Large-scale fermentation culture method of aeromonas veronii
A large-scale fermentation method for Aeromonas veronii using specific conditions in stainless steel fermenters enhances bacterial concentration and reduces costs, addressing inefficiencies in shake flask culture for industrial production.
Patent Information
- Application Number
- CN202411498330.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2024-12-13
AI Technical Summary
Current shake flask culture methods for Aeromonas veronii fermentation are unsuitable for large-scale industrial production due to low bacterial concentration, high raw material consumption, and long fermentation times, making them costly and inefficient.
A large-scale fermentation method using 50L and 100L stainless steel stirred fermenters with specific conditions: temperature 26-30°C, 4-5 hours feeding start, 18-33 times fermentation volume per hour glucose feed, 10-15g/L tryptone, 5-7.5g/L beef extract, 5-7.5g/L sodium chloride, 100-300g/L glucose, 55-75% liquid filling, 40-60% dissolved oxygen, and pH 6.8-7.8, achieving 2×10 10 CFU/ml bacterial concentration.
The method significantly increases bacterial concentration, reduces production costs, and shortens fermentation time, enabling transformation of laboratory research results into cost-effective industrial production.
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Figure BDA0005101866910000041 
Figure BDA0005101866910000042
Abstract
Description
Technical Field
[0001] The invention belongs to the field of bacterial fermentation, and in particular relates to a large-scale fermentation and cultivation method of Aeromonas veronii. Background Art
[0002] Aeromonas veronii is a Gram-negative facultative anaerobic bacillus belonging to the family Aeromonadaceae and the genus Aeromonas. Aeromonas veronii is an important pathogen of fish. The bacterial sepsis caused by this bacterium is an acute infectious disease that is recognized to harm the most types of fish, the largest age range of fish, the widest epidemic area, the longest epidemic season, the most types of fish farming waters, and the most serious losses in the history of fish farming in my country. At present, vaccines for Aeromonas veronii bacterial sepsis in fish are in the laboratory research stage, and most of them are inactivated vaccines. Since the unit price of aquatic seedlings is different from that of poultry and livestock, the unit price of each tail is very low, so the cost control requirements for aquatic vaccines are very strict. Increasing the total amount of bacteria in the fermentation process of a single batch of bacterial vaccines and reducing the amount of raw materials consumed and the fermentation reaction time are the keys to low cost of vaccines.
[0003] At present, the fermentation process of Aeromonas vermifuge is shake flask culture. This culture technology has the problems of low concentration of prepared bacterial solution, high consumption of raw materials and long fermentation reaction time. It is only suitable for research and development process development in scientific research institutes and is difficult to support large-scale industrial production. Therefore, it is urgent to develop a method with good effect, simple operation, low cost and large-scale culture. Summary of the invention
[0004] The object of the present invention is to provide a large-scale fermentation and cultivation method of Aeromonas veronii.
[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:
[0006] A large-scale fermentation and cultivation method for Aeromonas vermifuge comprises the following steps: fermenting at a temperature of 26 to 30°C, starting feeding 4 to 5 hours after the start of fermentation, adjusting the flow rate of a carbon source feeding pump, adding 18 to 33 times the fermentation volume per hour, and replenishing the feeding nutrients within 1 to 2 hours.
[0007] Furthermore, the basic culture medium components used in the culture method include: tryptone: 10-15g / L, beef extract:
[0008] 5-7.5g / L, glucose: 5-7.5g / L, sodium chloride: 5-7.5g / L.
[0009] Furthermore, the carbon source feed medium component used in the culture method is glucose with a concentration of 100 to 300 g / L.
[0010] Furthermore, the inoculation amount of the fermentation method is 4-6%, and the concentration is 3-6×10 9 CFU / ml.
[0011] Furthermore, the liquid filling coefficient of the fermentation method is 55-75%.
[0012] Furthermore, the dissolved oxygen content of the fermentation method is required to be 40-60%.
[0013] Furthermore, the pH of the culture medium in the fermentation method is maintained at 6.8 to 7.8.
[0014] Furthermore, in the fermentation method, clean compressed air is used to maintain 0.1-0.6 MPa in the fermentation tank.
[0015] Furthermore, the viable bacterial content of Aeromonas verumii GZ15-2 obtained by the fermentation method is 2×10 10 CFU / ml.
[0016] Compared with the prior art, the present invention has the following advantages:
[0017] The Aeromonas vernix culture process of the present invention can achieve 1.1-2×10 10 The CFU / ml live bacterial concentration can also be simply adjusted according to the hardware conditions of different fermentation tanks, reducing the cost of vaccine preparation and the difficulty of operation, and improving the conversion rate of research results of the laboratory's Aeromonas verumii inactivated vaccine.
