CN111705092B - Feed supplement for improving fermentation titer of spectinomycin and preparation method and use method thereof - Google Patents
Feed supplement for improving fermentation titer of spectinomycin and preparation method and use method thereof Download PDFInfo
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Abstract
The invention provides a supplement for improving the fermentation titer of spectinomycin, and a preparation method and a use method thereof, belonging to the technical field of bio-pharmaceutical fermentation, wherein the supplement comprises the following raw materials for preparation: starch, soybean oil, potassium dihydrogen phosphate, amylase and water. In the fermentation process of spectinomycin, the total sugar of the feed liquid of the fermentation tank can be controlled to be maintained in a certain range by supplementing the feed supplement disclosed by the invention, so that a carbon source becomes a restrictive matrix, thalli enter a secondary metabolite synthesis stage, and meanwhile, the total sugar bottom limit value is controlled, and the phenomenon that hyphae autolysis advances due to insufficient nutrition of mycelia to cause reduction of product synthesis amount is avoided. The feed supplement provided by the invention can reduce power consumption in the fermentation process of spectinomycin, improve the fermentation level, reduce the fermentation cost and has an obvious cost competitive advantage.
Description
Technical Field
The invention relates to the technical field of bio-pharmaceutical fermentation, in particular to a supplement for improving the fermentation titer of spectinomycin and a preparation method and a use method thereof.
Background
Spectinomycin (Sptectinomycin) is an aminoglycoside antibiotic found in the sixties of the last century, also known as spectinomycin, a broad-spectrum antibiotic produced by Streptomyces spectabilis (Streptomyces spectabilis), and its hydrochloride is a white or off-white crystalline powder. Spectinomycin has activity against most gram-positive and gram-negative bacteria, is effective against infections caused by these bacteria, and has good inhibitory effect against pneumonia bacillus, influenza bacillus, and mycoplasma. Besides, spectinomycin has low toxicity and broad spectrum, and can be used as a feed additive to promote the growth of various animals.
As the requirement of dissolved oxygen is very high in the fermentation process of spectinomycin, batch fermentation is adopted at present, and required nutrient substances are added into a culture medium at one time before fermentation until the batch fermentation is finished. However, the nutrition is too rich in the early stage of fermentation, so that the thalli grow too fast, the nutrition is insufficient in the later stage, and the problems of premature senility and insufficient aftereffect of the strain exist. Therefore, feeding treatment is required in the fermentation process of the spectinomycin.
In the prior art, spectinomycin fermentation feed supplement liquid mainly comprises a rapid carbon source and a nitrogen source, wherein the carbon source comprises the following components in percentage by weight: liquefied starch and maltodextrin, etc., nitrogen source: corn steep liquor, ammonium sulfate, ammonia water and the like. The current feed supplement has high requirements on the power conditions of a fermentation tank, and causes the problems of high comprehensive cost, low fermentation titer and the like.
Disclosure of Invention
The invention aims to provide a supplement for improving the fermentation titer of spectinomycin, and a preparation method and a use method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a feed supplement for improving the fermentation titer of spectinomycin, which comprises the following raw materials for preparation in percentage by mass: 12 to 20 percent of starch, 0.7 to 1.2 percent of soybean oil, 0.07 to 0.12 percent of monopotassium phosphate, 0.008 to 0.015 percent of amylase and the balance of water.
Preferably, the feed comprises the following raw materials for preparation in percentage by mass: 15% of starch, 1% of soybean oil, 0.1% of monopotassium phosphate, 0.01% of amylase and the balance of water.
Preferably, the amylase comprises a thermostable alpha-amylase.
The invention also provides a preparation method of the supplement in the scheme, which comprises the following steps:
and mixing the starch, the soybean oil, the monopotassium phosphate, the amylase and the water, and liquefying at 80-100 ℃ for 35-45 min to obtain the supplementary material.
Preferably, after the liquefaction, the method further comprises sterilizing and cooling the liquefied feed liquid; the sterilization temperature is 130-140 ℃, and the sterilization time is 3-5 min.
