CN105671184B - Method for measuring concentration of thalli in culture medium - Google Patents
Method for measuring concentration of thalli in culture medium Download PDFInfo
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- CN105671184B CN105671184B CN201610185344.3A CN201610185344A CN105671184B CN 105671184 B CN105671184 B CN 105671184B CN 201610185344 A CN201610185344 A CN 201610185344A CN 105671184 B CN105671184 B CN 105671184B
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to an improved method for determining thallus concentration in a composite culture medium. The method comprises the steps of fermentation liquor heat preservation, enzymolysis, TCA extraction, absorbance determination, calculation and the like. The improvement points of the invention are mainly as follows: the method better avoids the interference of nucleic acid or analogues contained in materials such as soybean meal, peanut meal and the like in solid components when extracting the thallus nucleic acid from the culture medium by proper enzymolysis treatment of the fermentation liquor, can more accurately and fully extract the thallus nucleic acid in the fermentation liquor, thereby more truly reflecting the thallus amount in the fermentation liquor, providing reference and guidance for fermentation production better and showing better practical value and popularization and application significance.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to an improved method for determining the concentration of thalli in a composite culture medium.
Background
The determination of the thallus concentration is an important index for reflecting the growth and metabolism conditions of microorganisms, and each production process can be well controlled only by accurate data. In the existing industrialized microbial fermentation process, a fermentation culture medium containing a large amount of solid components, such as soybean cake powder, peanut cake powder and the like, is often used, and the solid components are mixed with thalli, so that the measurement of the concentration of the thalli in a fermentation liquid becomes a complicated and difficult problem, and the problem is remained for a long time.
At present, most antibiotic factories generally adopt a loading volume Method (Cell bulk measuring Method, centrifugal compression Cell volume Method, The Packed-Cell volume Method) for measuring The concentration of The bacteria in fermentation liquor. However, in the actual process, the method is greatly influenced by the solid raw material components in the fermentation liquor, and when the method is used in the stable period of the thallus growth, the increase of the concentration value of the thallus is mainly the result of the synthesis of some lipids and secondary metabolites, and the change of the thallus concentration cannot be really represented. Therefore, the value obtained by the volumetric filling method is the result of the addition of two variables, i.e., the cell concentration and the solid medium component, and does not reflect the actual cell concentration. Moreover, data measured by the method has no obvious rule in the whole fermentation process, and the real growth rule of the thalli cannot be reflected.
In the existing method for measuring the concentration of the bacteria, another more accurate and commonly used method is to measure the concentration of the nucleic acid contained in the bacteria in a culture medium, the amount of the nucleic acid is used as an index for measuring the concentration of the bacteria, and the extraction of the nucleic acid in the bacteria is mainly obtained by a TCA extraction method. However, in actual use, the method is greatly influenced by the components of the culture medium. For example, if the fermentation medium contains insoluble components such as soybean meal, peanut meal, etc., it may contain a small amount of nucleic acids or nucleic acid analogs, and these substances are extracted into TCA together with the nucleic acids in the cells, thereby interfering with the measurement to some extent; especially when the culture medium uses germ powder as nutrient component, it contains high content of nucleic acid, which often causes serious interference to the measured value. Therefore, the method needs to be further improved to obtain more accurate thallus concentration data.
Disclosure of Invention
The invention aims to provide an improved TCA (ternary content addressable memory) measuring method for measuring the concentration of nucleic acid in fermentation liquor, so that the concentration data of bacteria can be reflected more accurately.
The technical scheme adopted by the invention is detailed as follows.
