CN103436521A - High cephalosporin C yield bacterial breeding method - Google Patents
High cephalosporin C yield bacterial breeding method Download PDFInfo
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- CN103436521A CN103436521A CN2013102680541A CN201310268054A CN103436521A CN 103436521 A CN103436521 A CN 103436521A CN 2013102680541 A CN2013102680541 A CN 2013102680541A CN 201310268054 A CN201310268054 A CN 201310268054A CN 103436521 A CN103436521 A CN 103436521A
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Abstract
The present invention discloses a high cephalosporin C yield bacterial breeding method, and relates to the field of microorganism bacterial breeding. According to the present invention, a metabolic regulation breeding method is adopted to overcome a saturation effect of bacterial on a mutagen so as to easily achieve mutation bacterial generation; a semi-continuous fermentation method is adopted to make bacterial with excellent characteristics rapidly establish growth advantages so as to increase chances of screening of excellent bacterial; and the bacterial breeding method is simple, fast and efficient.
Description
Technical field
The present invention relates to microorganism strains seed selection field, especially a kind of selection of cephalosporin bacterial strain.
Background technology
Cephalosporin is a kind of important medicine intermediate, through chemical method or enzymatic cleavage, be important cephalosporin analog antibiotic parent nucleus---7-amino-cephalosporanic acid (being 7-ACA), it can synthesize the cynnematin series products that a series of toxicity is little, effect is good, as ceftriaxone sodium, Cefixime Micronized, cephalofruxin, Cefamandole etc.Semi-synthetic cephalosporins developed into for the 4th generation now, and the clinical demand amount is large, and the huge market space is arranged.
Cephalosporin is to be produced by fermentation by microorganisms such as cephalosporium acremoniums, this bacterium is the most separated early than 1945, fermentation level has improved more than 4000 times, this too busy to get away strain improvement worker's hard working more than having brought up to 40000ug/mL by initial 10ug/mL.The selection of cephalosporin bacterial classification comprises traditional method and molecular biology method.Ultraviolet mutagenesis in traditional method, microwave irradiation, chemical mutagen (acridine orange, nitrosoguanidine etc.) mutagenesis etc. are widely used in the cephalosporin strain selection, improved fermentation level, but along with the raising of bacterial strain to traditional mutagenic compound resistance, effect is more and more poorer, has affected the seed selection process of bacterial classification; The molecular biology method purpose is strong, and effect is better, but the method complicated operation spends highlyer, and the gene imported in fermentation production process is easily lost, and has influence on fermentation yield.
Summary of the invention
A kind of method that the purpose of this invention is to provide easy, bacterial strain of metabolic regulation seed selection fast and efficiently.
The objective of the invention is to be achieved through the following technical solutions:
A kind of cephalosporin superior strain selection comprises the following steps:
1) get-80 ℃ of freezing mycelia of cephalosporium acremonium, 20～40 ℃ of quick-thawings, the inoculum size with 1～6.7% is inoculated in seed culture medium, shaking table shaking culture 2～6 days;
2) be inoculated in fermention medium shaking table shaking culture 4～10 days with 1～33.3% inoculum size;
3) with 1～33.3% inoculum size, be inoculated in fermention medium again, shaking table shaking culture 4～10 days, this process repeats 7 times;
4) take off shaking flask from shaking table, with physiological saline, carry out 10 times of serial gradient dilutions, with red-cell count plate counting, find cephalosporium acremonium arthrospore quantity 10
6～10
8extent of dilution 10
-4;
5) centered by the extent of dilution of step 4, choose 10
-2, 10
-3, 10
-4, 10
-5, 10
-6totally 5 gradient bacteria suspensions evenly are coated with lipase and identify dull and stereotypedly, and each gradient arranges 3 repetitions, and flat board is placed at 20～30 ℃ of temperature to standing cultivation 10～20 days;
6) cultivation is placed in cultured flat board under the 350nm ultraviolet lamp after finishing, with toothpick picking fluorescent ring, be of moderate size and sharply marginated bacterium colony, be inoculated on normal flat board, at 20～30 ℃ of temperature, standing cultivation is 10～20 days, prepares the lawn of 1～2cm * 4～6cm;
7) previously prepared cephalosporin sensitive organism plate culture medium is melted, accessing wherein final concentration when temperature is down to 45 ℃ of left and right is 10
8cFU/mL left and right sensitive organism, pour plate, solidify rear standby;
8) punch tool that is 5mm with diameter punches on the lawn of step 6 preparation, prepares agar column, with this agar column of transfering loop picking, is inoculated on the flat board of step 7, is placed at the temperature of 30～40 ℃ standing cultivation 1～2 day;
9) after cultivate finishing, with vernier callipers, measure antibacterial circle diameter, select bacterial strain that diameter is greater than 35mm and carry out another flat board of taking turns and sieve again test;
10) after too much the flat board of round sieves again, filter out 10 larger bacterial strains of inhibition zone and prepare freezing mycelia, carrying out shaking flask according to the method for step 1 and step 2 sieves again, measure the content that fermentation culture finishes cephalosporin in secondary fermentation liquid, carry out repeatedly that inclined-plane goes down to posterity, multiple batches of bacterial strain genetic stability and production performance test, filter out bacterial strain 1～2 strain that fermentation level is the highest.