The invention also provides a use method of the feed supplement in the scheme, which comprises the following steps:
1) fermenting by utilizing actinomycetes to produce spectinomycin for 30-40 h, and supplementing the supplementary material for the first time when the total sugar in the fermentation material is less than 3.5g/100 ml;
2) fermenting for 50-70 h, and supplementing the supplementary material for the second time when the total sugar in the fermentation material is less than 2.5g/100 ml;
3) and fermenting for 80-100 hours, and supplementing the supplementary material for the third time when the total sugar in the fermentation material is less than 1.5g/100 ml.
Preferably, when the total sugar in the fermentation material in the step 1) is lower than 3g/100ml, the feeding material is supplemented for the first time; the volume ratio of the supplementary material to the fermentation material is 3-5: 100.
preferably, when the total sugar in the fermentation material in the step 2) is lower than 1.5g/100ml, the supplementary material is supplemented for the second time; the volume ratio of the supplementary material to the fermentation material is 3-5: 100.
preferably, the volume ratio of the fed materials to the fermented materials in the step 3) is 1.5-3: 100.
the invention has the beneficial effects that: the invention provides a feed supplement for improving the fermentation titer of spectinomycin, which comprises the following raw materials for preparation: starch, soybean oil, potassium dihydrogen phosphate, amylase and water. In the feed supplement, soybean oil and starch are used as carbon sources; starch is liquefied and hydrolyzed by amylase to generate micromolecular carbon sources such as glucose, maltose and the like, so that hyphae can be conveniently and quickly utilized, the viscosity of fermentation liquid and the concentration of thalli can be improved, and a quick carbon source can be provided for the generation of a target product; soybean oil is used as a slow carbon source and plays two roles in the fermentation process of spectinomycin, and firstly, the soybean oil is decomposed into short-chain fatty acid by lipase for the growth of mycelium and the synthesis and utilization of products; secondly, play the defoaming effect in the fermentation process, control fermentation cylinder operation volume, improve equipment coefficient of charging and utilization ratio. In the fermentation process of spectinomycin, the total sugar of the feed liquid of the fermentation tank can be controlled to be maintained in a certain range by supplementing the feed supplement disclosed by the invention, so that a carbon source becomes a restrictive matrix, thalli enter a secondary metabolite synthesis stage, and meanwhile, the total sugar bottom limit value is controlled, and the phenomenon that hyphae autolysis advances due to insufficient nutrition of mycelia to cause reduction of product synthesis amount is avoided. The feed supplement provided by the invention can reduce power consumption in the fermentation process of spectinomycin, improve the fermentation level, reduce the fermentation cost and has an obvious cost competitive advantage. In the fermentation process of the spectinomycin, the fermentation titer of the spectinomycin can be effectively improved by supplementing the supplement disclosed by the invention, so that the fermentation level (the generation amount of the spectinomycin) of the spectinomycin is improved by 10-15% compared with that of a non-supplement process, and the production cost is reduced by 5-8%.
Drawings
FIG. 1 shows the effect of different soybean oil concentrations on the potency of spectinomycin in the tank;
FIG. 2 is a graph showing the effect of different starch concentrations on the potency of spectinomycin in the tank;
FIG. 3 shows the effect of different potassium dihydrogen phosphate concentrations on the tank-out potency of spectinomycin;
FIG. 4 shows the effect of different supplement materials on the titer of spectinomycin in the tank.
Detailed Description
The invention provides a feed supplement for improving the fermentation titer of spectinomycin, which comprises the following raw materials for preparation in percentage by mass: 12 to 20 percent of starch, 0.7 to 1.2 percent of soybean oil, 0.07 to 0.12 percent of monopotassium phosphate, 0.008 to 0.015 percent of amylase and the balance of water; preferably, the feed comprises the following raw materials for preparation in percentage by mass: 15% of starch, 1% of soybean oil, 0.1% of monopotassium phosphate, 0.01% of amylase and the balance of water.
According to the invention, the addition combination of starch and amylase can liquefy and hydrolyze starch to generate micromolecular carbon sources such as glucose and maltose, so that hyphae can be conveniently and rapidly utilized, the viscosity of fermentation liquid and the concentration of thalli can be improved, and a rapid carbon source can be provided for the generation of a target product; the amylase is preferably a high temperature resistant alpha-amylase from Shandong Kete enzyme preparation, Inc. The invention has no special requirements on the types of the starch, and in the specific implementation process of the invention, the starch is corn starch.
