JPH0638753B2 - Vitamin B by culturing microorganisms at high concentration - Google Patents

Description

BACKGROUND OF THE INVENTION [Technical Field of the Invention The present invention relates to novel vitamin B ₁₂ production method using a high concentration cells cultivation of the microorganism producing vitamin B _12.

[Prior art and its problems]

Vitamin B ₁₂ is a factor necessary for in vivo metabolism of carbohydrates, fats, proteins, nucleic acids and the like, and is increasingly used as a drug such as known as an antineoplastic anemia factor and a therapeutic drug for pernicious anemia. There is. It is also widely used as a medicine or feed additive for livestock, and its importance is increasing more and more.

Currently, for the industrial production of vitamin B _12, the structure of vitamin B ₁₂ is complex, especially for chemical synthesis is extremely difficult to stereoisomers, fermentation production exclusively utilizing the action of microorganisms The law is taking place. Most of the fermentation production methods employ a method of collecting the produced and accumulated vitamin B ₁₂ by batch-culturing microorganisms. By the way, most of the microorganisms that produce vitamin B ₁₂ are in a medium in which all the necessary nutrients such as a nitrogen source and a carbon source are filled under the optimal culture conditions (temperature, PH, anaerobic level, etc.). When cultivated, when the number of bacterial cells of the bacterium is small, that is, when the bacterial cell concentration is low, it grows logarithmically at the maximum growth rate of the bacterium, but when it grows to a certain degree, the growth rate slows down and eventually stops growing. Will end up. The reason for this is vitamin B ₁₂
The producing bacterium metabolizes nutrients for growth and produces one or more metabolites other than vitamin B ₁₂ peculiar to the bacterium, and the concentration of this metabolite in the culture solution is the bacterium. This is because when the body growth exceeds a certain level, the metabolites inhibit the growth.

Thus, according to the conventional batch culture method, only a certain (1 to 5 g dry cell / -broth) bacterial cell concentration is obtained in the culture solution. Also, most of the high vitamin B ₁₂ production capacity microorganism to produce a vitamin B ₁₂ intracellularly, in the conventional Such batch culture method, be only vitamin B ₁₂ corresponding to the degree of cell concentration Production Can not.

However, in the past, in order to improve the productivity of vitamin B ₁₂ , it is necessary to find a microorganism having a high vitamin B ₁₂ producing ability, to produce it by cell fusion, or to examine the culture medium and culture conditions. It has often been aimed only at whether microbial cells produce vitamin B ₁₂ . Therefore, the present inventors have completed the present invention by paying attention to the fact that the productivity of vitamin B ₁₂ can be dramatically improved by culturing the vitamin B _12- producing bacterium at a high concentration by a culture method involving an excess.

[Outline of Invention]

In the present invention, a high concentration of vitamin B ₁₂ producing bacteria is ensured while the concentration of metabolites of vitamin B ₁₂ producing bacteria other than vitamin B ₁₂ (produced outside the cells) in the culture solution does not exceed a certain level. It is intended to obtain bacterial cells and to easily produce a large amount of vitamin B ₁₂ industrially. According to the studies and experiments by the present inventors, a metabolite that inhibits bacterial growth (extracellular) In order to prevent the concentration in the culture solution of () generated in the above) from increasing, a culture solution containing continuously growing cells is continuously passed through a material such as a hollow fiber type microfilter module or ceramic, The liquid containing the bacterial cells is separated into a liquid containing the bacterial cells and a liquid containing no bacterial cells, and the liquid containing the bacterial cells is returned to the culture tank and the latter liquid in which the metabolites are dissolved is taken out to supply a fresh medium. , high vitamin B ₁₂ producing bacteria by supplementing It has been found that the degree cells is obtained.

Thus although not a high concentration of vitamin B ₁₂ was obtained in high concentration cell culture of vitamin B ₁₂ producing bacteria, at the same time continuing the culture further involve excessive as described above, the vitamin B ₁₂ producing bacteria If a high-concentration cell culture solution is continuously extracted separately from the cell-free solution, and a fresh medium of the same volume is additionally supplied continuously, it is possible to obtain a high level of the extracted vitamin B _12- producing cells. High-concentration vitamin B ₁₂ can be continuously collected from the concentrated bacterial culture. Here, the bacterial cell concentration is diluted by the amount of high-concentration bacterial cells removed, but metabolites that cause growth inhibition due to excess are removed, so the bacterial cells can continue to grow. ,
A high-concentration bacterial cell culture solution can be collected at a dilution rate that is in a level that is in equilibrium with the growth rate.

