Large scale industrialized multi-cellular animal fermentation culture method

Abstract

The present application provides a large-scale industrialized multi-cellular animal fermentation culture method, which overcomes the problem that the current culture method of nematodes cannot realize the way of industrialized production of fermentation products, by stably controlling the temperature in the fermentation tank, adjusting the temperature to the most suitable temperature 20~25℃ for the growth of nematodes, and establishing a fermentation system suitable for the growth of nematodes, thereby technically breaking through the process of industrialized large-scale fermentation culture of multi-cellular animal nematodes and breaking the bottleneck of laboratory solid culture medium and small-volume liquid culture. 12 The present application can obtain a large number of nematode individuals at one time, about 10 12 individuals, and this technology can meet the development of nematodes into a eukaryotic system carrier for protein expression and purification, upgrade the past lower single-cell tool carrier system to a living eukaryotic higher multi-cellular animal system, and produce fermentation products with biological activity, especially proteins and protein complexes with functional activity and easy maintenance activity.

Description

Technical Field

[0001] The invention belongs to the technical field, and in particular relates to a method for large-scale fermentation and culture of eukaryotic multicellular animals using a large fermentation tank. Background Art

[0002] Caenorhabditis elegans is a determinate nematode, consisting of pharyngeal, excretory, and reproductive systems. It is characterized by a simple structure, a fixed cell number, a small size, rapid development, and a short life cycle. Its life cycle is approximately 18-22 days, with development from egg to adult in just 3-5 days. Most C. elegans are self-fertilizing hermaphrodites. C. elegans is a non-parasitic nematode that primarily feeds on bacteria, making it experimentally safe. Each adult worm lays approximately 300 eggs, allowing for rapid production in the laboratory. Experimental nematodes can be cultured in large quantities in either solid or liquid culture medium containing Escherichia coli. Solid culture involves inverted culture on agar plates supplemented with nematode food in a 20°C constant-temperature biochemical incubator. Liquid culture of nematodes is also limited to conical flasks, and due to limited conditions, the quantity of nematodes obtained is extremely limited.

[0003] Current industrial fermentation and cultivation processes have many limitations: 1. They are only used for the cultivation of single-cell systems such as bacteria, yeast, and tool cells; 2. Since the above-mentioned culture objects that have been modified by genetic means are single-celled lower organisms or cells, in many cases it is difficult to meet the biological activity of the fermentation products. For example, if the fermentation product is a protein, there may be protein folding errors and inactivity, resulting in problems such as low activity of the fermented product; 3. Multicellular animals are a whole entity with complete physiological systems, which is very conducive to the production and maintenance of product activity, but there is currently no fermentation and cultivation technology involving multicellular animals.

[0004] The model animal nematode is a multicellular animal commonly used in laboratories. Although its structure is simple, its nervous system, digestive system, immune system and other functions are very similar to those of mammals. However, due to the lack of breakthroughs in technical difficulties, the cultivation of nematodes is currently limited to solid culture medium NGM culture and small volume liquid culture. Neither of these two culture methods can be realized as a way to industrially produce fermentation products. Summary of the Invention

[0005] In order to solve the above technical deficiencies, we invented a large-scale multi-cellular animal fermentation culture technology. This invention fundamentally solves a series of problems brought by single-cell fermentation system. Taking the fermentation product as an example, the protein complex with biological activity can be purified after the multi-cellular animal nematode is fermented and cultured; at the same time, the fermentation tank is upgraded, and a low-temperature water circulation system is connected to stably control the temperature in the fermentation tank, facilitate the adjustment of the temperature to the most suitable temperature 20~25℃ for the growth of nematodes, and establish a fermentation system suitable for the growth of nematodes. The industrialized large-scale fermentation culture technology of multi-cellular animal nematodes is technically broken through, and the bottleneck of laboratory solid culture medium and small-volume liquid culture is broken.

