阅读说明：本技术 一种提高鸟苷发酵菌株产率的方法 (Method for improving yield of guanosine fermentation strain ) 是由 陆可 王海波 严盟 谢畅丰 张小兰 于 2020-12-28 设计创作，主要内容包括：本发明属于食品微生物、发酵技术领域,具体涉及一种提高鸟苷发酵菌株产率的方法。步骤顺序为确立菌种耐高温的目标温度值；选取出发菌液；出发菌液采用变温式定向培育；尤其是重复1～5次变温式定向培育,每次温度升高0.5～1.0℃,直到5L罐最高温度达菌种耐高温的目标温度值,选取24～80小时的发酵液作终段出发菌液；即选取发酵产率较高批次、发酵糖酸转化率最高时间段的发酵液作为出发菌液,利用与生产方法高度融合的培养环境,通过定向改变育种环境温度,获得菌种特性更接近野生型菌株、耐高温的高产鸟苷菌株应用于鸟苷发酵生产,菌种对变温式调控方法适应能力更强,大大提高鸟苷发酵菌株产率。(The invention belongs to the technical field of food microorganism and fermentation, and particularly relates to a method for improving the yield of a guanosine fermentation strain. The sequence of the steps is to determine a high-temperature resistant target temperature value of the strain; selecting starting bacterial liquid; the starting bacterial liquid is directionally cultured in a temperature-variable manner; particularly, repeating 1-5 times of temperature-variable directional cultivation, raising the temperature by 0.5-1.0 ℃ every time until the highest temperature of a 5L tank reaches the high-temperature-resistant target temperature value of the strain, and selecting 24-80 hours of fermentation liquor as a final starting bacterial liquid; the fermentation broth with a higher fermentation yield and the highest fermentation saccharic acid conversion rate time period is selected as the starting bacterial liquid, the culture environment highly fused with the production method is utilized, the breeding environment temperature is directionally changed, the strain characteristics are closer to those of wild strains, the high-temperature-resistant high-yield guanosine strains are obtained and applied to guanosine fermentation production, the adaptability of the strains to a variable temperature regulation and control method is stronger, and the yield of the guanosine fermentation strains is greatly improved.)

1. A method for improving the yield of a guanosine fermentation strain comprises the following steps:

a: determining the high-temperature resistant target temperature of the strain to be 40-42 ℃;

b: in the fermentation production process of taking bacillus amyloliquefaciens as guanosine producing bacteria, selecting fermentation liquor with a fermentation saccharic acid conversion rate time period of 42 hours as a primary starting bacterial liquid;

c: b, taking the primary starting bacterial liquid obtained in the step B, streaking the primary starting bacterial liquid in a solid eggplant bottle slant base, culturing the eggplant bottle slant base for 20-24 hours at 32-35 ℃, eluting eggplant bottle slant seeds into a fermentation culture medium of a 5L fermentation tank by using 100ml of normal saline, fermenting, and selecting 24-80 hours of fermentation liquor as a starting bacterial liquid for the next stage of temperature-variable directional culture;

d: operating according to the step C, repeating the temperature-variable directional culture for 1-5 times, raising the temperature by 0.5-1.0 ℃ every time until the highest temperature of a 5L tank reaches the high-temperature-resistant target temperature value of the strain, and selecting the fermentation liquor for 24-80 hours as the final starting bacterial liquid;

e: selecting 1ml of the final-stage starting bacterial liquid obtained in the step D, and diluting the final-stage starting bacterial liquid to 10 degrees by using 0.85 percent normal saline in a gradient manner^－8Respectively take 10^－4、10^－5、10^－6、10^－7、10^－8Coating the diluent on a solid culture medium plate, coating 5 diluent plates for each time to prepare plate seeds, and culturing at the temperature of 32-35 ℃ for 36-72 hours until the diameter of a single colony reaches 0.8-1.5 mm; selecting single colonies, respectively inoculating the single colonies in a solid culture medium inclined plane, and performing shake flask fermentation to obtain a single strain with high yield;

f: and E, starting from the high-yield single strain selected in the step E, streaking the strain in a solid eggplant bottle slant medium, culturing for 20-24 hours at 32-35 ℃, eluting the eggplant bottle slant medium with normal saline into a fermentation medium of a fermentation tank, and culturing until tank placing parameters are met.