That is, according to this method, it is possible to continuously collect high-concentration vitamin B _{12 and} thereby achieve a dramatic improvement in the production of vitamin B ₁₂ , as well as to reduce the size of production equipment and continuous production. From the viewpoint of energy saving, it is possible to realize extremely effective production of vitamin B ₁₂ .

Therefore, the present invention is a method for culturing a vitamin B ₁₂ producing bacterium in a culture medium in a liquid medium to produce a high-concentration microbial cell, in which a culture solution containing the microbial cell is continuously hollowed before growth is reduced or stopped. liquor returned to the culture tank containing spent through the yarn-type micro filter module or timber such as a ceramic is separated into a liquid containing no liquid and vitamin B ₁₂ production cells containing vitamin B ₁₂ production cells bacterial cells Then, the liquid containing no bacterial cells is extracted, and a fresh liquid medium having the same volume as the extracted liquid is supplied to the vitamin B ₁₂ producing bacterium culture solution to continue overculturing, whereby a high concentration of vitamin B ₁₂ producing bacteria is obtained. While obtaining the bacterial cells and further culturing with the above-mentioned excess, the obtained high-concentration bacterial cell culture solution of vitamin B _12- producing bacteria containing a high concentration of vitamin B ₁₂ was used as a liquid containing no bacterial cells. Separately and continuously withdrawal By newly continuously supply additional fresh medium volume, vitamins, characterized in that to produce a continuously high concentration of vitamin B ₁₂ from the high-density cell cultures of vitamin B ₁₂ producing bacteria withdrawn B _It provides ₁₂ production methods.

[Specific Description of the Invention]

 The present invention will be described in more detail.

In the method of the present invention, a microorganism having a high vitamin B ₁₂ producing ability is used as a control. As the vitamin B ₁₂ producing bacterium, bacteria and actinomycetes are mainly known. Specifically, specifically, Propionibacterium genus, Streptomyces, Micromonospora, Pseudomonas, Nocardia, Corynebacterium, Clostridium, Butyribacterium,
Micrococcus genus, Arthrobacter genus, Paracoccus genus, Bacillus genus, Protaminobacterium genus, Rhodopseudomonas genus and the like are listed as typical bacteria.

The medium used for the cultivation of these vitamin B ₁₂ producing bacteria, nitrogen sources, is optional if the medium filled with nutrients necessary for the growth of vitamin B ₁₂ producing bacteria, including a carbon source, a vitamin B ₁₂ a small amount of cobalt salt for producing bacteria (COCl ₂ -6H ₂ O _or, COSO 4 _-7H 2 O)
Add. The normal liquid medium is used, the medium of appropriate composition selected and used by the vitamin B ₁₂ producing bacteria of interest.

In the present invention, when culturing such a vitamin B ₁₂ -producing bacterium in a medium, a culture solution containing continuously growing cells is cultivated while being passed through a suitable material. First, preculture, bacterial inoculation, and PH control batch culture are performed. The following will be described in order.

First, the vitamin B ₁₂ producing bacterium is batch-cultured in the previously sterilized liquid medium under the optimum conditions, and this is pre-cultured for the culture of the present invention (main culture) to obtain an initial inoculum.

Next, the same liquid medium is put into the main culture tank and sterilized, and then the precultured bacteria are inoculated into the medium. At this time, the preculture liquid may be added as it is, but if necessary, the preculture liquid is aseptically centrifuged and centrifuged at 8,000 to 13,000 rpm for 3 times.
The cells are collected by centrifugation for 10 minutes, and the collected cells are suspended in a fresh homogenized medium that has been sterilized in advance and added to the main culture medium. At that time, the initial bacterial cell concentration of the main culture solution may be extremely low, but it is desirable that the culture solution containing the bacterial cells have an absorbance (turbidity) at 570 nm, that is, O.D. D (= Optical Densi
ty) 570 value is preferably about 0.01 to 0.3.
The actual cell concentration in the culture solution is usually determined from this absorbance. That is, the absorbance of the liquid containing the bacterial cells was measured to determine the O. The D570 value is obtained, and the dry cell weight in a constant liquid volume can be known from the value using a fixed mathematical formula. The cell density level is indirectly represented by the D570 value itself.

