Method for screening optimal proportion of calcium carbonate from fermentation liquor and application thereof
Technical Field
The invention belongs to the technical field of antibiotic medicines, and particularly relates to a method for screening an optimal proportion of calcium carbonate from fermentation liquor and application thereof.
Background
The main function of calcium carbonate in the fermentation medium is a pH buffering agent, and organic acid and inorganic acid can be neutralized to ensure the stability of the pH of the whole system, and calcium exists in an ionic state after neutralization. In order to maintain the pH stability during fermentation, calcium carbonate is often added as an excess of pH buffer to the fermentation medium. At present, oxalic acid is often used for removing calcium ions in the pretreatment of fermentation liquor after fermentation is finished, and redundant calcium carbonate reacts with oxalic acid in the pretreatment of the fermentation liquor and is then filtered and removed together with bacterial residues. Therefore, the redundant calcium carbonate can be removed only by adding sufficient oxalic acid, the pretreatment cost of the fermentation liquor is greatly improved, the residual calcium can bring troubles to the subsequent product aftertreatment, and even the residue proportion of the finished product is increased.
How to determine the optimal proportion of calcium carbonate in a fermentation medium so as to improve the quality of fermentation products and reduce the fermentation cost is a significant research. In the prior art, in order to determine the optimal addition amount of calcium carbonate in fermentation liquor, an orthogonal experiment is required for optimization, generally, the addition amount of calcium carbonate is changed to perform a series of parallel tests, and a fermentation proportion is determined according to a fermentation result, so that the whole process is long in time consumption and high in cost, and the result is only suitable for the specific fermentation liquor. If the method for determining the optimal proportion of the calcium carbonate in the fermentation liquor, which is simple to operate, high in accuracy, short in time consumption and wide in applicability, can be developed, the method has important application value.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for screening the optimal proportion of calcium carbonate from fermentation liquor, which can simply and quickly determine the optimal proportion of calcium carbonate in the fermentation liquor, obviously reduce the addition of calcium carbonate in the obtained fermentation liquor, reduce the use amount of oxalic acid in the subsequent fermentation liquor pretreatment, and bring help for the subsequent deep treatment of the fermentation liquor and the improvement of the quality of finished products. The invention also provides two fermentation liquors prepared by the method.
A method for screening the optimal proportion of calcium carbonate from fermentation liquor comprises the following steps:
(1) filtering the fermentation liquor at the production end point to obtain clean filtrate;
(2) preparing an oxalic acid solution for titration of the filtrate, and calculating a theoretical value of the amount of oxalic acid required by the complete removal of calcium carbonate added in the fermentation formula;
(3) sequentially and progressively reducing and adding oxalic acid with different amounts according to a theoretical value, performing vacuum filtration on the obtained solution again, adding a proper amount of oxalic acid solution into the filtrate after the vacuum filtration, standing for at least 2 hours, and observing whether a precipitate is generated or not;
(4) and calculating the actual utilization amount of the calcium carbonate according to the condition of precipitation generation, and determining the optimal proportion of the calcium carbonate according to the actual utilization amount.
Preferably, the fermentation broth is kitasamycin fermentation broth or tylosin fermentation broth.
Preferably, the concentration of the oxalic acid solution is 5-15%. More preferably 10%.
The method for screening the optimal proportion of the calcium carbonate comprises the following steps:
in the step (1), the fermentation broth is a fermentation end-point tank-placing fermentation broth. The filtering method adopts filter paper filtration or vacuum filtration.
In the step (2), the oxalic acid in the oxalic acid solution is completely dissolved, and the solution is prepared according to the corresponding relation between the solubility of the oxalic acid and the temperature.
In the step (3), the solution added with the experimental amount of oxalic acid is filtered according to the process condition of the product. Clarifying the filtered filtrate, adding a proper amount of oxalic acid solution, and standing for observation. After the oxalic acid solution is added again, the mixture is placed for at least 2 hours to improve the accuracy of the screening conclusion.
In step (4), the amount of calcium carbonate added in the experiment should be slightly more than that obtained in the experiment in consideration of the possibility that calcium carbonate may participate in other synthesis reactions in the fermentation, and preferably, the optimum ratio of the calcium carbonate is higher than the calculated actual utilization amount of calcium carbonate. Preferably, in the step (4), the optimal ratio of the calcium carbonate is 1.45 to 1.85 times of the calculated actual utilization amount of the calcium carbonate. Through comparison experiments of fermentation culture, the use amount of calcium carbonate is 1.45-1.85 times of the actual use amount of the calculated calcium carbonate, the working procedure and cost of product post-treatment can be reduced, and the obtained fermentation liquor has higher biological value. When the dosage of calcium carbonate is further reduced, the fermentation process is affected, so that the biological value of the product is low, and the dosage of calcium carbonate is not easy to be too low.
The invention also provides kitasamycin fermentation liquor determined by the method for screening the optimal proportion of calcium carbonate, wherein the proportion of light calcium carbonate is 0.21-0.26 percent by weight.