[0006] First, the preparation of nematode food E. coli OP50, 2 days before starting the fermentation tank culture of nematodes, the required nematode food should be prepared

[0007] 1.1) inoculate a single E. coli colony into 300~500mL bacterial liquid medium, and cultivate at 37℃ on a shaker overnight.

[0008] 1.2) the next day, the shaken bacterial liquid is stored at 4℃, and 20μL or so is added to a clean agar plate to verify whether it will grow with miscellaneous bacteria.

[0009] 1.3) weigh 500g NaCl, 500g tryptone, and 250g yeast powder, add water to 50L, prepare 50L volume of bacterial liquid medium, sterilize in the fermentation tank (add 0.1%~1% proportion of defoaming agent before sterilization), and the sterilization operation is as follows:

[0010] 1.3.1) open the evaporator (the evaporator water inlet valve is in the open state), and slowly open the steam total valve when the pressure gauge rises to 0.4MPa.

[0011] 1.3.2) open the vent valve, tap water in, tap water out and blowdown valve, and other valves are in the closed state.

[0012] 1.3.3) turn on the automatic sterilization function on the control panel (set the temperature to 120℃ and the time to 20min).

[0013] 1.3.4) when the temperature rises to 80℃, close the blowdown valve.

[0014] 1.3.5) when the temperature rises to 100℃, close the vent valve.

[0015] 1.3.6) when the temperature rises to 110℃, perform pipeline sterilization: open the needle valve, half-open the steam valve, and slowly open the diaphragm valve to ensure that the filter pressure (not more than 0.2MPa) is higher than the tank pressure (0.05~0.1MPa).

[0016] 1.3.7) At the end of sterilization, open the air flow measurement (manual or automatic, only one of them), close the diaphragm valve and the steam valve (first), open the air valve (later), and close the needle valve after a few minutes.

[0017] 1.3.8) Cooling: Close the blow-off valve and open the cooling valve. During the cooling process, open the diaphragm valve to supply air and ensure that the pressure in the tank is positive (0.05-0.1 MPa). On the control panel, change the temperature control program to automatic. First, use tap water to cool to about 40°C, and then turn on the cooling water system.

[0018] 1.3.9) Inoculation: When the temperature in the tank drops to 37°C, use the vent valve to bring the pressure in the tank to 0.05 MPa, slowly unscrew the inoculation port, and insert alcohol-soaked cotton near the inoculation port. Ignite the flame at the moment the inoculation port is unscrewed, inoculate the verified OP50 into the fermenter, tighten the inoculation port knob, and use the vent valve to restore the pressure in the tank to 0.05-0.1 MPa.

[0019] 1.4) Ferment at 37°C, 120 rpm overnight.

[0020] 1.5) The next day, sterilize the fermenter outlet: open the evaporator (the evaporator inlet valve is open), slowly open the steam valve when the pressure gauge rises to 0.4 MPa. Then open the sampling sterilization valve and the outlet valve, and when steam comes out of the outlet, the sterilization is complete. First, close the sampling sterilization valve, and then close the evaporator.

[0021] 1.6) Take the bacterial solution from the fermenter outlet, take about 20 μL and add it to a clean agar plate to verify whether it will grow bacteria.

[0022] 1.7) Centrifuge to obtain bacterial precipitate and store at 4°C for future use.

[0023] 1.8) Clean the fermenter: first rinse with water, then sterilize.

[0024] Second: use 50-100 L of nematode liquid medium to culture nematodes, which requires about 100-120 L of nematode food bacteria solution.

[0025] Preparation of nematodes

[0026] 2.1) Use 10-12 90 mm agar plates with nematode food to culture nematodes.

[0027] 2.2) Cultivate until most nematodes are in the first 1-2 days of adulthood, lyse the eggs with lysis solution, and transfer to 2-3 6-well plates for liquid shaking culture.

[0028] 2.3) Inoculate 6-well plates with eggs from the 1st to 2nd day of adult stage, and then transfer to a 500-800 mL conical flask containing nematode liquid medium.

