a spectrum of the present invention;
Fig. 5 is that in the fermention medium of Methylotrophic genus bacillus CGMCC No.7485 bacterial strain of the present invention, glucose content affects lab diagram to acetoin output.
Embodiment
Embodiment 1: the screening of bacterial strain
High temperature Daqu (taking from Anhui two-wheel employment limited liability company) is ground to form powdery, accurately takes 25g and join in the Erlenmeyer flask of the stroke-physiological saline solution containing 200ml.The granulated glass sphere adding sterilizing is put into take out after 30min is broken up in concussion under shaking table (180r/min) room temperature and is left standstill 30min.Get 1mL supernatant liquor and add 9mL stroke-physiological saline solution, being mixed with the bacteria suspension that volumetric concentration is 10-1.The like, being mixed with volumetric concentration is 10
-3, 10
-410
-7bacteria suspension, (preparation method of isolation medium is: by extractum carnis 3g/L, peptone 10g/L, NaCl 5g/L to be spread evenly across the isolation medium of cooling overnight, agar strip 20g/L, water 1000mL, nystatin 0.01g/L mix, adjust pH to 7.0,121 DEG C, sterilizing 20min.) on flat board, the flat board inoculated is placed in 37 DEG C of constant incubators and cultivates 48h.Chosen by bacterial strains different for all bacterium colonies and be purified to purebred, 4 DEG C of refrigerator inclined-planes are preserved.
Embodiment 2: the fermentation of bacterial strain
One, the fermentation of bacterial strain
From picking bacterial strain inclined-plane to seed culture medium, (preparation method of seed culture medium is: by extractum carnis 20.0g, glucose 20.0g, K
2hPO
41.0g, (NH
4)
2sO
41.0g, MgSO
47H
2o 1.0g, NaCl 0.5g, FeSO
47H
2o0.01g mixes, and adding distil water is adjusted to 1L, adjusts pH to 7.0,121 DEG C, sterilizing 20min.) in be cultured to logarithmic phase, will cultivate the bacterial strain of logarithmic phase with seed culture medium, (preparation method of fermention medium is: by extractum carnis 20.0g, glucose 20.0g, K to join fermention medium by 5% inoculum size
2hPO
41.0g, (NH
4)
2sO
41.0g, MgSO
47H
2o1.0g, NaCl 0.5g, FeSO
47H
2o 0.01g mixes, and adding distil water is adjusted to 1L, adjusts pH to 7.0,121 DEG C, sterilizing 20min.) in, be then placed in 37 DEG C, shaking culture 72h under 150r/min condition.Collect fermented liquid in order to detecting.
Two, in fermention medium, the impact experiment of glucose content on acetoin output is as follows:
In seed culture medium (filling a prescription the same), logarithmic phase is cultured to from picking bacterial strain inclined-plane, the bacterial strain of logarithmic phase will be cultivated with seed culture medium, in the fermention medium joining containing different glucose by 5% inoculum size (except glucose, other component prescription is the same), wherein glucose concn is respectively 10g/L, 20g/L, 40g/L, 60g/L, 80g/L, 100g/L, is then placed in 37 DEG C, shaking culture 72h under 150r/min condition.Collect fermented liquid in order to detecting.As shown in Figure 5, when glucose concn is 40g/L, acetoin content is up to 15.71g/L.
Embodiment 3: the detection of acetoin
Gas chromatographic detection condition: the fermented liquid of each different strains embodiment 2 obtained dilutes 5 times respectively, gets 2 μ L sample introductions afterwards respectively and carries out gas chromatographic analysis, detect the output of acetoin.After testing, that acetoin output is the highest is 15.71g/L.
Gas chromatograph parameters: white wine chromatographic column specially CP-Wax 57C, internal diameter: 250.00 μm, length: 50.0m, thickness of liquid film: 0.20 μm, dead time: 2.983min, column temperature: 60 DEG C keep 5min, 5 DEG C/min temperature programmings to 140 DEG C to keep 2min, injector temperatures: 150 DEG C, detector temperature: 160 DEG C, carrier gas: N2, pressure 90kPa, tail blow before post: 30mL/min, splitting ratio: 1:30, air: 50kPa, hydrogen: 50kPa.