The formula of described seed culture medium is: corn steep liquor 3.0～5.0%, glucose 3.0～5.0%, sucrose 2.0～4.0%, calcium carbonate 0.2～1.0%, pH6～8.
Described fermention medium, its formula is: corn steep liquor 3.0～5.0%, groundnut meal 3.0～5.0%, sucrose 2.0～4.0%, calcium sulfate 0.2～1.0%, peptone 1.0～3.0%, ammonium sulfate 1.0～2.0%, ferrous sulfate 0.01～0.1%, calcium carbonate 0.2～0.5%, soya-bean oil 5～20%, pH6～8.
Described lipase is identified dull and stereotyped, its formula is: ammonium sulfate 0.05～1.0%, potassium primary phosphate 0.05～0.5%, vitriolate of tartar 0.1～0.5%, magnesium sulfate heptahydrate 0.01～0.2%, sweet oil 1.0～4.0%, rhodamine B 0.01～0.03%, polyvinyl alcohol 0.01～0.3%, agar 1.0～2.0%, pH6～8.
Described cephalosporin sensitive organism plate culture medium, its formula is: yeast extract paste 0.1～1.5%, peptone 0.5～3.0%, sodium-chlor 0.1～1.5%, agar 1.0～2.0%, pH6～8.
Described normal plate culture medium formula is: sucrose 1.0～3.0%, yeast extract paste 0.1～0.8%, peptone 0.1～0.5%, ferrous sulfate 0.001～0.01%, sal epsom 0.01～0.06%, agar 1.0～2.0%, pH6～8.
The lawn size of described normal flat board is 1～2cm * 4～6cm.
Superiority of the present invention is:
1) adopt the method for metabolic regulation breeding, overcome the saturation effect of bacterial strain to mutagenic compound, be conducive to the generation of mutant strain;
2) adopt the method for semicontinuous fermentation, make the bacterial strain Rapid Establishment growth vigor of good character, increased the probability that screens strain excellent;
3) in the semicontinuous fermentation process, cephalosporin can constantly produce, and has both saved the step of adding the cephalosporin crystal, is conducive to again maintain high-caliber meta-bolites, make the strain growth advantage of anti-feedback inhibition more obvious, be convenient to the screening of spontaneous mutation and anti-feedback inhibition bacterial strain;
4) because metabolism seed selection bacterial strain substratum used is fermention medium, soya-bean oil wherein can constantly activate the lipase system of bacterial strain, promotes the generation of the metabolism and growth product of bacterial strain, and the bacterial strain that lipase activity is high can be set up rapidly growth vigor;
5) adopt method easy, ultraviolet ray excited generation fluorescence fast to screen the bacterial strain that lipase activity is high; Adopt the dull and stereotyped dual test of sensitive organism and agar column method can screen in a large number, fast the bacterial strain that production performance is high;
6) through above processing, just can break the mutagenic compound saturation effect of bacterial strain, make the bacterial strain that production performance is high, feedback inhibition weak, the soya-bean oil utilization ratio is high obtain enrichment, greatly improved high-performance strain selection efficiency.
Embodiment
Embodiment
1) get some previously prepared good cephalosporium acremoniums (Cephalosporium acremonium) deep cooling pipe bacterial strain from-80 ℃ of cryogenic refrigerators, 20～40 ℃ of quick-thawings, that draws that 1～2mL is inoculated into sterilizing in advance is equipped with 30～100mL seed culture medium (corn steep liquor 3.0～5.0%, glucose 3.0～5.0%, sucrose 2.0～4.0%, calcium carbonate 0.2～1.0%, pH6～8) in 500mL triangular flask, be placed on the shaking table of 20～30 ℃ 150～300rpm shaking culture 2～6 days.
That 2) gets that above-mentioned bacteria suspension 1～10mL is inoculated into sterilizing in advance is equipped with 30～100mL fermention medium (corn steep liquor 3.0～5.0%, groundnut meal 3.0～5.0%, sucrose 2.0～4.0%, calcium sulfate 0.2～1.0%, peptone 1.0～3.0%, ammonium sulfate 1.0～2.0%, ferrous sulfate 0.01～0.1%, calcium carbonate 0.2～0.5%, soya-bean oil 5～20%, pH6～8), in 500mL triangular flask, be placed on the shaking table of 20～30 ℃ 150～300rpm shaking culture 4～10 days.