In the invention, the soybean oil is used as a slow carbon source and plays two roles in the fermentation process of spectinomycin, and firstly, the soybean oil is decomposed into short-chain fatty acid by lipase for mycelium growth and product synthesis; secondly, play the defoaming effect in the fermentation process, control fermentation cylinder operation volume, improve equipment coefficient of charging and utilization ratio. In the invention, the soybean oil is from Yihaijiali (Yanzhou) grain and oil industry Co.
The method takes the starch (fast carbon source) and the soybean oil (slow carbon source) after enzymolysis as the common carbon source, can control the growth rate of the hyphae after the mycelium enters the secondary metabolism, avoids the excessive growth of the hyphae, and simultaneously maintains a certain growth level of the hyphae.
In the invention, the monopotassium phosphate has the function of controlling the concentration of bacteria within a certain range and improving the ability of the bacteria to synthesize the spectinomycin.
The invention also provides a preparation method of the supplement in the scheme, which comprises the following steps:
mixing the starch, the soybean oil, the monopotassium phosphate, the amylase and water, and liquefying at 80-100 ℃ for 35-45 min to obtain a supplementary material; the temperature of the liquefaction is preferably 90 ℃; the time for liquefaction is preferably 40 min;
in the invention, the starch is liquefied for 35-45 min at 80-100 ℃, so that the proper liquefaction degree of the starch can be effectively controlled; on one hand, the excessively high liquefaction degree is avoided, the monosaccharide is more in the liquefied material liquid, the mycelium can quickly utilize the monosaccharide to grow and reproduce, the mycelium metabolism is easily transferred from the secondary metabolism to the primary metabolism, and the product synthesis is reduced; on the other hand, the conditions that the liquefaction degree is too low, the polysaccharide content in the feed liquid is more, part of polysaccharide decomposing enzyme is required to be synthesized by the hypha by utilizing the polysaccharide, the utilization rate is slow, the hypha gradually becomes autolysis, the bacterial concentration is low, and the titer is low are avoided.
After the liquefaction, the invention preferably further comprises feeding the liquefied feed liquidSterilizing and cooling; the sterilization mode is preferably high-temperature instant sterilization; the sterilization temperature is preferably 130-140 ℃, more preferably 135 ℃, the sterilization time is preferably 3-5 min, and the feeding flow is preferably 60-80 m3/h。
The invention also provides a use method of the feed supplement in the scheme, which comprises the following steps:
1) fermenting by utilizing actinomycetes to produce spectinomycin for 30-40 h, and supplementing the supplementary material for the first time when the total sugar in the fermentation material is less than 3.5g/100 ml;
2) fermenting for 50-70 h, and supplementing the supplementary material for the second time when the total sugar in the fermentation material is less than 2.5g/100 ml;
3) and fermenting for 80-100 hours, and supplementing the supplementary material for the third time when the total sugar in the fermentation material is less than 1.5g/100 ml.
Firstly, fermenting by utilizing actinomycetes to produce spectinomycin for 30-40 h, and supplementing the supplementary material for the first time when the total sugar in a fermentation material is less than 3.5g/100ml, preferably less than 3g/100 ml; the volume ratio of the fed materials to the fermented materials is preferably 3-5: 100, more preferably 4: 100, respectively; the process for producing the spectinomycin by fermenting the actinomycetes is not particularly limited, and the method for producing the spectinomycin by fermenting the actinomycetes in the field can be adopted; the invention has no special limitation on the fermentation material, and the conventional fermentation material in the field can be adopted. In the specific implementation process of the invention, the formula of the fermentation material is shown in table 1.
TABLE 1 basic fermentation material ratio
| Name of material | Mass percentage of |
| Starch | 7％～10％ |
| Amylase | 0.005％～0.01％ |
| Bean cake powder | 1.0％～2.0％ |
| Yeast powder | 1.0％～2.0％ |
| Glucose | 0.5％～1.0％ |
| Ammonium sulfate | 0.1％～0.2％ |
| Potassium dihydrogen phosphate | 0.1％～0.2％ |
| Soybean oil | 0.1％～0.2％ |
| GPE | 0.01％～0.02％ |
| Silicone | 0.01％～0.02％ |
The total sugar content in 1 fermentation batch is preferably tested every 24h according to the invention.