2. The method of increasing fermentation yield of a guanosine strain according to claim 1 wherein: and the fermentation process in the step B is controlled under the conditions that the initial temperature is 34-35 ℃, the temperature reaches 36-37 ℃ in 12 hours, the temperature reaches 24-80 hours, and the temperature reaches 37-42 ℃.

3. The method of increasing fermentation yield of a guanosine strain according to claim 2 wherein: and B, after fermenting for 24 hours, aseptically extracting 10-30 ml of fermentation liquor with different periods every 6 hours, calculating the saccharic acid conversion rate of the fermentation, and synchronously sampling and measuring the guanosine content, the reducing sugar content and the volume of the fermentation liquor.

4. The method of increasing fermentation yield of a guanosine strain according to claim 2 wherein:

the formula of the solid eggplant bottle culture medium in the step C is as follows: 3.0 +/-0.5% of soluble starch, 1.0 +/-0.5% of yeast powder, 0.5 +/-0.1% of peptone, 2.0 +/-0.2% of agar and 7.0 of PH;

the formula of the fermentation medium of the 5L fermentation tank in the step C is as follows: 12% of glucose, 2 +/-0.5% of yeast powder, 3.0 +/-0.5% of ammonium sulfate, 0.3 +/-0.1% of monopotassium phosphate, 0.002% of ferrous sulfate and 7.0% of PH.

5. The method of increasing fermentation yield of a guanosine strain according to claim 1 wherein: and E, performing shake flask fermentation in the step E, namely selecting single bacterial colonies, respectively inoculating the single bacterial colonies into a solid culture medium inclined plane, culturing for 24 hours at 32-35 ℃, inoculating the single bacterial colonies into a shake flask fermentation medium, culturing for 62-72 hours at 7.5cm stroke, 110-120 rpa and 37-40 ℃ of a reciprocating shaking table, and selecting shake flask fermentation to obtain the single bacterial strains with high yield.

6. The method of increasing fermentation yield of a guanosine strain according to claim 1 wherein: the formula of the solid culture medium in the step E is as follows: 3.0 +/-0.5% of soluble starch, 1.0 +/-0.5% of yeast powder, 0.5 +/-0.1% of peptone, 2.0 +/-0.2% of agar and 7.0 of PH;

the formula of the shake flask fermentation medium in the step E is as follows: 12% of glucose, 2 +/-0.5% of yeast powder, 3.0 +/-0.5% of ammonium sulfate, 0.3 +/-0.1% of monopotassium phosphate, 0.002% of ferrous sulfate, 3.0% of light calcium carbonate and 7.0% of PH.

7. The method of increasing fermentation yield of a guanosine strain according to claim 1 wherein: the step F comprises the following fermentation process control conditions: the initial fermentation temperature is 34-35 ℃, the temperature reaches 36-37 ℃ in 12 hours, and the temperature reaches 37-42 ℃ in 24-80 hours.

8. The method of increasing fermentation yield of a guanosine strain according to claim 1 wherein: and C, selecting the fermentation liquor for 42 hours as starting bacteria liquid for the next stage of variable-temperature oriented cultivation.

9. The method of increasing fermentation yield of a guanosine strain according to claim 1 wherein: and D, selecting the fermentation liquor for 42 hours as a final-stage starting bacterial liquid.

Technical Field

The invention belongs to the technical field of food microorganism and fermentation, and particularly relates to a method for improving the yield of a guanosine fermentation strain.

Background

Guanosine is a raw material for producing guanylic acid, is also a precursor of ribavirin and acyclovir serving as antiviral drugs, is widely applied to the fields of food, medicine, chemical industry and the like, and has good development prospect.

