CN113444670A - Screening method and culture method of high-activity acarbose producing strain - Google Patents
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Abstract
The invention provides a screening method and a culture method of high-activity acarbose producing bacteria, belonging to the technical field of microbial culture. The invention transfers a single colony of actinoplanes SE50 to a plate culture medium, cultures for 5-7 d at 25-30 ℃, selects a strain which does not bloom, has an elliptical colony shape and a long axis of 5-10 cm as a target strain, and obtains the high-activity acarbose producing strain. After a single colony which does not bloom, is elliptical in colony shape and has a long axis of 5-10 cm is inoculated to a seed bottle, the concentration of the seed bacteria is higher, the growth condition is better, the titer in a fermentation bottle is highest, and the titer can reach 6280 u/ml. The invention can select the high-activity strains in the glycerol tube for subsequent seed culture and fermentation culture.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a screening method and a culture method of high-activity acarbose producing bacteria.
Background
Acarbose chemical name: 0-4, 6-dideoxy-4- [ [ (1S, 4R, 5S, 6S)4,5, 6-trihydroxy-3- (hydroxymethyl) -2-cyclohexen-1-yl]Amino group]- α -D glucopyranosyl (1 → 4) -D-glucopyranose, formula: c25H43NO18Molecular weight: 645.63, acarbose is a novel oral hypoglycemic agent, competitively inhibits the enzyme glucoside hydrolase in the intestinal tract. Acarbose can inhibit polysaccharide and sucrose from decomposing into glucose, slow down sugar absorption, and reduce postprandial blood sugar. Acarbose mainly reduces postprandial hyperglycemia, and if the carbohydrate in the diet of a user accounts for more than 50%, the blood sugar reducing effect is more obvious, so that the acarbose is suitable for people taking carbohydrate as staple food. Acarbose is the first glycosidase inhibitor on the market, and is the only drug with the indication of impaired glucose tolerance at present, so that the risk of diabetes mellitus is reduced by 36% in patients with impaired glucose tolerance.
The acarbose raw material is obtained by a microbial fermentation method, the strain is actinoplanes SE50, and the strain is given certain fermentation conditions to obtain the production of the secondary metabolite acarbose, the biosynthesis process is completed under the catalysis of a series of enzymes coded by an Acb gene group, and the synthesis process comprises the synthesis of 42 amino 24,62 dideoxy glucose and the subsequent glycosylation to generate acarbose. The existing strain screening and culturing method is that a glycerol tube preserved at the temperature of minus 80 ℃ is naturally thawed, then a sterile straw is used for absorbing bacteria liquid for inclined plane coating and transferring, the inclined plane is cultured for 7 days at the temperature of 28 ℃, bacterial colonies grow over the inclined plane to form bacterial lawn, then the inclined plane bacterial lawn is selected for seed bottle inoculation, the seed bottle is cultured for 2-3 days, and finally the bacterial lawn is inoculated into a fermentation bottle for fermentation of acarbose under proper conditions. However, in the storage process of the glycerol tube, part of thalli is degenerated and the activity is reduced, high-activity, low-activity and dead strains exist in strains in the glycerol tube at the same time, after the strains are transferred to the inclined plane, the high-activity strains and the low-activity strains grow on the inclined plane and cannot be distinguished, when inclined plane lawn is picked for shake flask transfer, the low-activity strains and the high-activity strains are transferred to the next step for culture, and finally the problems of uneven fermentation index or generally low index are caused.
Disclosure of Invention
The invention aims to provide a screening method and a culture method of high-activity acarbose producing bacteria.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a screening method of high-activity acarbose producing bacteria, which comprises the following steps:
transferring a plurality of single colonies of actinoplanes SE50 to a plate culture medium, culturing for 5-7 d at 25-30 ℃, and selecting strains which do not bloom, have elliptical colony shapes and long axes of 5-10 cm as target strains to obtain the high-activity acarbose producing strains.
Preferably, the selected conditions further comprise that the color is dark yellow and/or the short axis is 4-8 cm.
The invention also provides a culture method of the high-activity acarbose producing strain, which comprises the following steps:
1) screening according to the screening method in the scheme to obtain high-activity acarbose producing bacteria;
2) transferring the high-activity acarbose producing strain to an eggplant bottle culture medium for amplification culture to obtain thalli;
3) transferring the thalli obtained by the amplification culture to a seed culture medium, and performing seed culture to obtain a seed solution in a logarithmic phase;
4) transferring the seed liquid in the logarithmic phase to a fermentation medium, and performing fermentation culture to obtain a fermentation product; the fermentation product comprises acarbose.