In the invention, when fermentation is carried out for 50-70 h, and the total sugar in the fermentation material is less than 2g/100ml, preferably less than 1.5g/100ml, the supplementary material is supplemented for the second time; the volume ratio of the fed materials to the fermented materials is preferably 3-5: 100, more preferably 4: 100.
the supplementary material is supplemented for the third time when fermentation is carried out for 80-100 hours and the total sugar in the fermentation material is lower than 1.5g/100 ml; the volume ratio of the supplementary material to the fermentation material is preferably 1.5-3: 100, more preferably 2 to 2.5: 100.
according to the method, the fermentation titer and the fermentation level of the spectinomycin can be effectively improved by strictly controlling the material supplementing time and the material supplementing amount, and the charging coefficient and the utilization rate of equipment are improved.
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
A use method for improving fermentation titer of spectinomycin comprises adding fermentation feed of spectinomycin comprising starch, soybean oil, potassium dihydrogen phosphate, amylase and water; wherein, 4500kg of starch, 300kg of soybean oil, 30kg of monopotassium phosphate, 3kg of alpha-amylase, and the total amount of spectinomycin fermentation feed supplement: 13m3。
The using method comprises the following steps:
(1) conveying the spectinomycin fermentation feed supplement liquid into a mixing tank, heating to 80-100 ℃ for liquefying for 40min, then heating to 130-140 ℃ through a continuous sterilizer for sterilizing for 3min, and cooling to 50-60 ℃ to obtain the sterilized liquefied feed supplement for later use.
(2) When the fermentation period of the fermentation tank is 35h and the total sugar in the fermentation tank is less than 3.3g/100ml, 5.0m is supplemented3The volume of the sterilized liquefied feed liquid and the fermentation liquid before supplementation is 100m3(ii) a The fermentation period is 60h, and when the total sugar in the fermentation tank is less than 2.2g/100ml, 5.0m is supplemented3The liquefied feed liquid after sterilization; the fermentation period is 84h, and when the total sugar in the fermentation tank is less than 1.4g/100ml, 3.0m is supplemented3The pot-out titer of the sterilized liquefied feed solution after the feed was 7943u/ml, which was 1115u/ml higher than that of the fermentor (comparative example 1) without any treatment.
The titer calculation formula is as follows:
in the formula: pT-the potency of the test product; a. theT-estimating the titer; d-ratio of the concentration of the standard substance to the concentration of the sample (range: + -2%, i.e. between 0.98 and 1.02)
T2-the sum of the zone diameters caused by the high dose of test article; t is1-the sum of the zone diameters caused by the low dose of test article;
S2-the sum of the zone of inhibition diameters due to the high dose standard; s1-the sum of the zone diameters caused by the low dose standard;
i-common logarithm of the ratio of high dose to low dose concentration (lg2 ═ 0.3010).
Comparative example 1
Mixing required spectinomycin fermentation tank basic materials, and putting into a tank, wherein the fermentation materials consist of starch, amylase, bean cake powder, yeast powder, glucose, ammonium sulfate, potassium dihydrogen phosphate, soybean oil, GPE, organic silicon and water; wherein 10000kg of starch, 10kg of amylase, 1000kg of bean cake powder, 1500kg of yeast powder, 500kg of glucose, 200kg of ammonium sulfate, 150kg of monopotassium phosphate, 200kg of soybean oil, 20kg of GPE20kg and 20kg of organic silicon, and the total amount of spectinomycin fermentation liquor is as follows: 100m3。
The sterilization and culture method comprises the following steps:
1. mixing the required spectinomycin fermentation tank base materials, putting into a tank, beating the materials to a specified volume, heating to 120-124 ℃ by steam, sterilizing for 25min, cooling to 30-35 ℃, and waiting for seed transfer.
2. After the fermentation tank is transferred into the spectinomycin seed liquid, the air flow, the stirring speed and the tank pressure are adjusted and cultured according to the process until the fermentation liquid is put into the tank, and no material supplementing operation is carried out in the culture process.