In the last 60 th century, Zalkin et al, Japanese academician, began to study the regulation mechanism of the nucleotide metabolic pathway, and now has elucidated the regulation mechanism of the purine metabolic pathway, which is subject to strict feedback regulation. Currently, the common methods for improving the fermentation yield of guanosine comprise strain breeding and method optimization. The common strain breeding method has extremely low probability of spontaneous mutation of high-yield guanosine strains in the nature due to the strict feedback regulation and control effect; the traditional mutagenesis method has large workload and low efficiency; the genetic engineering breeding has the defects of possibly causing ecological crisis, great technical difficulty and limitation of application by clients forbidding use of genetic strains; the breeding of single plants has the defects of poor adaptability to method regulation and control and the like in large-scale production application.

Disclosure of Invention

In view of the above technical drawbacks, the present invention provides a method for increasing the productivity of guanosine-fermenting strains.

In order to solve the technical problems, the technical scheme of the invention is as follows: a: determining a high-temperature-resistant target temperature value of the strain: setting a high-temperature-resistant target temperature of the strain according to the highest high-temperature-resistant temperature value of the strain plus 1 ℃ of a production method, and in the invention, determining the high-temperature-resistant target temperature of the strain to be 40-42 ℃;

b: selecting starting bacterial liquid: in the fermentation production process of taking bacillus amyloliquefaciens as guanosine producing bacteria, selecting fermentation liquor of batches with higher fermentation yield, aseptically extracting 10-200 ml of fermentation liquor with different periods every 6 hours, temporarily storing the fermentation liquor in a refrigerator at 2-8 ℃, synchronously sampling and measuring guanosine content, reducing sugar content and fermentation liquor volume in addition, calculating fermentation saccharic acid conversion rate, and selecting the fermentation liquor with the highest fermentation saccharic acid conversion rate time period as a first starting bacterial liquid.

The method for calculating the saccharic acid conversion rate of fermentation in the step B comprises the following steps: the guanosine content in the fermentation liquor is multiplied by volume, and the sugar consumption is divided into the conversion rate of fermentation saccharic acid.

C: the starting bacterial liquid adopts temperature-variable directional cultivation: taking out the primary fermentation liquid, streaking the primary fermentation liquid in a solid eggplant bottle slant base, culturing the solid eggplant bottle slant base at the temperature of 32-35 ℃ for 20-24 hours, and eluting the eggplant bottle slant base seed into a fermentation culture medium of a 5L fermentation tank by using 100ml of normal saline. Controlling conditions in the fermentation process: the temperature is 34-35 ℃ for 0 hour, 36-37 ℃ for 12 hours and 37-42 ℃ for 24-80 hours. And starting to extract 10-30 ml of fermentation liquor with different periods at intervals of 6 hours in an aseptic mode after 24 hours, temporarily storing the fermentation liquor in a refrigerator with the temperature of 2-8 ℃, synchronously sampling and measuring the guanosine content, the reducing sugar content and the volume of the fermentation liquor, calculating the conversion rate of the fermentation saccharic acid, and selecting the fermentation liquor with the time of 24-80 hours as starting bacterial liquid for the next stage of temperature-variable directional culture.

The formula of the solid eggplant bottle culture medium in the step C is as follows: 3.0 plus or minus 0.5 percent of soluble starch, 1.0 plus or minus 0.5 percent of yeast powder, 0.5 plus or minus 0.1 percent of peptone, 2.0 plus or minus 0.2 percent of agar and 7.0 percent of PH.

The formula of the fermentation medium of the 5L fermentation tank in the step C is as follows: 12% of glucose, 2 +/-0.5% of yeast powder, 3.0 +/-0.5% of ammonium sulfate, 0.3 +/-0.1% of monopotassium phosphate, 0.002% of ferrous sulfate and 7.0% of PH.

And (4) operating according to the step C, repeating the temperature-variable directional culture for 1-5 times, wherein the temperature is increased by 0.5-1.0 ℃ every time until the highest temperature of the 5L tank reaches the high-temperature-resistant target temperature value of the strain. And selecting 24-80 hours of fermentation liquor as a final-stage starting bacterial liquid.