[0018] The present invention starts from actual production, uses production-type 50L and 100L stainless steel stirred fermenters to expand the culture of Aeromonas veronii in order to obtain high-yield Aeromonas veronii and reduce production costs, and proposes a feeding process for high-density fermentation of Aeromonas veronii. The feeding process can quickly reproduce the bacteria in the fermentation process, and obtain a bacterial concentration 2 to 4 times that of ordinary shake flask culture under the conditions of low feeding amount, short feeding time and fermentation time, thereby realizing the transformation of laboratory research results and cost control. DETAILED DESCRIPTION
[0019] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.
[0020] The raw materials and reagents used in the present invention are all commercially available medicines and reagents.
[0021] Example 1
[0022] The culture bacteria were live Aeromonas veronii GZ15-2, the carbon source feed medium was glucose 200 g / L, and the medium was sterilized by high pressure steam at 116° C. for 15 min.
[0023] The sterilized carbon source feed medium is connected to the fermentation tank through a silicone tube and controlled by a feed pump; the prepared sterile NaOH solution is connected to the fermentation tank through an alkali feed pump to adjust the pH of the basal culture medium. Generally, a small tank below 10L uses a concentration of 1M, and 50-100L uses 2-10M; the prepared sterile HCl solution is connected to the fermentation tank through an acid feed pump to adjust the pH of the culture medium, and the concentration used is 7%.
[0024] Before inoculation, set the fermentation temperature to 28°C, the tank pressure to 0.3Mpa, and the pH to 7.4. When all parameters are stable, inoculate. The seed inoculation amount is 6%, and the concentration is 6×10 9 CFU / ml. After the seed solution is added, the filling factor in the fermenter is 60%.
[0025] During the fermentation, NaOH solution or HCl solution was added using an alkali feed pump or an acid feed pump to maintain the pH at 7.4±0.1.
[0026] The dissolved oxygen content was maintained at 50% by adjusting the speed and clean air ventilation.
[0027] Using the above-mentioned culture medium and fermentation method, fermentation was carried out at 28°C. Feeding began 4.5 hours after the start of fermentation. The flow rate of the carbon source feed pump was adjusted to 23×fermentation volume ml / L (fermentation volume unit L) per hour, unit ml. The feed medium was replenished within 1.5 hours, and the dissolved oxygen content was maintained at 40-50% during the feeding period.
[0028] Harvest at the 10th hour of fermentation to obtain 2×10 10 CFU / ml bacterial solution containing the live bacteria content of Aeromonas viridis GZ15-2.
[0029] Example 2
[0030] The culture bacteria were live Aeromonas veronii GZ15-2, the carbon source feed medium was 300 g / L glucose, and the medium was sterilized by high pressure steam at 116° C. for 15 min.
[0031] Before inoculation, set the fermentation temperature to 30°C, the tank pressure to 0.6Mpa, and the pH to 7.5. When all parameters are stable, inoculate. The seed inoculation amount is 4% and the concentration is 6×10 9 CFU / ml. After the seed solution is added, the filling factor in the fermenter is 60%.
[0032] During the fermentation, NaOH solution or HCl solution was added using an alkali feed pump or an acid feed pump to maintain the pH at 7.5±0.1.
[0033] The dissolved oxygen content was maintained at 30% by adjusting the speed and clean air ventilation.
[0034] Using the above-mentioned culture medium and fermentation method, fermentation was carried out at 30°C. Feeding began 5 hours after the start of fermentation. The flow rate of the carbon source feed pump was adjusted to 17×fermentation volume per hour (fermentation volume in L), in ml. The feed medium was replenished within 2 hours, and the dissolved oxygen content was maintained at 30% during the feeding period.
[0035] Harvest at the 10th hour of fermentation to obtain 1.8×10 10 CFU / ml bacterial solution containing the live bacteria content of Aeromonas viridis GZ15-2.
[0036] Example 3
[0037] The culture bacteria were live Aeromonas veronii GZ15-2, the carbon source feed medium was glucose 100 g / L, and the medium was sterilized by high pressure steam at 116° C. for 15 min.
[0038] Before inoculation, set the fermentation temperature to 26°C, the tank pressure to 0.1Mpa, and the pH to 7.0. When all parameters are stable, inoculate. The seed inoculation amount is 5%, and the concentration is 3×10 9 CFU / ml. After the seed solution is added, the filling factor in the fermenter is 60%.
[0039] During the fermentation, NaOH solution or HCl solution was added using an alkali feed pump or an acid feed pump to maintain the pH at 7.0±0.1.
[0040] The dissolved oxygen content was maintained at 50% by adjusting the speed and clean air ventilation.
[0041] Using the above-mentioned culture medium and fermentation method, fermentation was carried out at 26°C. Feeding began 4 hours after the start of fermentation. The flow rate of the carbon source feed pump was adjusted to 33×fermentation volume per hour (fermentation volume in L), in ml. The feed medium was replenished within 1 hour, and the dissolved oxygen content was maintained at 50% during the feeding period.