A method for measuring the concentration of thalli in a culture medium specifically comprises the following steps:
(1) taking fermentation liquor to be tested, and preserving the heat of the fermentation liquor at 45-50 ℃ for 20-40 min, preferably at 50 ℃ for 30 min; after the heat preservation of the fermentation liquor is finished, the viscosity of the fermentation liquor is reduced, and the fluidity is good;
then adding cellulase and pectinase into the fermentation liquor after the heat preservation is finished, and uniformly mixing to carry out enzymolysis on the fermentation liquor, wherein the fermentation liquor can be reversely and uniformly mixed every 5 min;
the fermentation liquid can be, for example, an aureomycin fermentation liquid, specifically, an aureomycin fermentation liquid which is normally fermented for 0-120 h or an aureomycin seed liquid which is cultured for 0-36 h;
the total addition amount of the cellulase and the pectinase is 2-4% of the mass of the fermentation liquor; wherein the mass ratio of the cellulase (the enzyme activity is more than or equal to 25 ten thousand U/g) to the pectinase (the enzyme activity is more than or equal to 10 ten thousand U/g) is 1: 1.5-2.0, the temperature is controlled to be 35-50 ℃ during enzymolysis, the enzymolysis time is more than or equal to 1h, but not more than 5h, so that the influence on the quality of the fermentation liquor due to too long enzymolysis time is avoided;
(2) centrifuging the fermentation liquor obtained in the step (1) after the enzymolysis at 0 ℃ at 3000 Xg for 10min, discarding the supernatant, and retaining the precipitate;
(3) adding 5% (V/V) TCA (trichloroacetic acid) solution at the temperature of 2-4 ℃ into the precipitate in the step (2), fully stirring and washing the precipitate, centrifuging at the temperature of 0 ℃ and 3000 Xg for 10 minutes, and removing the supernatant; repeating the steps once;
the volume ratio of the TCA solution to the sample solution (i.e., the fermentation broth to be tested obtained in step (1)) is 1:1 to 1.5; the preferred ratio is 1:1.5, but because the consumption of TCA is larger at the moment, the TCA can be properly adjusted according to the actual condition;
(4) adding 5% (V/V) TCA into the precipitate obtained in the step (3), fully stirring, stirring and extracting in a water bath at 100 ℃ for 10-15 min, then placing in ice water for cooling, centrifuging at 0 ℃ and 3000 Xg for 10min, taking the supernatant, and removing the precipitate;
(5) the supernatant from step (4) was diluted appropriately with 5% (V/V) TCA, OD260The absorbance was measured and the nucleic acid concentration was calculated according to the following formula:
in the formula: OD260nmThe absorbance of the TCA extract at 260nm is measured; n is the dilution multiple of TCA extract; 0.022 represents the absorbance at a nucleic acid concentration of 1. mu.g/ml, which is a constant.
The main technical thought of the invention is as follows: the addition of cellulase and pectinase destroys cell walls (biological enzyme has no influence on microbial cells) of part of plant materials (soybean meal, corn meal, peanut meal and the like) in the composite culture medium, so that nucleic acid in plant cells is released out of the cells, and released plant contents are removed along with other impurities through subsequent steps of centrifugation, supernatant removal and the like, so that the interference of the nucleic acid and analogues in the raw materials of the culture medium on measurement results is avoided.
In general, the present invention belongs to an improved TCA method for measuring the concentration of nucleic acid in fermentation liquor, and the improvement point is mainly as follows: the method better avoids the interference of nucleic acid or analogues contained in materials such as soybean meal, peanut meal and the like in solid components when extracting the thallus nucleic acid from the culture medium by proper enzymolysis treatment of the fermentation liquor, can more accurately and fully extract the thallus nucleic acid in the fermentation liquor, thereby more truly reflecting the thallus amount in the fermentation liquor, providing reference and guidance for fermentation production better and showing better practical value and popularization and application significance.
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FIG. 1 is a comparison graph of the measurement results of the present invention and the existing measurement method for the nucleic acid concentration of different fermentation time in a batch of fermentation broth of chlortetracycline.
Detailed Description
The present invention is further illustrated by the following examples. Before describing the specific embodiments, some of the test equipment and materials of the present invention are briefly described as follows.
The preparation process of the aureomycin fermentation liquor in the following examples is provided by Huazhong Zhengzhong Dai Co., Ltd, in Lima shop, and is prepared by adopting streptomyces aureofaciens, and the preparation process of the aureomycin fermentation liquor is roughly as follows:
(1) preparing slant hypha, namely culturing streptomyces aureofaciens spores stored in a sandy soil tube in a slant culture mode at the temperature of 32 +/-1 ℃ for 3-5 days to prepare slant hypha of a mother bottle;
(2) preparing seed liquid, namely inoculating the slant hypha in the mother bottle in the step (1) into a culture bottle, and performing shake culture at the temperature of 26-32 ℃ at 230 rpm for 18-24 h to prepare the seed liquid;
(3) preparing a chlortetracycline fermentation liquid, namely inoculating the seed liquid in the step (2) into a fermentation tank, and culturing at 26-30 ℃ and under the condition of pH 5.4-6.5 to obtain the chlortetracycline fermentation liquid;
the fermentation medium of the streptomycin in the fermentation tank comprises the following specific components (1L): 20g of peanut cake powder, 18g of soybean cake powder, 80g of corn starch, 7.0g of yeast powder, 12g of corn steep liquor, 3g of soybean oil, 0.1g of amylase, 2.0g of sodium chloride, 6g of ammonium sulfate, 7.0g of calcium carbonate, 0.2g of magnesium sulfate and 0.3g of potassium dihydrogen phosphate, and the pH value is natural.