The temperature is one of the key factors influencing the growth and metabolism of microorganisms, the optimal temperature for strain growth is not necessarily the optimal temperature for fermentation, nor equal to the optimal temperature for accumulation of metabolites, and the adoption of temperature-variable culture is more beneficial to improving the yield. Since a plurality of enzyme reactions in organisms can be influenced by temperature change, the high-temperature resistant strains obtained by singly increasing the breeding temperature in the prior art often have the problem of poor applicability of large-scale production with unappreciated yield. In order to establish each temperature rise range, a series of temperature rise range tests are carried out, and finally the optimal temperature of 0.5-1.0 ℃ is established. By adopting the method, the applicability of the selected high-temperature resistant strain in mass production is obviously improved, and the aim of improving the yield is achieved.

E: selecting a high-temperature resistant single strain with high yield of guanosine: taking the final stage starting bacterium selected in the step D1ml of the solution was diluted to 10% with a gradient of 0.85% physiological saline^－8Respectively take 10^－4、10^－5、10^－6、10^－7、10^－8The dilutions were plated on solid medium plates, 5 for each dilution, to make plate seeds. Culturing at 32-35 ℃ for 36-72 hours until the diameter of a single colony reaches 0.8-1.5 mm.

And (3) selecting single bacterial colonies, respectively inoculating the single bacterial colonies into a solid culture medium inclined plane, culturing for 24 hours at the temperature of 32-35 ℃, inoculating the single bacterial colonies into a shaking flask fermentation medium, culturing for 62-72 hours at the temperature of 37-40 ℃ and in a reciprocating shaking table with 7.5cm stroke, and selecting a single bacterial strain with high shaking flask fermentation yield to apply to the step F.

The formula of the solid culture medium in the step E is as follows: 3.0 plus or minus 0.5 percent of soluble starch, 1.0 plus or minus 0.5 percent of yeast powder, 0.5 plus or minus 0.1 percent of peptone, 2.0 plus or minus 0.2 percent of agar and 7.0 percent of PH.

The formula of the shake flask fermentation medium in the step E is as follows: 12% of glucose, 2 +/-0.5% of yeast powder, 3.0 +/-0.5% of ammonium sulfate, 0.3 +/-0.1% of monopotassium phosphate, 0.002% of ferrous sulfate, 3.0% of light calcium carbonate and 7.0% of PH.

F: the fermentation adopts a temperature-variable control method: and E, starting from the high-yield single strain selected in the step E, streaking the strain in a solid eggplant bottle slant base, culturing for 20-24 hours at 32-35 ℃, and eluting the eggplant bottle slant base with a proper amount of normal saline into a fermentation culture medium of a fermentation tank according to the requirement of production inoculation amount. Controlling conditions in the fermentation process: the temperature is 34-35 ℃ for 0 hour, 36-37 ℃ for 12 hours and 37-42 ℃ for 24-80 hours. Culturing until the requirement of tank placing is met.

Compared with the prior art, the invention has the innovation points that: the fermentation broth with a higher fermentation yield and the highest fermentation saccharic acid conversion rate time period is selected as the starting bacterial liquid, the culture environment highly fused with the production method is utilized, the breeding environment temperature is directionally changed, the strain characteristics are closer to those of wild strains, and the high-temperature-resistant high-yield guanosine strains are applied to guanosine fermentation production.

Detailed Description

The present invention is further illustrated by the following specific examples. The following procedures, which are not described in detail, can be performed according to the molecular biology laboratory manual.

Example (b):

a: determining a high-temperature-resistant target temperature value of the strain: the high temperature resistant target temperature of the strain is set to be 41 ℃ according to the production method and the highest temperature value plus 1 ℃ for the high temperature resistance of the strain.

B: selecting starting bacterial liquid: in the fermentation production process of taking bacillus amyloliquefaciens as guanosine producing bacteria, selecting fermentation liquor with high fermentation yield at the present stage, aseptically extracting 10-200 ml of fermentation liquor with different periods every 6 hours, temporarily storing the fermentation liquor in a refrigerator at 2-8 ℃, synchronously sampling and measuring guanosine content, reducing sugar content and fermentation liquor volume in addition, calculating fermentation saccharic acid conversion rate, and selecting the fermentation liquor with the highest fermentation saccharic acid conversion rate time period (42 hours) as starting bacteria liquid.