Preferably, the temperature of the amplification culture in the step 2) is 25-30 ℃; the time of the amplification culture is 5-7 d.
Preferably, the temperature of the seed culture in the step 3) is 25-30 ℃; the time for seed culture is 42-48 h.
Preferably, the pH value of the seed liquid in the step 3) is 6.5-7.5; the mass concentration of thalli in the seed liquid is 20-30%; the seed liquid is dark yellow in color.
Preferably, the volume ratio of the seed liquid to the fermentation medium in the step 4) is 1: (8-12).
Preferably, the fermentation culture in the step 4) is carried out at the temperature of 25-30 ℃ under the oscillation condition; the fermentation culture time is 6-8 d; the oscillation frequency is 200-300 rpm; the amplitude of the oscillation is 45-55 mm.
Preferably, when the fermentation culture in the step 4) is carried out for 48-50 hours, the glucose aqueous solution is supplemented for the first time; when the fermentation culture is carried out for 98-100 hours, supplementing the glucose aqueous solution for the second time; the volume ratio of the glucose aqueous solution supplemented each time to the fermentation medium is independently (1-3): 25; the mass concentration of the glucose aqueous solution is 50%.
Preferably, the pH value of the fermentation product in the step 4) is 6.4-6.8; the mass concentration of the thalli in the fermentation product is 28-32%.
The invention provides a screening method of high-activity acarbose producing bacteria. The invention transfers a single colony of actinoplanes SE50 to a plate culture medium, cultures for 5-7 d at 25-30 ℃, selects a strain which does not bloom, has an elliptical colony shape and a long axis of 5-10 cm as a target strain, and obtains the high-activity acarbose producing strain. After a single colony which does not bloom, is elliptical in colony shape and has a long axis of 5-10 cm is inoculated to a seed bottle, the concentration of the seed bacteria is higher, the growth condition is better, the titer in a fermentation bottle is highest, and the titer can reach 6280 u/ml.
Drawings
FIG. 1 is a single colony from the secondary plate single colony screen of example 1;
FIG. 2 is a mycelioscopic image of the seed flask of example 1.
Detailed Description
The invention provides a screening method of high-activity acarbose producing bacteria, which comprises the following steps:
transferring a single colony of Actinoplanes SE50 to a plate culture medium, culturing at 25-30 ℃ for 5-7 days, and selecting a strain which does not bloom, has an oval colony shape and a long axis of 5-10 cm as a target strain to obtain the high-activity acarbose producing strain.
In the invention, the selected conditions preferably further comprise that the color is dark yellow and/or the short axis is 4-8 cm.
In the present invention, the single colony of Actinoplanes SE50 is preferably obtained by culturing the following: transferring the actinoplanes SE50 bacterial liquid preserved by the frozen glycerin tube to a blank plate culture medium, performing gradient streaking, and culturing for 4-6 days at the temperature of 25-30 ℃. The invention carries out plate streak culture on actinoplanes SE50 bacterial liquid preserved by a frozen glycerin tube to obtain small single bacterial colonies, thereby achieving the purpose of pure culture.
In the present invention, the formulation of the plating medium is shown in Table 1.
TABLE 1 plate Medium formulation
The screening method of the invention can obtain high-activity strains and exclude low-activity strains.
The invention also provides a culture method of the high-activity acarbose producing strain, which comprises the following steps:
1) screening according to the screening method in the scheme to obtain high-activity acarbose producing bacteria;
2) transferring the high-activity acarbose producing strain to an eggplant bottle culture medium for amplification culture to obtain thalli;
3) transferring the thalli obtained by the amplification culture to a seed culture medium, and performing seed culture to obtain a seed solution in a logarithmic phase;
4) transferring the seed liquid in the logarithmic phase to a fermentation medium, and performing fermentation culture to obtain a fermentation product; the fermentation product comprises acarbose.
According to the invention, the high-activity acarbose producing strain is obtained by screening according to the screening method in the scheme.
After the high-activity acarbose producing strain is obtained by screening, the high-activity acarbose producing strain is transferred to an eggplant bottle culture medium for amplification culture to obtain thalli.
In the invention, the formula of the eggplant bottle culture medium is the same as that of a flat culture medium.
The invention performs enlarged culture on the high-activity strains through the enlarged culture of the eggplant bottles, ensures that the strains inoculated into the seed bottles are all high-activity strains, eliminates the difference between different bottle batches, and simultaneously meets the strain supply of subsequent large-scale production to a large number of seed bottles.
In the invention, the temperature of the amplification culture is preferably 25-30 ℃, and more preferably 28 ℃; the time for the amplification culture is preferably 5-7 d, and more preferably 6 d.