Further, the paint specifically comprises the following components in parts by weight:
the invention also provides a production method of kitasamycin, which adopts the kitasamycin fermentation liquor for fermentation production.
The invention also provides tylosin fermentation liquor determined by the method for screening the optimal proportion of calcium carbonate, and the proportion of light calcium carbonate is 0.19-0.21 percent in percentage by weight.
Further specifically, the paint comprises the following components in parts by weight:
the invention also provides a production method of tylosin, which comprises the following steps: the tylosin fermentation liquor is adopted for fermentation production.
According to the invention, the filtrate obtained by directly filtering the fermentation liquor is titrated by oxalic acid to determine the utilization condition of calcium carbonate in the fermentation medium; and simultaneously, performing a biological potency comparison test of fermentation culture on the screened calcium carbonate to determine the optimal fermentation ratio of the calcium carbonate.
Compared with the prior art, the invention has the beneficial effects that: the invention adopts the utilization condition of calcium carbonate to optimize the proportion, and the method can more accurately use individual ions such as calcium carbonate and the like, different from the orthogonal optimization experimental scheme of a basic culture medium adopted in a fermentation culture medium. Because calcium carbonate is used in formulations primarily to buffer the pH of the solution, excessive use of calcium carbonate does not result in increased metabolites, but rather, excessive calcium carbonate adds cost and unnecessary trouble to the product's post-processing. The screening method of the invention improves the adding precision of calcium carbonate, reduces the working procedure and cost of product post-treatment, and improves the biological value of the end-point fermentation liquor to a certain extent.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
The utilization conditions of the gentamicin and the tylosin calcium carbonate in the end-point fermentation liquor are respectively determined by an oxalic acid titration method, and are specifically as follows:
1. calcium carbonate utilization assessment of kitasamycin
Taking kitasamycin fermentation liquor to directly filter, collecting filtrate, taking 100ml of filtrate each time, adding an experimental amount of prepared 10% oxalic acid solution (6.6 ml of oxalic acid solution is the maximum value of calcium ion removal added theoretically), then adjusting the pH value to be 3.1 by using 10% sodium hydroxide, carrying out vacuum filtration on the fermentation liquor again, adding oxalic acid into the filtered filtrate again, standing for 2 hours, and observing whether precipitates are generated or not, wherein the specific conditions are as follows:
from the above three consecutive experiments, it was shown that there is a certain excess of calcium carbonate added in the fermentation medium, about 1/2 excess, the amount of calcium carbonate added in the original formulation of kitasamycin is 0.31%, the actual utilization amount is about 0.14%, considering that calcium carbonate may participate in other reactions of fermentation metabolism, and considering that pH fluctuation may be affected when the production is abnormal, resulting in low biological value of the product, the experiment was conducted with 0.26% (1.85 times of actual utilization amount) and 0.31%, respectively.
2. Evaluation of calcium carbonate utilization of tylosin
Directly carrying out vacuum filtration on tylosin fermentation liquor, collecting filtrate, taking 100ml of filtrate each time, adding an experimental amount of prepared 10% oxalic acid solution (2.7 ml of oxalic acid solution is the maximum value of calcium ion removal added theoretically), carrying out vacuum filtration on the fermentation liquor again, adding oxalic acid into the filtered filtrate again, standing for 2 hours, observing whether a precipitate is generated or not, and specifically:
from the above three consecutive experiments, it was shown that there was a certain excess of calcium carbonate added to the fermentation medium, at least 1/3 excess, the amount of calcium carbonate added in the original formulation of tylosin was 0.22%, the actual utilization was about 0.13%, and the experiments were conducted with 0.19% (about 1.46 times the actual utilization) and 0.22% respectively.
Example 1:
the experimental group and the control group in the kitasamycin fermentation medium are mainly characterized in that the proportion of light calcium carbonate is different and is respectively 0.26 percent and 0.31 percent. The mixture ratio of other substances is as follows:
name of raw materials
|
Proportioning raw materials%
|
Name of raw materials
|
Proportioning raw materials%
|
Corn starch
|
1
|
Protein powder
|
0.21
|
Wheat germ powder
|
1.15
|
Oleic acid methyl ester
|
1.128
|
Bean pulp
|
0.94
|
Potassium dihydrogen phosphate
|
0.073
|
H8 natural killer
|
0.019
|
Manganese chloride
|
0.052
|
Glucose
|
1.50
|
Zinc sulfate
|
0.00625
|
N-propanol
|
0.30
|
Soybean oil
|
2.4 |
And respectively carrying out biological titer detection on fermentation liquor at the fermentation end point, wherein the experimental group and the control group are 10850u/ml and 10784u/ml respectively.