[0029] 2.4) Inoculate the conical flask with the 1st to 2nd day of adult stage.

[0030] Third, large-scale culture of nematodes in a 50-100 L fermenter

[0031] Prepare the ingredients required for 50-100 L nematode liquid medium S-Medium:

[0032] NaCl 0.5%~0.6%, EDTA 0.15%~0.2%, K2PO40.55%~0.65%, KH2PO40.8%~0.12%, 0.22‰~0.28‰ cholesterol (prepared with anhydrous ethanol), 0.14~0.15‰ CaCl2, 0.12~0.14‰ MgSO4, 0.65~0.7‰ FeSO4 . 7H2O, 0.18~0.22‰ MnCl2 . 4H2O, 0.25~0.3‰ ZnSO4 . 7H2O, 0.022~0.028‰ CuSO4 . 5H2O.

[0033] 3.1) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter.

[0034] 3.2) Weigh NaCl, EDTA, K2PO4 and KH2PO4 into the fermenter, add water to 50 L, and then add 0.1%~1% antifoam agent, sterilize in the fermenter, and sterilize as above.

[0035] 3.3) When the temperature in the tank drops below 50°C, use the vent valve to bring the pressure in the tank to 0~0.05 MPa, slowly unscrew the inoculation port, and insert alcohol-soaked cotton near the inoculation port. Light the flame at the moment the inoculation port is unscrewed, add sterilized CaCl2 and MgSO4, and then add filtered sterilized 0.65~0.7‰ FeSO4 . 7H2O, 0.18~0.22‰ MnCl2 . 4H2O, 0.25~0.3‰ ZnSO4 . 7H2O, 0.022~0.028‰ CuSO4 . 5H2O, extinguish the flame, and add 0.22‰~0.28‰ cholesterol (prepared with anhydrous ethanol).

[0036] 3.4) When the inoculation port knob is tightened, the pressure in the fermentor is restored to 0.05-0.1 MPa by using the vent valve.

[0037] 3.5) When the inoculation port knob is tightened, the pressure in the fermentor is restored to 0.05-0.1 MPa by using the vent valve.

[0038] 3.6) The pH range is controlled to be 6.5-7.6, and the dissolved oxygen content is controlled to be 30%-70%.

[0039] 3.7) The temperature of the liquid in the fermentor is lowered to 20-25°C by using the cold water cooling system, and the stirring system is started to mix the liquid in the fermentor.

[0040] 3.8) The nematodes in the conical flask are lysed to obtain eggs, and the eggs are washed several times with sterile M9 buffer.

[0041] 3.9) The nematodes are inoculated into the fermentor: the pressure in the fermentor is reduced to 0-0.05 MPa by using the vent valve, the inoculation port knob is slowly loosened, alcohol-soaked cotton is inserted near the inoculation port, a flame is ignited at the moment when the inoculation port knob is loosened, and the obtained nematode eggs are poured in. When the inoculation port knob is tightened, the pressure in the fermentor is restored to 0.05-0.1 MPa by using the vent valve.

[0042] 3.10) The next day, the nematodes have hatched in the fermentor, and the nematode food is added, and the obtained bacteria are resuspended with sterile M9 buffer, and the bacteria are introduced into the fermentor through the inoculation port (the inoculation step is the same as above).

[0043] 3.11) Daily inspection: the nematode growth temperature is 20-25°C, the pH is 6.5-7.6, and the stirring speed is 100-160 rpm.

[0044] 3.12) On the 8th-9th day of growth in the fermentor, sampling and observation should be performed, and the second generation of nematodes has grown to the adult stage, and the sample can be collected.

[0045] The beneficial effects of the present application: using the fermentor to culture nematodes in a large scale, a large number of nematode individuals can be obtained at one time, about 10 12 ~10 15 The most direct beneficial effect of this technology is that it is easy to produce fermentation products with biological activity, especially functional proteins, and it is easy to maintain the activity. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The results of the protein absorption activity detection of the large-scale fermentation and purification of the multicellular animal nematodes of Example 1.