Example 1
The method for measuring the concentration of the bacterial cells in the culture medium provided by the embodiment specifically comprises the following steps:
(1) taking 5mL of fermentation broth of aureomycin which is fermented for 0h (i.e. before the sterilization of the fermentation medium of the aureomycin in the fermentation tank), and preserving the temperature of the fermentation broth for 30min at 50 ℃; after the heat preservation is finished, the viscosity of the fermentation liquid is reduced, the fluidity is good, and a large amount of precipitation does not occur within 3 min.
It is to be explained that the viscosity difference of the fermentation liquid at different fermentation time is large, the viscosity is within 1s when the fermentation liquid is not inoculated, the viscosity of the fermentation liquid in the normal fermentation process is about 1-15 s, and the viscosity of the fermentation liquid is detected by adopting a capillary viscometer.
Then adding cellulase and pectinase, mixing uniformly to carry out enzymolysis on the fermentation liquor, and reversing and mixing uniformly every 5 min;
the total adding amount of the cellulase and the pectinase is 2.6 percent of the mass of the fermentation liquid, wherein the using amounts of the cellulase (the enzyme activity =25 ten thousand U/g) and the pectinase (the enzyme activity =10 ten thousand U/g) are 0.05 g and 0.08g respectively, the enzymolysis temperature is controlled at 40 ℃, and the enzymolysis time is 3 hours;
(2) centrifuging the fermentation liquor obtained in the step (1) after the enzymolysis at 0 ℃ at 3000 Xg for 10min, and removing the supernatant;
(3) adding 5mL of 5% (V/V) TCA solution at 4 ℃ into the precipitate in the step (2), fully stirring and washing the precipitate, centrifuging at 0 ℃ and 3000 Xg for 10 minutes, and removing the supernatant; repeating the steps once;
(4) adding 5mL of 5% TCA into the precipitate obtained in the step (3), fully stirring, stirring and extracting in a water bath at 100 ℃ for 15min, then placing in ice water for cooling, centrifuging at 0 ℃ and 3000 Xg for 10min, taking the supernatant, and removing the precipitate;
(5) the supernatant of step (4) was diluted appropriately with 5% TCA, OD260The absorbance was measured and the nucleic acid concentration was calculated according to the following formula:
the calculation shows that the nucleic acid concentration in the aureomycin fermentation liquor is 0.17 mg/mL. At this time, the fermentation liquor is not inoculated with the aureomycin fermentation strain, so that the solid materials such as peanut cake powder, soybean cake powder and the like in the culture medium contain trace nucleic acid substances, and the determination of the nucleic acid concentration in the subsequent fermentation process is easily influenced to a certain extent.
Comparative example
In the same manner, the nucleic acid concentration in the same batch of the aureomycin fermentation medium was measured without the need for the incubation and the enzymatic hydrolysis (i.e., without the above step (1)), and the result showed that the nucleic acid concentration was 2.73 mg/mL.
As can be seen from the detection results of the present embodiment and the comparative example, the influence of nucleic acid in the original fermentation broth can be greatly reduced after the enzymolysis treatment, and a better guarantee can be provided for the real detection of the thallus concentration in the production process.
Example 2
The concentration of nucleic acid in the same batch of aureomycin fermentation broth was determined in the same manner as in example 1, except that the incubation temperature of the fermentation broth in step (1) was adjusted to 45 ℃ and the incubation time was adjusted to 30 min. The final calculation showed that the concentration of nucleic acid in the mycelium was 0.29 mg/mL.