The method for calculating the saccharic acid conversion rate of fermentation in the step B comprises the following steps: the guanosine content in the fermentation liquor is multiplied by volume, and the sugar consumption is divided into the conversion rate of fermentation saccharic acid.

C: the starting bacterial liquid adopts temperature-variable directional cultivation: taking out the fermentation liquid, streaking in solid eggplant bottle slant medium, culturing at 33 deg.C for 24 hr, and eluting eggplant bottle slant seed with 100ml physiological saline into fermentation medium of 5L fermenter. Controlling conditions in the fermentation process: the temperature is 0-12 hours, 34-12-24 hours, 36-24-80 hours and 38 ℃. And (4) starting 24 hours, aseptically extracting 20ml of fermentation liquor with different periods every 6 hours, temporarily storing the fermentation liquor in a refrigerator at the temperature of 2-8 ℃, synchronously sampling and measuring the guanosine content, the reducing sugar content and the volume of the fermentation liquor, calculating the fermentation saccharic acid conversion rate, and selecting the fermentation liquor with the highest fermentation saccharic acid conversion rate time period (54 hours) of 24-80 hours as the starting bacterial liquid of the step D.

The formula of the solid eggplant bottle culture medium in the step C is as follows: 3.0 plus or minus 0.5 percent of soluble starch, 1.0 plus or minus 0.5 percent of yeast powder, 0.5 plus or minus 0.1 percent of peptone, 2.0 plus or minus 0.2 percent of agar and 7.0 percent of PH.

The formula of the fermentation medium of the 5L fermentation tank in the step C is as follows: 12% of glucose, 2 +/-0.5% of yeast powder, 3.0 +/-0.5% of ammonium sulfate, 0.3 +/-0.1% of monopotassium phosphate, 0.002% of ferrous sulfate and 7.0% of PH.

D: and (4) operating according to the step C, repeating the temperature-variable directional cultivation, and raising the temperature by 0.5-1.0 ℃. Taking out the fermentation liquid, streaking in solid eggplant bottle slant medium, culturing at 33.5 deg.C for 23 hr, and eluting eggplant bottle slant seed with 100ml physiological saline into fermentation medium of 5L fermenter. Controlling conditions in the fermentation process: the temperature is 0-12 hours, 34.5-12-24 hours, 37-24-80 hours and 39 ℃. And (3) starting 24 hours, aseptically extracting 20ml of fermentation liquor with different periods every 6 hours, temporarily storing the fermentation liquor in a refrigerator at the temperature of 2-8 ℃, synchronously sampling and measuring the guanosine content, the reducing sugar content and the volume of the fermentation liquor, calculating the fermentation saccharic acid conversion rate, and selecting the fermentation liquor with the highest fermentation saccharic acid conversion rate time period (48 hours) of 24-80 hours as the starting bacterial liquid of the step E.

E: and D, repeating the temperature-variable directional cultivation, wherein the temperature is increased by 0.5-1.0 ℃. Taking out the fermentation liquid, streaking in solid eggplant bottle slant medium, culturing at 34 deg.C for 22 hr, and eluting eggplant bottle slant seed with 100ml physiological saline into fermentation medium of 5L fermenter. Controlling conditions in the fermentation process: the temperature is 0-12 hours, 35-12 hours, 24 hours, 37.5-24 hours, 40 ℃ for 24-80 hours. And (3) starting 24 hours, aseptically extracting 20ml of fermentation liquor with different periods every 6 hours, temporarily storing the fermentation liquor in a refrigerator at the temperature of 2-8 ℃, synchronously sampling and measuring the guanosine content, the reducing sugar content and the volume of the fermentation liquor, calculating the fermentation saccharic acid conversion rate, and selecting the fermentation liquor with the highest fermentation saccharic acid conversion rate time period (48 hours) of 24-80 hours as the starting bacterial liquid of the step F.