[0047] Figure 2 Results of the large scale fermentation of the purified protein complex of the multicellular nematode of the present invention. DETAILED DESCRIPTION

[0048] Example 1

[0049] Large scale culture of nematodes, purification of short wavelength light absorbing protein

[0050] Implementation steps:

[0051] Ingredients of 50L nematode liquid culture S Medium:

[0052] NaCl 292.5g, EDTA 93g, K2PO4 300g, KH2PO4 50g

[0053] 5 mg / ml Cholesterol (prepared with anhydrous ethanol) 50 mL, 1 M CaCl2 50 mL, 1 M MgSO4 50 mL, 500x metal stock solution 100 mL;

[0054] 1) Weigh 292.5g NaCl, 93g EDTA, 300g K2PO4 and 50g KH2PO4 in the fermenter, add water to 50L, then weigh 5g of defoamer, sterilize in the fermenter, and sterilize as above.

[0055] 2) Add 50 mL of sterilized 1 M CaCl2 and 1 M MgSO4, and then add 100 mL of sterilized 500x metal stock solution, extinguish the fire ring, and add 50 mL of 5 mg / ml Cholesterol (prepared with anhydrous ethanol).

[0056] 3) The pressure in the tank should be 0 when inoculating, and the acid-base pump should be connected to the prepared 1 M NaOH and 1 M HCl.

[0057] 4) Set the PH to 7.2 and the dissolved oxygen to 50%.

[0058] 5) Continue to use the cold water cooling system to reduce the temperature to 20°C, and turn on the stirring system to mix the liquid in the tank.

[0059] 6) Lyse the short wavelength light absorbing protein transgenic nematodes in the conical flask that have been cultured to the first 2 days of adulthood, obtain eggs, and wash several times with sterile M9 buffer.

[0060] 7) Inoculate nematodes into the fermenter.

[0061] 8) The next day, the nematodes have hatched in the fermenter, add nematode food, and connect the tank from the inoculation port (the inoculation steps are the same as above).

[0062] 9) Daily inspection: the temperature of nematode growth is 20℃, the PH is 7.2~7.5, and the stirring speed is 120rpm.

[0063] 10) On the 8th~9th day of growth in the fermenter, samples should be taken for observation, and the second generation of nematodes has grown to the adult stage, and the samples can be collected.

[0064] The implementation results are shown in Table 1. Figure 1 As can be seen from the table, the protein light absorption activity provided by Example 1 is more than 200 times higher than that of the traditional method, and the method provided by the present application better ensures the functional activity of the protein.

[0065] Example 2

[0066] Large-scale culture of nematodes and purification of mechanosensory protein complex

[0067] Implementation steps:

[0068] Composition of 50L nematode liquid culture medium S Medium:

[0069] NaCl 292.5g, EDTA 93g, K2PO4 300g, KH2PO4 50g

[0070] 5 mg / ml Cholesterol (prepared with anhydrous ethanol) 50mL, 1 M CaCl2 50mL, 1M MgSO4 50mL, 500×metal stock solution 100mL

[0071] 1) Weigh 292.5g of NaCl, 93g of EDTA, 300g of K2PO4 and 50g of KH2PO4 in the fermenter, add water to 50L, and then weigh 5g of defoamer and sterilize in the fermenter. The sterilization operation is the same as above.

[0072] 2) Add 50mL of sterilized 1 M CaCl2 and 1 M MgSO4, and then add 100mL of filtered sterilized 500×metal stock solution, extinguish the fire ring, and add 50mL of 5 mg / ml Cholesterol (prepared with anhydrous ethanol).

[0073] 3) When inoculating, the pressure in the tank should be 0, and an acid-base pump should be connected to prepare 1M NaOH and 1M HCl.

[0074] 4) Set the PH to 7.2 and the dissolved oxygen content to 50%.

[0075] 5) Continue to use the cold water cooling system, and the temperature is reduced to 20℃, and the stirring system is started to mix the liquid in the tank.