TABLE 1 comparison of the pot Titers of example 1 and comparative example 1
| 66h | 90h | 114h | Can valence (u/ml) | |
| Example 1 | 3586 | 5677 | 6837 | 7943 |
| Comparative example 1 | 3015 | 4897 | 5768 | 6828 |
Example 2
The sterilization and culture methods were the same as in example 1
And (3) supplementing material proportioning: 3600kg of starch, 210kg of soybean oil, 21kg of monopotassium phosphate, 3kg of alpha-amylase and the total amount of spectinomycin fermentation feed supplement: 13m3。
The fermentation period of the fermentation tank is 32h, and when the total sugar in the fermentation tank is less than 3.4g/100ml, 5.0m is supplemented3The volume of the sterilized liquefied feed liquid and the fermentation liquid before supplementation is 100m3(ii) a The fermentation period is 56h, and when the total sugar in the fermentation tank is less than 2.3g/100ml, 5.0m is supplemented3The liquefied feed liquid after sterilization; the fermentation period is 80h, and when the total sugar in the fermentation tank is less than 1.2g/100ml, 3.0m is supplemented3The sterilized liquefied feed liquid has a tank discharge titer of 7789u/ml after feeding, and the tank discharge titer is 826u/ml higher than that of a fermentation tank without any treatment (the method is the same as that of the treatment method of the comparative example 1). See table 2.
Table 2 comparison of example 2 with control can Titer data
| 66h | 90h | 114h | Can valence (u/ml) | |
| Example two | 3358 | 5346 | 6541 | 7789 |
| Control | 3167 | 4785 | 5872 | 6963 |
Example 3
The sterilization and culture methods were the same as in example 1
And (3) supplementing material proportioning: 3000kg of starch, 150kg of soybean oil, 15kg of monopotassium phosphate, 1.5kg of alpha-amylase and spectinomycin fermentation supplementIn total: 13m3。
When the fermentation period of the fermentation tank is 38h and the total sugar in the fermentation tank is less than 3.2g/100ml, 5.0m is supplemented3The volume of the sterilized liquefied feed liquid and the fermentation liquid before supplementation is 100m3(ii) a The fermentation period is 62h, and when the total sugar in the fermentation tank is less than 2.4g/100ml, 5.0m is supplemented3The liquefied feed liquid after sterilization; the fermentation period is 86h, and when the total sugar in the fermentation tank is less than 1.3g/100ml, 3.0m is supplemented3The tank-discharging titer of the sterilized liquefied material liquid after being supplemented is 7833u/ml, and is 957u/ml higher than that of a fermentation tank without any treatment.
Table 3 example 3 and control can Titer data
| 66h | 90h | 114h | Can valence (u/ml) | |
| Example 3 | 3487 | 5576 | 6975 | 7833 |
| Control | 2897 | 4675 | 5763 | 6876 |
Example 4
Selecting a fermentation tank, adopting spectinomycin fermentation feed supplements with different starch concentrations, spectinomycin fermentation feed supplements with different soybean oil concentrations and spectinomycin fermentation feed supplements with different potassium dihydrogen phosphate to feed in the middle and later periods of fermentation, and respectively measuring the tank placing potency.
The results are shown in tables 4 to 6, wherein the table 4 shows the influence curve of the spectinomycin fermentation feed with different soybean oil concentrations (the mass percentage contents are 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.2% and 1.5%) on the tank-discharging titer by feeding in the middle and later fermentation periods of the fermentation tank respectively according to the using method of the invention, and the table 4 shows that the feed with the soybean oil concentration of 0.5-1% has the titer of spectinomycin reaching the peak value, and the tank-discharging titer is the highest when the soybean oil concentration is 1%;
table 5 shows the influence curve diagram of the feeding in the middle and later periods of fermentation of the fermentation tank on the tank-discharging titer of the spectinomycin fermentation feeding materials with different starch concentrations (the mass percentage contents are 5%, 10%, 12%, 15%, 18%, 20%, 22% and 25%) respectively according to the use method of the invention, and as can be seen from Table 5, the feeding with the starch concentration of 10% -20% achieves the peak value of the tank-discharging titer of the spectinomycin, and the tank-discharging titer is highest when the starch concentration is 15%.