When the main culture tank is inoculated with the bacterium, main culture is performed while maintaining optimal conditions such as temperature, pH, and anaerobic degree. For example, make the main culture tank a jacket type and circulate constant temperature water at the optimum temperature in the jacket tank to maintain the optimum temperature conditions. Also, use alkaline solution such as ammonia water or sodium hydroxide, or acid solution such as hydrochloric acid. Automatically add drops to maintain optimum PH conditions. Also, stir the liquid at an appropriate speed so that the bacterial cells do not precipitate, or in the case of facultative anaerobic bacteria, supply an appropriate amount of sterile nitrogen gas to keep the facultative anaerobic state, and in the case of obligate anaerobic bacteria It is necessary to ventilate sterile nitrogen gas into an airtight culture tank to keep the culture tank at a positive pressure while gradually exhausting it to maintain an anaerobic state. Further, in the case of aerobic bacteria, sterile air, and oxygen gas if necessary, are aerated in the culture medium so that the aerobic state is maintained.

When cultured in the main culture tank as described above, vitamin B ₁₂
Producing bacteria continue to grow logarithmically at the maximum growth rate, but when the bacterial concentration level reaches a certain level, vitamin B ₁₂
Other than its own metabolites (produced outside the cells), the growth is inhibited, the growth rate of the cells gradually decreases, and finally the growth stops. The level is, for example, O. The D570 value is about 10. It is preferable that such a level be examined in advance by batch culture for each vitamin B ₁₂ producing bacterium.

In the present invention, the culture is continued in this way, and before the growth is reduced or stopped, preferably immediately before, that is, in the latter stage of logarithmic growth, each nutrient solution in the main culture tank is continuously supplied to the material. Start the operation. The material may be a hollow fiber (hollow fiber) type micro filter module, a ceramic material or the like, but it is particularly preferable to use a hollow fiber (hollow fiber) type micro filter module. This consists of a number of hollow fiber-shaped precision membranes made of synthetic resin precision membranes (microfilters) that have a small and sharp pore size distribution that does not allow cells to pass through. In use, the liquid is pumped into the hollow part of the hollow fiber membrane, and the stock solution is flowed in parallel to the inner surface of the hollow fiber membrane.
The so-called cross-flow method is used.

As such a hollow fiber type micro filter module, for example, MICROZA PW-303 and PW-103 commercially available from Asahi Kasei Corporation can be used. These polyolefin hollow fibers are made of hollow membranes with an inner diameter of several mm or less, and have an average pore diameter of 0.1 μm.
Is. It can withstand a temperature of 80 ° C, can be used in a pH range of 1 to 14, and has good chemical resistance. Polypropylene hollow fibers and copolymer type hollow fibers of halogenated hydrocarbons and polyolefins can also be used. The average pore size of the membrane used is preferably 0.05 to 1 μm.

In this way, before the growth rate is reduced or the growth is stopped, the nutrient solution in the main culture tank is supplied to the hollow fiber type microfilter module as described above, and the solution containing the bacterial cells and the bacterial cells containing no bacterial cells are passed. The liquid containing the cells is returned to the main culture tank, and the solution containing no cells, that is, the solution containing the metabolite causing the growth inhibition is sequentially withdrawn. Then, a fresh liquid medium having the same volume as the extracted liquid can be continuously supplied to the culture tank to further continue the overculture without receiving growth inhibition.

As a result, the bacterial cells continue to grow logarithmically at the maximum growth rate without being inhibited by the metabolites, and a high concentration of bacterial cells can be obtained. However, as the cell concentration increases, the amount of liquid to be extracted increases. In other words, it is necessary to increase the dilution rate with fresh medium. Otherwise, the metabolite concentration will rise too high and growth will stop at that point. Therefore, a small amount of liquid may be withdrawn at the time of over-start, but it is necessary to take care to increase the amount of liquid withdrawal stepwise, preferably continuously, as the overculture progresses. Here, as described above, the hollow fiber-shaped microfilter module used in the present invention has an extremely large membrane area as compared with the module volume, and thus is superior to other types of microfilters in supercapacity. If the ratio of the total volume of the culture solution to the surface area of the module membrane is appropriate even after continuous continuous filtration, a sufficient amount of solution is always withdrawn in order to increase the solution step by step and continuously. be able to.