[0076] 6) The mechanical sensilla gene modified nematodes in the first 2 days of the adult stage in the split conical flask are lysed to obtain eggs, and the eggs are washed several times with sterile M9 buffer.

[0077] 7) The nematodes are inoculated into a fermentation tank.

[0078] 8) The next day, the nematodes have hatched in the fermentation tank, and the nematode food is added into the tank from the inoculation port (the inoculation step is the same as above).

[0079] 9) Daily inspection: the growth temperature of the nematodes is 20℃, the PH is 7.2-7.5, and the stirring speed is 120 rpm.

[0080] 10) On the 8th-9th day of the growth in the fermentation tank, the sample should be observed, and the second generation of nematodes has grown to the adult stage, and the sample can be collected.

[0081] Figure 2 For the detection results of the large-scale fermentation of the protein complex of the multicellular nematode of the present application, three proteins that have not been reported to form a complex with the mechanical sensilla are found, and it can be seen that the method provided by the present application can better preserve the structure and function of the protein and further purify the protein complex.

Claims

1. A large-scale industrial fermentation culture method for multicellular animals, characterized in that: The method comprises the following steps: 1) Calibrate the pH and dissolved oxygen electrodes in the fermenter and prepare the following ingredients for the nematode liquid culture medium (S-Medium): NaCl 0.5%–0.6%, EDTA 0.15%–0.2%, K2PO4 0.55%–0.65%, KH2PO4 0.8%–0.12%, 0.22‰–0.28‰ cholesterol, 0.14–0.15‰ CaCl2, 0.12–0.14% MgSO4, 0.65–0.7% FeSO4·7H2O, 0.18–0.22‰ MnCl2·4H2O, 0.25–0.3‰ ZnSO4·7H2O, and 0.022–0.028‰ CuSO4·5H2O. 2) Weigh NaCl, EDTA, K2PO4 and KH2PO4 in a fermenter, add water, and then add 0.1-1% of the total weight of a defoamer, and sterilize in the fermenter; 3) When the temperature inside the tank drops below 50°C, reduce the pressure inside the tank to 0-0.05MPa, slowly loosen the inoculation port, plug cotton soaked in alcohol near the inoculation port, ignite the flame the moment the inoculation port is unscrewed, add sterilized CaCl2 and MgSO4, and then add filtered and sterilized metal mother liquors FeSO4·7H2O, MnCl2·4H2O, ZnSO4·7H2O, and CuSO4·5H2O, extinguish the flame ring, and add cholesterol; 4) When the pressure in the tank is 0 during inoculation, connect the acid and alkali pump and connect the prepared 1-10M NaOH and 1-10M HCl; 5) Tighten the inoculation port knob and use the vent valve to restore the pressure in the tank to 0.05-0.1 MPa; 6) Control the pH range to 6.5-7.6 and the dissolved oxygen content to 30%-70%; 7) Cool down the liquid in the tank to 20-25°C, start stirring, and mix the liquid in the tank; 8) Lyse the nematodes grown in a conical flask to the 1st to 2nd day adult stage, obtain the eggs, and wash them several times with sterile M9 buffer; 9) Inoculate nematodes into the fermentation tank: Use the vent valve to reduce the tank pressure to 0-0.05 MPa. Place a cotton ball soaked in alcohol near the inoculation port. Light a flame the moment you open the inoculation port, pour in the nematode eggs, tighten the inoculation port knob, and use the vent valve to restore the tank pressure to 0.05-0.1 MPa. 10) After the nematodes have hatched in the fermenter, add nematode food and resuspend the resulting cells in sterile M9 buffer. Inoculate the cells into the fermenter through the inoculation port, following the same sterility control procedure as in step 9. 11) Daily inspection: Control the nematode growth temperature at 20-25°C, pH at 6.5-7.6, and stirring speed at 100-160 rpm; 12) The second generation of nematodes can be collected when they grow to the adult stage.

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