[0042] Harvest at the 10th hour of fermentation to obtain 1.2×10 10 CFU / ml bacterial solution containing the live bacteria content of Aeromonas viridis GZ15-2.
[0043] Comparative Example 1
[0044] The high-density fermentation of Aeromonas veronii was carried out according to the fermentation method of Example 3, except that no carbon source was added during the fermentation culture process, and finally the Aeromonas veronii fermentation liquid was obtained.
[0045] Comparative Example 2
[0046] According to the fermentation method of Example 3, high-density fermentation of Aeromonas veroni was carried out, except that the total amount of carbon source added in Example 3 was reduced to half of that in Example 3, that is, the concentration of glucose was 50 g / L, the amount added per hour was 33×fermentation volume (fermentation volume unit L), unit ml, and the feed medium was replenished within 1 hour. Finally, Aeromonas veroni fermentation broth was obtained.
[0047] Comparative Example 3
[0048] The high-density fermentation of Aeromonas veroni was carried out according to the fermentation method of Example 3, except that the carbon source added in Example 3 was directly added to the basal culture medium, so that the total amount of carbon source added was half of that in Example 3. However, the fermentation culture process was no longer supplemented with carbon source, and finally Aeromonas veroni fermentation broth was obtained.
[0049] Comparative Example 4
[0050] The high-density fermentation of Aeromonas veroni was carried out according to the fermentation method of Example 3, except that the carbon source added in Example 3 was directly added to the basal culture medium to ensure that the total amount of carbon source added during the fermentation process was consistent with that in Example 3. However, the carbon source was no longer added during the fermentation culture process, and finally the Aeromonas veroni fermentation liquid was obtained.
[0051] Example 2
[0052] The viable bacterial concentration of the fermentation broth was tested by plate count method using the parameter conditions of 3 in Example 1 and the Aeromonas veronii bacterial broth obtained in Comparative Examples 1 to 3. The results are shown in Table 1.
[0053] Table 1 Summary of the concentration results of Aeromonas verumii bacterial solution prepared in each embodiment
[0054]
[0055] As can be seen from Table 1, the use of appropriate glucose concentration and flow rate in parameter 1 in Example 1 can maximize the final concentration of the fermentation broth, reaching 2.0×10 10 CFU / ml. When a higher concentration of carbon source and a higher total amount of carbon source feed were used in parameter condition 3, the final concentration of the fermentation broth was slightly lower than that in parameter condition 1; when a lower concentration of carbon source and a lower total amount of carbon source feed were used in comparative example 2, the final concentration of the fermentation broth was similar to that in comparative example 3. It can be seen that the rate of feed and a certain concentration have an important influence on the growth of Aeromonas veronii.
[0056] 2. Effect of dissolved oxygen content on Aeromonas veronii fermentation
[0057] The culture medium was placed in a 50L fermenter, sterilized at 121°C for 15 min, cooled to 28°C, and inoculated with 5.3×109 CFU / ml concentration seed solution, 28±1℃ constant temperature stirring culture. Clean air was continuously introduced during the culture to maintain different dissolved oxygen content in each group. Samples were taken at the 10th hour of fermentation to determine the concentration of the bacterial solution.
[0058] Table 2 Effect of different dissolved oxygen conditions on the final fermentation concentration
[0059]
[0060] The results showed that when the dissolved oxygen content was greater than 40%, the final fermentation concentration could be significantly increased.
[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A large-scale fermentation culture method for Aeromonas veronii, characterized in that: The culture method comprises the following steps: fermenting at a temperature of 26 to 30° C., starting feeding 4 to 5 hours after the start of fermentation, adjusting the flow rate of the carbon source feeding pump, adding 18 to 33× the fermentation volume per hour, and replenishing the feeding nutrients within 1 to 2 hours.
2. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The basic culture medium components used in the culture method include: tryptone: 10-15 g / L, beef extract: 5-7.5 g / L, glucose: 5-7.5 g / L, and sodium chloride: 5-7.5 g / L.
3. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The carbon source feed culture medium component adopted in the culture method is glucose with a concentration of 100-300 g / L.
4. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The inoculation amount of the fermentation method is 4-6%, and the concentration is 3-6×10 9 CFU / ml.
5. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The liquid filling coefficient of the fermentation method is 55-75%.
6. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The dissolved oxygen content requirement of the fermentation method is 40-60%.
7. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The pH of the culture medium in the fermentation method is maintained at 6.8-7.
8.
8. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: In the fermentation method, clean compressed air is used in the fermentation tank to maintain 0.1-0.6Mpa.
9. The large-scale fermentation culture method of Aeromonas veronii according to claim 1, characterized in that: The viable bacterial content of Aeromonas verumii GZ15-2 obtained by the fermentation method is 2×10 10 CFU / ml.