If the overculturing is continued in this way, the bacterial cells continue to grow logarithmically and reach a high concentration up to the physical limit. For example, with a propionic acid bacterium, it is possible to obtain a high-concentration bacterial cell culture solution which is at least 50 times or more that of the conventional batch culture method.
So this way Once high density cell cultures of vitamin B ₁₂ producing bacteria is obtained, a high concentration cells of vitamin B ₁₂ producing bacteria that contain vitamin B ₁₂ obtained while further continued for over-culture to a high density Continuously withdraw the culture solution separately from the liquid,
By continuously supplying the same volume of fresh medium, it is possible to continuously produce high-concentration vitamin B ₁₂ from the high-concentration cell culture medium of the extracted vitamin B _12- producing bacterium.

Next, an example of an apparatus for carrying out the culture method of the present invention is shown in FIG. Referring to FIG. 1, reference numeral 1 denotes a double culture tank made of glass, in which a constant temperature water 3 having a constant temperature is circulated in an outer jacket portion 2 to cultivate an inner culture solution 4.
To maintain a constant temperature. The lid 5 is made of stainless steel, and a silicone rubber packing 6 is inserted between the lid 5 and the container. The lid 5 is tightened with a thumbscrew 7 to make it airtight and prevent contamination of various bacteria. This culture tank 1 is placed on a magnetic stirrer 8, and the magnet chip 9 at the bottom of the culture tank 1 is rotated to stir the culture solution 4.

10 is an air filter for nitrogen gas when anaerobic bacteria are cultured, and air or oxygen gas for aerobic bacteria 11.
Is a line for aseptically supplying to the culture solution 4 through
The whipping device 12 for breeding goldfish is attached to the tip thereof.
Reference numeral 13 is a line for exhausting the aerated gas, and a trap is attached to the line to prevent contamination of various bacteria. Reference numeral 14 denotes one of the contacts of a liquid level gauge which is provided so as to be always contained in the culture solution, and may be a stainless rod. Further, 15 is the other contact of the liquid level gauge set on the culture liquid level, which may also be a stainless rod.

Reference numeral 16 is a line for supplying a fresh culture medium 19 for supply, which is filled in a container 18 immersed in the constant temperature water 3 in the constant temperature water tank 17, to the culture solution 4, and the liquid is withdrawn due to excess and the liquid level is lowered to When the contact 15 separates from the liquid surface, the liquid level gauge 20 operates to move the microtube pump 21 to supply the fresh medium 19 to the culture solution 4.

22 is an alkaline solution or acid solution for controlling pH 2
3 is a line for supplying the culture solution 4 with the culture solution 4
When the pH of the above is decreased or rises above a certain value, the PH controller 25 connected to the PH electrode 24 operates to move the peristaltic pump 26 to supply the above alkaline solution or acid solution 23 to the culture solution 4. 27 is a line for taking out the sample.

28 is a hollow fiber type microfilter module 29 provided at the lower side of the culture tank 1 for extracting the culture solution 4.
Is a line for supplying from the opening 30 at one end thereof, and here, a roller pump 31, a flow meter 32, a pressure gauge 3
3 is provided. Reference numeral 34 is a liquid take-out line connected to the side opening 35 of the module 29.
6. A liquid adjusting cock 37 is provided, and a line 39 for returning the liquid taken out from the other end opening 38 of the module 29 to the culture tank 1 is connected. The line 39 is provided with a pressure gauge 40 and a ball valve 41. The solenoid valve 36 is connected to a nitrogen gas cylinder (not shown) by a line 42.

Reference numeral 43 denotes a circulation pump for supplying the constant temperature water 3 from the constant temperature water tank 17 to the jacket portion 2 of the culture solution tank 1 through the line 44. Each line is made of silicone rubber.

When setting up such a device and operating it,
PH measured in the PH electrode 24
Depending on the value of, the alkaline liquid or the acid liquid 23 is added, the magnetic stirrer 8 is operated to stir the culture liquid 4, and the culture liquid is supplied by supplying sterile nitrogen gas or sterile air, oxygen from the line 10 if necessary. Keep the culture at the optimum temperature, pH, anaerobic level, etc., and use line 27 whenever necessary.
A sample is taken and the absorbance is measured. When the absorbance becomes close to the value which causes the growth inhibition which is measured in advance, the roller pump 31 is operated to pass the culture solution 4 through the line 28 to the hollow fiber type micro filter module 29.
Supply to. The liquid (liquid) containing no passed bacterial cells and containing metabolites is taken out from the line 34, while the liquid containing bacterial cells is returned to the culture tank 1 via the line 39.