The thallus obtained by the amplification culture is transferred to a seed culture medium for seed culture, and the seed liquid in the logarithmic phase is obtained.
In the invention, the temperature of the seed culture is preferably 25-30 ℃, and more preferably 28 ℃; the time for seed culture is preferably 42-48 h, and more preferably 45 h.
In the present invention, the formulation of the seed culture medium is shown in table 2.
TABLE 2 seed culture Medium
In the invention, the pH value of the seed liquid is preferably 6.5-7.5, and more preferably 7.0; the mass concentration of the cells in the seed solution is preferably 20% to 30%, more preferably 25%; the color of the seed liquid is preferably dark yellow; the seed liquid has less thallus bifurcations.
After the seed liquid in the logarithmic growth phase is obtained, the seed liquid in the logarithmic growth phase is transferred to a fermentation culture medium for fermentation culture to obtain a fermentation product; the fermentation product comprises acarbose.
The invention inserts the seeds in logarithmic phase into the fermentation bottle, thereby ensuring that the subsequent fermentation bottle obtains high-efficiency index.
In the present invention, the volume ratio of the seed liquid to the fermentation medium is preferably 1: (8-12), more preferably 1: 10.
in the invention, the fermentation culture is preferably carried out at 25-30 ℃ under the oscillation condition; the fermentation culture time is preferably 6-8 d, and more preferably 7 d; the oscillation frequency is preferably 200-300 rpm, and more preferably 250 rpm; the amplitude of the oscillation is preferably 45-55 mm, and more preferably 50 mm.
In the present invention, the formulation of the fermentation medium is preferably as shown in Table 3.
TABLE 3 fermentation Medium
In the invention, when the fermentation culture is carried out for 48-50 h, the glucose aqueous solution is supplemented for the first time; when the fermentation culture is carried out for 98-100 hours, supplementing the glucose aqueous solution for the second time; the volume ratio of the glucose aqueous solution supplemented each time to the fermentation medium is preferably (1-3): 25, more preferably 2: 25; the aqueous glucose solution preferably has a mass concentration of 50%.
In the invention, the pH of the fermentation product is preferably 6.4-6.8, and more preferably 6.6; the mass concentration of the cells in the fermentation product is preferably 28% to 32%, more preferably 30%.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The process flow comprises the following steps: glycerol pipe, first-stage flat plate separation, second-stage flat plate screening, eggplant bottle expanding culture, seed bottle and fermentation bottle.
1. Separating single colony with the first-level plate; and (3) dipping the frozen glycerol tube bacterium liquid by using a flat-head toothpick under an aseptic condition, transferring the frozen glycerol tube bacterium liquid to a blank plate culture medium, carrying out gradient lineation, and culturing in a constant-temperature incubator at 25-30 ℃ for 4-6 days.
2. Screening single bacterial colonies of a secondary flat plate; and under the aseptic condition, a sharp-pointed toothpick is dipped in a plate, and the separated single colony is transferred to a blank plate culture medium and is cultured in a constant-temperature incubator at the temperature of 25-30 ℃ for 5-7 days. And selecting strains which do not bloom, have the diameter of 5-10 mm, are oval, are plump and have dark yellow colors, wherein the strains are shown as target single colonies in the figure 1.
A large number of single colonies in different states are selected, and then the single colonies are inoculated to a subsequent seed bottle and a subsequent fermentation bottle, and the concentration and the fermentation titer of the seed bacteria are counted, and the results are shown in a table 4.
TABLE 4 seed concentration and fermentation titer of single colonies at different states
As can be seen from Table 4, in the single colonies in different states, after the single colony which does not bloom, has a diameter of 10-15 mm, is irregular and has a dark yellow color is inoculated to a seed bottle, the concentration of the bacteria in the seed bottle is the highest, the growth condition is the best, but the titer in the fermentation bottle is not the highest. And after the single colony which does not bloom, has the diameter of 5-10 mm, is elliptical and is dark yellow is inoculated to the seed bottle, the concentration of the seed bacteria is higher, the growth condition is better, but the titer in the fermentation bottle is the highest, and the titer is 6280 u/ml.
3. Bacterial colony culture in eggplant bottles: under the aseptic condition, a single colony is picked from the flat single colony by using an inoculating loop and transferred to a blank eggplant bottle culture medium, the culture medium is uniformly coated, and the eggplant bottle culture medium is placed in a constant-temperature incubator at the temperature of 25-30 ℃ for culture for 5-7 days.