Example 2:
the experimental group and the control group in the kitasamycin fermentation medium are mainly characterized in that the proportion of light calcium carbonate is different, and is respectively 0.19 percent (about 1.36 times of the actual utilization amount) and 0.31 percent. The mixture ratio of other substances is as follows:
name of raw materials
|
Proportioning raw materials%
|
Name of raw materials
|
Proportioning raw materials%
|
Corn starch
|
1
|
Protein powder
|
0.21
|
Wheat germ powder
|
1.15
|
Oleic acid methyl ester
|
1.128
|
Bean pulp
|
0.94
|
Potassium dihydrogen phosphate
|
0.073
|
H8 natural killer
|
0.019
|
Manganese chloride
|
0.052
|
Glucose
|
1.50
|
Zinc sulfate
|
0.00625
|
N-propanol
|
0.30
|
Soybean oil
|
2.4 |
And respectively carrying out biological titer detection on fermentation liquor at the fermentation end point, wherein the experimental group and the control group are respectively 8159u/ml and 10893 u/ml.
The results of example 2 show that when the proportion of precipitated calcium carbonate is controlled to 1.36 times the amount actually used, the biological value of the product is greatly reduced, probably because calcium carbonate participates in other reactions of fermentation metabolism, and therefore, the proportion of precipitated calcium carbonate cannot be completely determined according to the amount actually consumed.
Example 3:
the experimental group and the control group in the kitasamycin fermentation medium are mainly characterized in that the proportion of light calcium carbonate is different, and is respectively 0.21% (about 1.46 times of the actual utilization amount) and 0.31%. The mixture ratio of other substances is as follows:
name of raw materials
|
Proportioning raw materials%
|
Name of raw materials
|
Proportioning raw materials%
|
Corn starch
|
1
|
Protein powder
|
0.21
|
Wheat germ powder
|
1.15
|
Oleic acid methyl ester
|
1.128
|
Bean pulp
|
0.94
|
Potassium dihydrogen phosphate
|
0.073
|
H8 natural killer
|
0.019
|
Manganese chloride
|
0.052
|
Glucose
|
1.50
|
Zinc sulfate
|
0.00625
|
N-propanol
|
0.30
|
Soybean oil
|
2.4 |
And respectively carrying out biological titer detection on fermentation liquor at the fermentation end point, wherein the experimental group and the control group are 10865u/ml and 10776u/ml respectively.
The results of example 3 show that when the proportion of light calcium carbonate is increased to 1.46 times the amount actually used, the fermentation level can be well secured.
Example 4:
the experimental group and the control group in the tylosin fermentation medium are mainly characterized in that the proportion of light calcium carbonate is different and is respectively 0.19 percent and 0.22 percent. The mixture ratio of other substances is as follows:
name of raw materials
|
Proportioning raw materials%
|
Name of raw materials
|
Proportioning raw materials%
|
Corn oil
|
2.8
|
Potassium chloride
|
0.11
|
Sodium hydroxide
|
0.033
|
Diammonium hydrogen phosphate
|
0.044
|
Corn flour
|
0.67
|
Nickel sulfate hexahydrate
|
0.00044
|
Fish meal
|
1.14
|
Cobalt chloride hexahydrate
|
0.00033
|
Soybean cake powder
|
1.0
|
|
|
And respectively carrying out biological titer detection on fermentation liquor at the fermentation end point, wherein the fermentation liquor at the experimental group and the fermentation end point at the control group are 10293u/ml and 10231u/ml respectively.
Example 5:
the experimental group and the control group in the tylosin fermentation medium are mainly characterized in that the proportions of precipitated calcium carbonate are different, namely 0.18% (about 1.38 times of the actual utilization) and 0.22%, respectively. The mixture ratio of other substances is as follows:
name of raw materials
|
Proportioning raw materials%
|
Name of raw materials
|
Proportioning raw materials%
|
Corn oil
|
2.8
|
Potassium chloride
|
0.11
|
Sodium hydroxide
|
0.033
|
Diammonium hydrogen phosphate
|
0.044
|
Corn flour
|
0.67
|
Nickel sulfate hexahydrate
|
0.00044
|
Fish meal
|
1.14
|
Cobalt chloride hexahydrate
|
0.00033
|
Soybean cake powder
|
1.0
|
|
|
And respectively carrying out biological titer detection on fermentation liquor at the fermentation end point, wherein the experimental group and the control group are respectively 8131u/ml and 9983 u/ml.
The results of example 5 show that when the proportion of precipitated calcium carbonate is controlled to 1.36 times the amount actually used, the biological potency of the product tylosin is greatly reduced, probably because calcium carbonate participates in other reactions of fermentation metabolism, and therefore, the proportion of precipitated calcium carbonate cannot be completely determined according to the amount actually used.
Example 6:
the experimental group and the control group in the tylosin fermentation medium are mainly different in the proportion of precipitated calcium carbonate, 0.21% (about 1.61 times of the actual utilization amount) and 0.22%, respectively. The mixture ratio of other substances is as follows:
and respectively carrying out biological value detection on fermentation liquor at the fermentation end point, wherein the biological value of the fermentation liquor at the fermentation end point is 10654u/ml and 10569u/ml respectively for an experimental group and a control group.