When the culture solution 4 is drawn out and the liquid level is lowered, the microtube pump 21 works to supply the fresh medium 19 for supply to the line 1.
It is supplied to the culture tank 1 via 6. Liquid level is contact point 1
When it reaches 5, the supply is stopped, and the same volume of fresh medium 19 as always withdrawn is supplied to the culture tank 1 to continue the overculture. At that time, by operating the liquid adjustment cock 37, the liquid withdrawal amount at the time of overstart is reduced,
Adjust so as to increase the sampling amount gradually or continuously. However, if the bacterial cell concentration becomes extremely high, it may happen that the liquid volume cannot be obtained as expected, so at that time, the flow rate of the roller pump 31 is increased and the module 2
9 to increase the flow rate of the culture solution in the hollow fiber,
The amount of liquid can be increased by squeezing the ball valve 41 of No. 1 to apply pressure. The module 29 is
The Microzer PW103 can normally withstand pressures up to about 2 atmospheres.

When the bacterial cells adhere to the hollow fiber membrane of the module 29 due to the progress of filtration, pressure is applied from the outside to the inside to remove them. Also in this case, the pressure can be applied to the pressure that the module 29 can withstand. In that case, the electromagnetic valve 36 is connected to a timer, and the line 34 is closed by the electromagnetic valve 36 at a constant time interval (about 1 second) at a constant time interval, and nitrogen gas is added from the line 42 so that gas pressure is applied. Of course, normally, the nitrogen gas line 42 is closed and the liquid drain line 34 is open.

When using a hollow fiber type microfilter, before passing through the culture solution, wash and sterilize with a 0.3% sodium hypochlorite aqueous solution in advance, and then sterilize water and sterilized at 80 ° C.
It is advisable to wash away sodium hypochlorite with hot water. When reusing the cells, immediately after using them, wash the cells thoroughly with water, and similarly sterilize the cells adhered to the hollow fiber membrane with a 0.3% sodium hypochlorite aqueous solution to lyse them. It is preferable to melt it and wash it.

In addition, the main culture tank should be airtight at the lid or the line connection part to prevent contamination of various bacteria. Also, the microtube pump 21, the peristaltic pump 26, and the roller pump 3 used here
No. 1 etc. is a pump based on the principle that the rotary roller squeezes the silicon tube to generate peristaltic action for the liquid (gas) in the tube, so the culture solution in the tube does not touch the outside air. There is no worry of contamination by various bacteria. By using such a pump, the method of the present invention can be carried out without fear of contamination of various bacteria.

Although not shown in the figure, when the high-concentration bacterial cell culture solution is continuously collected after the high-concentration bacterial cells are obtained, the culture tank 1
A line for extracting the culture solution 4 (which is a high-concentration bacterial cell) may be newly provided in the above, and a fixed amount may be extracted by a roller pump. At this time, the culture solution decreases, but since the level gauge works and the fresh medium is automatically supplied, the amount of the culture solution can be always kept constant.

〔Example〕

Strain: Propionibacterium Shermanii medium composition: polypeptone 1%, yeast extract 0.5%, glucose 2%, K ₂ HPO ₄ 0.05%, CoSO ₄ ·
7H ₂ O 15ppm PH6.5 Pre-culture 18ml Volume 10ml of each medium was put into 2 test tubes, cotton stoppered, sterilized by autoclave at 121 ℃ for 15 minutes (separate sterilization of glucose), inoculated with bacteria, and kept at 30 ℃ water bath. Two
The culture was allowed to stand for 4 hours.

Bacterial inoculation: 600 ml of the medium was placed in the main culture tank, and sterilized at 121 ° C for 15 minutes in an autoclave including the silicon tube line set in the main culture tank in advance. (Glucose was sterilized separately) After that, the preculture solution was added as it was to the medium in the main culture tank to inoculate the bacteria.

PH control batch culture Immediately after inoculating the medium in the main culture tank with the bacterium, the culture solution is maintained at 30 ° C., nitrogen gas is aerated, and the mixture is stirred (100 rpm).
Started. In addition, by operating the PH controller,
I tried to keep it at 6.5. From the result of pre-batch culture for PH control for 35 hours, this bacterium was found to be
~ 30 hours later, O. Since it is known that D570 values of 7 to 10 start to undergo growth inhibition by propionic acid and acetic acid, which are self-metabolites, over-culture was started after performing PH control batch culture for 27 hours. In addition, the culture solution was sampled every 3 to 8 hours during the culture,
Immediately after the D570 value was measured, it was stocked at -20 ° C to prepare a sample over time.