4. Hypha in a seed bottle: scraping 1 x 1cm of eggplant bottle slant under aseptic condition with inoculating shovel2Inoculating the large-size thallus into 50ml of seed culture medium, and culturing at constant temperature of 25-30 ℃ for 42And obtaining hypha of a seed bottle after 48 hours. And (3) performing sterile detection on the seed bottle, and performing microscopic examination to obtain a result shown in FIG. 2, wherein hyphae are deeply dyed, and have fewer branches and no mixed bacteria.
And simultaneously, indexes such as pH, bacterial concentration, glucose content and the like meet the requirements of the table 5.
TABLE 5 hypha requirements of seed bottles
5. And (3) shaking flask fermentation:
and 2.5mL of the grown seed solution is inoculated into 25mL of fermentation liquor, and is subjected to shaking culture at 250rpm, 50mm amplitude and 25-30 ℃. Culturing the fermentation liquor for 48h, and supplementing 2mL of 50% glucose; the fermentation broth was cultured for 100h, and 2mL of 50% glucose was added. The final pH value is 6.4-6.8, and the bacterial concentration is about 30%. The final titer is above 6000u/ml after the total fermentation period is 7 d.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for screening high-activity acarbose producing bacteria comprises the following steps:
transferring a plurality of single colonies of actinoplanes SE50 to a plate culture medium, culturing for 5-7 d at 25-30 ℃, and selecting strains which do not bloom, have elliptical colony shapes and long axes of 5-10 cm as target strains to obtain the high-activity acarbose producing strains.
2. The screening method according to claim 1, wherein the selected conditions further comprise a deep yellow color and/or a minor axis of 4 to 8 cm.
3. A method for culturing high-activity acarbose producing bacteria comprises the following steps:
1) screening according to the screening method of claim 1 or 2 to obtain high-activity acarbose producing bacteria;
2) transferring the high-activity acarbose producing strain to an eggplant bottle culture medium for amplification culture to obtain thalli;
3) transferring the thalli obtained by the amplification culture to a seed culture medium, and performing seed culture to obtain a seed solution in a logarithmic phase;
4) transferring the seed liquid in the logarithmic phase to a fermentation medium, and performing fermentation culture to obtain a fermentation product; the fermentation product comprises acarbose.
4. The culture method according to claim 3, wherein the temperature of the scale-up culture in the step 2) is 25 to 30 ℃; the time of the amplification culture is 5-7 d.
5. The culture method according to claim 3, wherein the temperature of the seed culture in the step 3) is 25-30 ℃; the time for seed culture is 42-48 h.
6. The culture method according to claim 3 or 5, wherein the pH value of the seed solution in step 3) is 6.5 to 7.5; the mass concentration of thalli in the seed liquid is 20-30%; the seed liquid is dark yellow in color.
7. The cultivation method according to claim 3, wherein the volume ratio of the seed solution to the fermentation medium in step 4) is 1: (8-12).
8. The culture method according to claim 3 or 7, wherein the fermentation culture in step 4) is performed under shaking conditions at 25 to 30 ℃; the fermentation culture time is 6-8 d; the oscillation frequency is 200-300 rpm; the amplitude of the oscillation is 45-55 mm.
9. The culture method according to claim 3 or 7, wherein the fermentation culture in step 4) is performed for 48-50 hours, and the glucose aqueous solution is supplemented for the first time; when the fermentation culture is carried out for 98-100 hours, supplementing the glucose aqueous solution for the second time; the volume ratio of the glucose aqueous solution supplemented each time to the fermentation medium is independently (1-3): 25; the mass concentration of the glucose aqueous solution is 50%.
10. The culture method according to claim 3 or 7, wherein the pH of the fermentation product in step 4) is 6.4 to 6.8; the mass concentration of the thalli in the fermentation product is 28-32%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249001A (en) * | 1997-02-28 | 2000-03-29 | 拜尔公司 | Acarbose (ACB) cluster from actinoplanes sp. SE 50/110 |
| TW201336992A (en) * | 2011-12-08 | 2013-09-16 | 拜耳智慧財產有限公司 | New actinomycete integrative and conjugative element from Actinoplanes sp. SE50/110 as plasmid for genetic transformation of related actinobacteria |
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- 2021-07-28 CN CN202110857524.2A patent/CN113444670A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249001A (en) * | 1997-02-28 | 2000-03-29 | 拜尔公司 | Acarbose (ACB) cluster from actinoplanes sp. SE 50/110 |
| TW201336992A (en) * | 2011-12-08 | 2013-09-16 | 拜耳智慧財產有限公司 | New actinomycete integrative and conjugative element from Actinoplanes sp. SE50/110 as plasmid for genetic transformation of related actinobacteria |
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