Table 6 shows the effect curve diagram of the feeding of spectinomycin fermentation feeds with different potassium dihydrogen phosphate concentrations (0.02%, 0.04%, 0.05%, 0.07%, 0.09%, 0.1%, 0.12% and 0.15% by weight) on the tank-discharging titer in the middle and later fermentation stages of the fermentation tank respectively according to the method of the present invention, and it can be seen from Table 6 that the feeding titer of spectinomycin reaches the peak value when the potassium dihydrogen phosphate concentration is 0.05% -0.1%, and the tank-discharging titer is the highest when the potassium dihydrogen phosphate concentration is 0.1%.
TABLE 4 Effect of different Soybean oil concentrations on spectinomycin tank Titer
| Can valence (u/ml) | 7321 | 7415 | 7543 | 7655 | 7689 | 7733 | 7587 | 7428 |
| Concentration of Soybean oil (%) | 0.1 | 0.3 | 0.5 | 0.7 | 0.9 | 1 | 1.2 | 1.5 |
TABLE 5 Effect of different starch concentrations on the tank titer of spectinomycin
| Can valence (u/ml) | 7276 | 7355 | 7450 | 7587 | 7501 | 7430 | 7400 | 7218 |
| Starch concentration (%) | 5 | 10 | 12 | 15 | 18 | 20 | 22 | 25 |
TABLE 6 Effect of different potassium dihydrogen phosphate concentrations on spectinomycin off-tank potency
| Can valence (u/ml) | 7289 | 7325 | 7465 | 7489 | 7541 | 7786 | 7652 | 7543 |
| Concentration of monopotassium phosphate (%) | 0.02 | 0.04 | 0.05 | 0.07 | 0.09 | 0.1 | 0.12 | 0.15 |
The optimal concentrations of the starch, the soybean oil and the potassium dihydrogen phosphate are determined through the experiments, then the three materials are mixed according to the optimal concentrations (the starch is 15%, the soybean oil is 1.0%, the potassium dihydrogen phosphate is 0.1%, and the mixture is called), the three materials are compared with the two materials which are only supplemented, the final tank-placing titer is determined, and the test result is shown in figure 4. From FIG. 4, it can be confirmed that the best feeding effect can be achieved when the mass percentage of starch, the mass percentage of soybean oil and the mass percentage of potassium dihydrogen phosphate are 15%, 1.0% and 0.1%, respectively.
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 (4)
1. A using method of a feed supplement for improving fermentation titer of spectinomycin comprises the following steps:
fermenting by utilizing actinomycetes to produce spectinomycin for 30-40 h, and supplementing the supplementary material for the first time when the total sugar in the fermentation material is less than 3g/100 ml; the volume ratio of the supplementary material to the fermentation material is 3-5: 100, respectively;
fermenting for 50-70 h, and supplementing the supplementary material for the second time when the total sugar in the fermentation material is less than 1.5g/100 ml; the volume ratio of the supplementary material to the fermentation material is 3-5: 100, respectively;
fermenting for 80-100 hours, and supplementing the supplementary material for the third time when the total sugar in the fermentation material is less than 1.5g/100 ml; the volume ratio of the supplementary material to the fermentation material is 1.5-3: 100, respectively;
the supplementary material comprises the following raw materials for preparation in percentage by mass: 12-20% of starch, 0.7-1.2% of soybean oil, 0.07-0.12% of potassium dihydrogen phosphate, 0.008-0.015% of amylase and the balance of water;
the amylase is an alpha-amylase.
2. The use method according to claim 1, wherein the feed is composed of the following raw materials for preparation in percentage by mass: 15% of starch, 1% of soybean oil, 0.1% of monopotassium phosphate, 0.01% of amylase and the balance of water.
3. The use of claim 1, wherein the feed is prepared by a method comprising the steps of:
and mixing the starch, the soybean oil, the monopotassium phosphate, the amylase and the water, and liquefying at 80-100 ℃ for 35-45 min to obtain the supplementary material.
4. The use of claim 3, further comprising, after said liquefying, sterilizing and cooling the liquefied feed solution; the sterilization temperature is 130-140 ℃, and the sterilization time is 3-5 min.
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