Embodiment 4: the qualification of bacterial strain
Select the bacterial strain (namely output is the bacterial strain of 15.71g/L) that in embodiment 3, acetoin output is the highest, its flat-plate bacterial colony photo, gramstaining photo and spore staining photo are respectively see Fig. 1 to Fig. 3.With its genome for template, the universal primer (16S27f and 16S1492r) of the 16SrDNA of bacterium is utilized to carry out pcr amplification.By amplified production with 1% agarose gel electrophoresis carry out genomic dna separation, there is a bright band in the pcr amplification product of this bacterial strain, its molecular size range is about 1500bp.See Fig. 4.Amplification is delivered to Sangon Biotech (Shanghai) Co., Ltd. check order, sequencing result is shown in sequence 1 in sequence table.Above-mentioned sequence is contrasted by blast program and Genbank nucleotide database, finds that the 16Sr DNA sequence dna similarity of itself and Methylotrophic genus bacillus (Bacillus methylotrophicus strain) is 99%.
It is delivered to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation on April 18th, 2013, and deposit number is: CGMCC No.7485.
As can be seen from (Japanese plum ripple etc., microorganisms producing 3-Hydroxybutanone progress [J], biological processing, 2011:9 (6) in bibliographical information; Wang Meng, produces the preliminary study [D] of acetoin subtilis metabolic engineering, University Of Tianjin, 2012.6; Yin Minghao, the seed selection [D] of high yield 3-Hydroxybutanone subtilis, University Of Qingdao, 2011.5), major part reported acetoin producing strains (bacterial strain had be through condition optimizing or breeding transformation) throughput lower than or be equivalent to Methylotrophic genus bacillus CGMCC No.7485 of the present invention.Methylotrophic genus bacillus CGMCCNo.7485 in the present invention is except having carried out except initial optimization glucose concn, and other culture condition and substratum composition are all the conventional selections of genus bacillus, also not yet transform through breeding.Can infer thus, Methylotrophic genus bacillus of the present invention is after further breeding transformation and condition optimizing, and acetoin output will be further enhanced, and therefore, Methylotrophic genus bacillus of the present invention has broad application prospects.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
<110> Anhui Polytechnic University
<120> Methylotrophic genus bacillus and application thereof
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1459
<212> DNA
<213> Methylotrophic genus bacillus
<400> 1
gcggagtctg ctgacatatc tgtcacgtcg tagcgagaca gatgggagct tgctccctga 60
tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 120
cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt 180
ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240
ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360
tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420
gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540
gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600
gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660
tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720
gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780
cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840
acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960
caggtcttga catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt 1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200
cacacgtgct acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca 1260
caaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct 1320
agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380
tcacaccacg agagtttgta acacccgaag tcggtgaggt aactagaagg agccagcgcg 1440
ctatatagag acaaccccg 1459
Claims (7)
1. a Methylotrophic genus bacillus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is: CGMCC No.7485.
2. the application of the Methylotrophic genus bacillus described in claim 1 in preparation acetoin.
3., according to the application described in claim 2, it is characterized by: described in the concentration of acetoin for preparing be 15.71g/L.
4. according to the application described in claim 2, it is characterized by: the step of described application is: be that fermenting substrate Methylotrophic genus bacillus is to produce acetoin with glucose.
5. according to the application described in claim 4, it is characterized by: the concentration of described glucose is 20-60g/L.
6. according to the application described in claim 5, it is characterized by: the step of described application is: be that the seed culture medium of Methylotrophic genus bacillus containing 20-60g/L glucose is cultured to logarithmic phase, the Methylotrophic genus bacillus being cultured to logarithmic phase is seeded in the fermention medium containing 20-60g/L glucose, carries out shaking culture.
7. the application according to claim 5 or 6, is characterized by: in described fermention medium, the concentration of glucose is 40g/L.
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| CN104017750B (en) * | 2014-04-14 | 2016-03-02 | 青岛海大生物集团有限公司 | A kind of salt tolerance Methylotrophic Bacillus strain LJ and application thereof |
| CN105112327A (en) * | 2015-08-20 | 2015-12-02 | 郑磊 | Method for separating bacilli and method for manufacturing fermented tea by aid of bacilli |
| CN110923163B (en) * | 2019-11-22 | 2021-06-25 | 郑州轻工业大学 | Bacillus methylotrophicus and application thereof |
| CN111235067B (en) * | 2020-03-16 | 2021-10-01 | 河南仰韶酒业有限公司 | White spirit enhanced yeast for making hard liquor, preparation method and application thereof |
| CN113430147B (en) * | 2021-07-30 | 2022-11-01 | 千禾味业食品股份有限公司 | Bacillus villagens QH-20011 with low pH tolerance and application thereof |
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