3) the fermented liquid 1～10mL that gets step 2 is inoculated in the 500mL triangular flask that 30～100mL fermention medium is housed of sterilizing in advance, is placed on the shaking table of 20～30 ℃, and 150～300rpm shaking culture 4～10 days, this step repeats 7 times.
4) after cultivating end, from shaking table, take off shaking flask, with physiological saline, carry out 10 times of serial gradient dilutions, with red-cell count plate counting, find cephalosporium acremonium arthrospore quantity 10
6～10
8extent of dilution 10
-4.
5), centered by the extent of dilution of step 4, choose 5 gradients (10
-2, 10
-3, 10
-4, 10
-5, 10
-6) bacteria suspension evenly is coated with lipase and identifies dull and stereotyped (ammonium sulfate 0.05～1.0%, potassium primary phosphate 0.05～0.5%, vitriolate of tartar 0.1～0.5%, magnesium sulfate heptahydrate 0.01～0.2%, sweet oil 1.0～4.0%, rhodamine B 0.01～0.03%, polyvinyl alcohol 0.01～0.3%, agar 1.0～2.0%, pH6～8), each gradient arranges 3 repetitions, and flat board is placed at 20～30 ℃ of temperature to standing cultivation 10～20 days.
6) cultivation is placed in cultured flat board under the 350nm ultraviolet lamp after finishing, with toothpick picking fluorescent ring, be of moderate size and sharply marginated bacterium colony, be inoculated into normal flat board (sucrose 1.0～3.0%, yeast extract paste 0.1～0.8%, peptone 0.1～0.5%, ferrous sulfate 0.001～0.01%, sal epsom 0.01～0.06%, agar 1.0～2.0%, pH6～8) on, be placed at 20～30 ℃ of temperature standing cultivation 10～20 days, prepare the lawn of 1～2cm * 4～6cm.
7) by previously prepared cephalosporin sensitive organism plate culture medium (yeast extract paste 0.1～1.5%, peptone 0.5～3.0%, sodium-chlor 0.1～1.5%, agar 1.0～2.0%, pH6～8) melt, accessing wherein final concentration when temperature is down to 45 ℃ of left and right is 10
8the sensitive organism of CFU/mL left and right, pour plate, solidify rear standby.
8) punch tool that is 5mm with diameter punches on the lawn of step 6 preparation, prepares agar column, with this agar column of transfering loop picking, is inoculated on the flat board of step 7, is placed at the temperature of 30～40 ℃ standing cultivation 1～2 day.
9) after cultivate finishing, with vernier callipers, measure antibacterial circle diameter, select bacterial strain that diameter is greater than 35mm and carry out another flat board of taking turns and sieve again test.
10) after too much the flat board of round sieves again, filter out 10 larger bacterial strains of inhibition zone and prepare freezing mycelia, carrying out shaking flask according to the method for step 1 and step 2 sieves again, measure the content that fermentation culture finishes cephalosporin in secondary fermentation liquid, carry out repeatedly that inclined-plane goes down to posterity, multiple batches of bacterial strain genetic stability and production performance test, filter out bacterial strain 1～2 strain that fermentation level is the highest.
After processing by above test, the metabolic pathway of bacterial strain can change, and bacterial strain can obviously be strengthened the adaptive faculty of end product, to obvious raising of utilization ratio meeting of soya-bean oil, metabolism fails to be convened for lack of a quorum and flows towards the direction that is conducive to end product formation, and the bacterial strain of excellent property will be out screened.
The above; be only the present invention's embodiment preferably, but protection scope of the present invention is not limited to this, anyly is familiar with in technical scope that those skilled in the art disclose in the present invention; the variation that can expect easily or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Claims (6)
1. a cephalosporin superior strain selection comprises the following steps:
1) get-80 ℃ of freezing mycelia of cephalosporium acremonium, 20～40 ℃ of quick-thawings, the inoculum size with 1～6.7% is inoculated in seed culture medium, shaking table shaking culture 2～6 days;
2) be inoculated in fermention medium shaking table shaking culture 4～10 days with 1～33.3% inoculum size;
3) with 1～33.3% inoculum size, be inoculated in fermention medium again, shaking table shaking culture 4～10 days, this process repeats 7 times;
4) take off shaking flask from shaking table, with physiological saline, carry out 10 times of serial gradient dilutions, with red-cell count plate counting, find cephalosporium acremonium arthrospore quantity 10
6～10
8extent of dilution 10
-4; (we did test of many times, 10
-4during this extent of dilution, the arthrospore amount is 10
6～10
8, these data are clear to be determined.)