F: and E, repeating the temperature-variable directional cultivation, wherein the temperature is increased by 0.5-1.0 ℃. Taking out the fermentation liquid, streaking in solid eggplant bottle slant medium, culturing at 35 deg.C for 20 hr, and eluting eggplant bottle slant seed with 100ml physiological saline into fermentation medium of 5L fermenter. Controlling conditions in the fermentation process: the temperature is 0-12 hours, 35.5-12 hours, 38.5-24-72 hours and 41 ℃. And (3) starting 24 hours, aseptically extracting 20ml of fermentation liquor with different periods every 6 hours, temporarily storing the fermentation liquor in a refrigerator at the temperature of 2-8 ℃, synchronously sampling and measuring the guanosine content, the reducing sugar content and the volume of the fermentation liquor, calculating the fermentation saccharic acid conversion rate, and selecting the fermentation liquor with the highest fermentation saccharic acid conversion rate time period (42 hours) of 24-72 hours as the starting bacterial liquid of the step G.

G: selecting a high-temperature resistant single strain with high yield of guanosine: taking 1ml of the starting bacterial liquid selected in the step F, and diluting the starting bacterial liquid to 10 degrees by using 0.85 percent normal saline in a gradient manner^－8Respectively take 10^－4、10^－5、10^－6、10^－7、10^－8The dilutions were plated on solid medium plates, 5 for each dilution, to make plate seeds. Culturing at 35 ℃ for 36 hours until the diameter of a single colony reaches 0.8-1.5 mm.

Selecting single colonies, respectively inoculating the single colonies into a solid culture medium inclined plane, culturing for 22 hours at 34 ℃, inoculating the inclined plane, looping the inclined plane into a shake flask fermentation medium, culturing for 62-72 hours at 7.5cm stroke of a reciprocating shaking table and at the temperature of 110-120 rpa and 37-41 ℃, and selecting the single strain M201 with the highest shake flask fermentation yield through detection.

H: the high-yield strain is applied to guanosine fermentation by adopting a temperature-variable control method: and (3) starting from the high-yield strain M201 selected in the step G, streaking the strain in a solid eggplant bottle slant base, culturing for 22 hours at 34 ℃, eluting the eggplant bottle slant base with a proper amount of normal saline according to the requirement of production inoculation amount, culturing for 10 hours, transferring into a fermentation tank, and mixing the seed tank and the fermentation tank with culture mediums according to the original production ratio. Controlling conditions in the fermentation process: the temperature is 34-35 ℃ for 0 hour, 36-37 ℃ for 12 hours and 37-42 ℃ for 24-80 hours. Culturing for 80 hours, and measuring fermentation liquor guanosine yield and other tank placing parameters according to production requirements.

The patent implements the result:

1) and the genetic characteristic verification of the strain is carried out on the high-yield guanosine strain M201 bred by the method, and as a result, the strain is subjected to slant passage for 8 times by using a solid culture medium, the growth level of the F1 → F8 strain and the shake flask fermentation level do not show a descending trend, and the stability of the genetic characteristic of the strain is good.

2) And verifying the large production level of the high-yield guanosine strain M201 selectively bred by the method according to the temperature-variable fermentation control method. As a result, the strain is applied in a 12m3 tank, the yield of the fermentation guanosine reaches more than 70g/l, compared with the original strain method, the fermentation yield is improved by more than 16%, and the yield of the guanosine in a single batch is obviously improved.

The detection method comprises the following steps:

paper chromatography: accurately sucking 20 mul of supernatant by using a microsyringe, and spotting the supernatant on No. 3 Xinhua filter paper, wherein the chromatographic solution is ammonia water: water 1: 4. spreading in a spreading cylinder until the front edge of the chromatographic solution reaches above 2/3 of the chromatographic paper, taking out, drying in the air, fumigating with concentrated hydrochloric acid fume for 5 min, drawing purple spots corresponding to the standard sample under a 253nm ultraviolet lamp, cutting off the spots, soaking in 10ml of 0.05mol/l NaOH solution for 60 min, and measuring the absorbance A at a wavelength of 260nm under an 752 ultraviolet spectrophotometer. Substituting the value A into the formula guanosine content g/l-A value x 12.5327 x 10.

The standard is as follows: 5% guanosine standard solution

The instrument comprises the following steps: a paper layer chromatography cylinder, an ultraviolet detector and a 752 ultraviolet spectrophotometer.

It should be understood that the examples are merely for illustrative purposes and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.