Example 3
The same as example 1, the nucleic acid concentration in the same batch of aureomycin fermentation liquor is measured, only the proportion of the biological enzyme during enzymolysis is adjusted, and the specific dosage of the biological enzyme is as follows: 0.07g of cellulase and 0.13g of pectinase, and the final calculated nucleic acid concentration is 0.06 mg/mL. From this result, it can be seen that the amount of nucleic acid originally in the fermentation broth at this time had little influence on the subsequent measurement.
According to the operation method, the thallus concentration in the fermentation liquor of the chlortetracycline fermentation liquor after different fermentation time is measured, and meanwhile, the measurement is carried out according to the existing (namely, the operation method in the comparative example), and the related measurement results are shown in figure 1. As can be seen from FIG. 1, the improved TCA assay method provided by the invention can better solve the defect of inaccurate concentration measurement of strains at the early stage of fermentation culture, can better reflect the real growth rule of thalli, and can better guide fermentation production.
Claims (2)
1. A method for measuring the concentration of bacteria in a culture medium, which is characterized by comprising the following steps:
(1) taking fermentation liquor to be tested, and preserving the temperature of the fermentation liquor for 20-40 min at 45-50 ℃; then adding cellulase and pectinase to carry out enzymolysis on the fermentation liquor;
the total addition amount of the cellulase and the pectinase is 2-4% of the mass of the fermentation liquor;
the mass ratio of cellulase to pectinase is =1 to 1.5-2.0, wherein the enzyme activity of the cellulase is more than or equal to 25 ten thousand U/g, and the enzyme activity of the pectinase is more than or equal to 10 ten thousand U/g;
the enzymolysis temperature is controlled to be 35-50 ℃, and the enzymolysis time is more than or equal to 1h but not more than 5 h;
the fermentation liquor is chlortetracycline fermentation liquor;
in the aureomycin fermentation liquor, the aureomycin fermentation culture medium comprises the following specific components in each L: 20g of peanut cake powder, 18g of soybean cake powder, 80g of corn starch, 7.0g of yeast powder, 12g of corn steep liquor, 3g of soybean oil, 0.1g of amylase, 2.0g of sodium chloride, 6g of ammonium sulfate, 7.0g of calcium carbonate, 0.2g of magnesium sulfate and 0.3g of potassium dihydrogen phosphate, and the pH value is natural;
(2) centrifuging the fermentation liquor obtained in the step (1) after the enzymolysis is finished, discarding the supernatant, and keeping the precipitate;
(3) adding 5% volume fraction TCA solution at 2-4 ℃ into the precipitate in the step (2), washing the precipitate, centrifuging, and discarding the supernatant; repeating the steps once;
the volume ratio of the TCA solution to the fermentation liquor to be detected in the step (1) is 1:1 to 1.5;
(4) adding 5% volume fraction of TCA solution into the precipitate in the step (3), stirring, extracting in a water bath at 100 ℃ for 10-15 min, cooling in ice water, centrifuging, taking supernatant, and removing the precipitate;
(5) diluting the supernatant obtained in step (4) with 5% TCA solution, OD260The absorbance was measured and the nucleic acid concentration was calculated according to the following formula:
in the formula: OD260The absorbance of the TCA extract at 260nm is measured; n is the dilution factor of the TCA extract.
2. The method for measuring the concentration of bacteria in a culture medium according to claim 1, wherein the aureomycin fermentation broth is specifically aureomycin fermentation broth fermented for 0-120 h.
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| US4969741A (en) * | 1989-07-21 | 1990-11-13 | Massachusetts Institute Of Technology | Measurement of solid particle concentration in presence of a second particle type |
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| US4969741A (en) * | 1989-07-21 | 1990-11-13 | Massachusetts Institute Of Technology | Measurement of solid particle concentration in presence of a second particle type |
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| 复合培养基中菌体浓度的测定研究-总核酸测定法;李树瑞等;《华东化工学院学报》;19821231(第2期);第177页倒数第2段至第186页第1段 * |
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Denomination of invention: A method for measuring bacterial concentration in culture medium Effective date of registration: 20230324 Granted publication date: 20200512 Pledgee: Industrial Bank Co.,Ltd. Zhumadian Branch Pledgor: ZHUMADIAN HUAZHONG CHIA TAI Co.,Ltd. Registration number: Y2023980036173 |