5), centered by the extent of dilution of step 4, choose 5 gradients (10
-2, 10
-3, 10
-4, 10
-5, 10
-6) bacteria suspension evenly is coated with lipase and identifies dull and stereotypedly, each gradient arranges 3 repetitions, and flat board is placed at 20～30 ℃ of temperature to standing cultivation 10～20 days;
6) cultivation is placed in cultured flat board under the 350nm ultraviolet lamp after finishing, with toothpick picking fluorescent ring, be of moderate size and sharply marginated bacterium colony, be inoculated on normal flat board, at 20～30 ℃ of temperature, standing cultivation is 10～20 days, prepares the lawn of 1～2cm * 4～6cm;
7) previously prepared cephalosporin sensitive organism plate culture medium is melted, accessing wherein final concentration when temperature is down to 45 ℃ of left and right is 10
8cFU/mL left and right sensitive organism, pour plate, solidify rear standby;
8) punch tool that is 5mm with diameter punches on the lawn of step 6 preparation, prepares agar column, with this agar column of transfering loop picking, is inoculated on the flat board of step 7, is placed at the temperature of 30～40 ℃ standing cultivation 1～2 day;
9) after cultivate finishing, with vernier callipers, measure antibacterial circle diameter, select bacterial strain that diameter is greater than 35mm and carry out another flat board of taking turns and sieve again test;
10) after too much the flat board of round sieves again, filter out 10 larger bacterial strains of inhibition zone and prepare freezing mycelia, carrying out shaking flask according to the method for step 1 and step 2 sieves again, measure the content that fermentation culture finishes cephalosporin in secondary fermentation liquid, carry out repeatedly that inclined-plane goes down to posterity, multiple batches of bacterial strain genetic stability and production performance test, filter out bacterial strain 1～2 strain that fermentation level is the highest.
2. method according to claim 1, is characterized in that described seed culture medium, and its formula is: corn steep liquor 3.0～5.0%, glucose 3.0～5.0%, sucrose 2.0～4.0%, calcium carbonate 0.2～1.0%, pH6～8.
3. method according to claim 1, it is characterized in that described fermention medium, its formula is: corn steep liquor 3.0～5.0%, groundnut meal 3.0～5.0%, sucrose 2.0～4.0%, calcium sulfate 0.2～1.0%, peptone 1.0～3.0%, ammonium sulfate 1.0～2.0%, ferrous sulfate 0.01～0.1%, calcium carbonate 0.2～0.5%, soya-bean oil 5～20%, pH6～8.
4. method according to claim 1, it is characterized in that described lipase evaluation is dull and stereotyped, its formula is: ammonium sulfate 0.05～1.0%, potassium primary phosphate 0.05～0.5%, vitriolate of tartar 0.1～0.5%, magnesium sulfate heptahydrate 0.01～0.2%, sweet oil 1.0～4.0%, rhodamine B 0.01～0.03%, polyvinyl alcohol 0.01～0.3%, agar 1.0～2.0%, pH6～8.
5. method according to claim 1, is characterized in that described cephalosporin sensitive organism plate culture medium, and its formula is: yeast extract paste 0.1～1.5%, peptone 0.5～3.0%, sodium-chlor 0.1～1.5%, agar 1.0～2.0%, pH6～8.
6. method according to claim 1, it is characterized in that described normal plate culture medium, its formula is: sucrose 1.0～3.0%, yeast extract paste 0.1～0.8%, peptone 0.1～0.5%, ferrous sulfate 0.001～0.01%, sal epsom 0.01～0.06%, agar 1.0～2.0%, pH6～8.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357051A (en) * | 1999-06-18 | 2002-07-03 | 抗生素有限公司 | Method for producing T-aminodesacetoxycephalosporanic acid (7-ADCA) |
| CN102690859A (en) * | 2012-04-16 | 2012-09-26 | 广西中医学院 | Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria |
-
2013
- 2013-06-28 CN CN2013102680541A patent/CN103436521A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357051A (en) * | 1999-06-18 | 2002-07-03 | 抗生素有限公司 | Method for producing T-aminodesacetoxycephalosporanic acid (7-ADCA) |
| CN102690859A (en) * | 2012-04-16 | 2012-09-26 | 广西中医学院 | Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria |
Non-Patent Citations (4)
| Title |
|---|
| 吴晓良: "顶头孢霉的遗传育种方法初步研究和头孢菌素C发酵行为突变筛选", 《 中国优秀硕士学位论文全文数据库》 * |
| 孟国庆等: "头孢菌素C高产菌株的推理选育", 《中国抗生素杂志》 * |
| 袁丽容等: "头孢菌素C产生菌顶头孢霉理性化筛选的研究", 《中国抗生素杂志》 * |
| 郭元昕等: "豆油对头孢菌素C发酵影响的初步研究", 《中国抗生素杂志》 * |
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Application publication date: 20131211 |