* The tip of the line of the main culture tank (the connection to the excess line and the fresh supply medium) was completely tightened with a pinch cock or screw cock to prevent contamination of various bacteria.

Preparation of over-cultivation Fresh medium 10 for feeding before over-culture is started
Put each in 5 glass containers, sterilize by autoclave for 15 minutes at 121 ℃ (separate sterilization for glucose), then 1
The book was placed in a constant temperature water bath of 30 ° C. and kept at 30 ° C.

As a hollow fiber type micro filter module,
Asahi Kasei Microza PW-103 was used. As Microza PW-103, use one that has been sterilized and washed with 0.3% sodium hypochlorite aqueous solution in advance, and then rinsed off sodium hypochlorite with sterilized water and sterilized hot water at 80 ° C. did.

Overculture start After culturing for 27 hours by PH control batch culture, connect the microline PW-103 with the overline attached to the main culture tank, connect all the lines necessary for overculture such as the fresh medium supply line, The roller pump was operated to extract the culture solution from the main culture tank, and Microuser PW-103 (average pore size 0.1 μm, membrane area 0.2)
m ² ) and separated into a liquid containing cells and a liquid containing no cells, and the liquid containing no cells, that is, the liquid was continuously withdrawn from the line. At this time, the amount of liquid was adjusted by squeezing the cock. At the same time, the liquid level gauge worked and the same volume of fresh medium as the liquid extracted by the microtube pump was supplied to the main culture liquid. In addition, the flow rate of the roller pump is about 40 / h, and since the main culture tank is smaller than the volume of the line this time, the volume of the culture solution including the overline is increased from 600 ml at the time of batch start to 800 ml at the time of batch start. It was The culture solution was sampled every 2 to 4 hours and the O. Immediately after the D570 value was measured, the sample was stocked at −20 ° C. to obtain a sample over time, and the liquid amount was measured every 1 to 4 hours. The stocked microbial cell culture solution over time was separated into the microbial cells and the supernatant together with the sample from the batch culture by a centrifuge (8,000-
The supernatant was collected at 13,000 rpm for 5 to 10 minutes), and glucose, propionic acid, and acetic acid were quantified. Glucose was quantified by the Somogene Nelson method, and propionic acid and acetic acid were quantified by gas chromatography. Also,
Vitamin B ₁₂ is a sample of bacterial cell culture liquid,
After heat extraction with KCN, centrifugation (10000
The supernatant was collected at 0 to 15,000 rpm for 5 to 10 minutes) to obtain a vitamin B ₁₂ sample in a form converted into a cyano form. Vitamin B ₁₂ was quantified by a bioassay using a vitamin B ₁₂ auxotrophic Escherichia coli mutant strain (Esherichia coli 215) as a quantification strain.

Culturing results The results of culturing as described above are shown in Tables 1, 2 and 3.
It is shown in the table and FIG. 2 (a), FIG. 2 (b), and FIG.

・ O. Relationship between D value and dry cell weight (dry cell weight per one) O. D value x 0.289 = dry cell weight (g /) after 69h dry cell weight 227g / As is clear from this result, by over-culturing the propionic acid bacterium using the hollow fiber type microfilter module, 75 times or more of the bacterial cell concentration (about 10 in OD570 value) obtained by ordinary batch culture ( O.D57
It was possible to obtain a high microbial cell concentration of 0 value 787), and the vitamin B ₁₂ concentration was also increased as the microbial cell concentration increased, and finally 52.4 mg / -broth could be obtained. Here, this propionic acid bacterium contained about 250 μg of vitamin B ₁₂ per 1 g of the dried microbial cell, but the amount thereof did not change even when the bacterial cell had a high concentration.

[Effect]

Thus, according to the present invention, a high-concentration bacterial cell of vitamin B ₁₂ producing bacteria can be obtained, and a large amount of vitamin B ₁₂ can be easily produced industrially. Therefore, a higher concentration of vitamin B ₁₂ can be obtained by culturing a bacterium having a higher vitamin B ₁₂ content per bacterium, in other words, a bacterium having a high vitamin B ₁₂ producing ability.

[Brief description of drawings]

FIG. 1 is a systematic diagram of an example of an apparatus for carrying out the method of the present invention, and FIGS. 2 (a), (b) and FIG. 3 are graphs showing the results obtained by the examples. Fig. (A) is a graph showing the relationship between the culture time and the liquid amount, and Fig. 2 (b) is a graph showing the relationship between the culture time and the absorbance of the cell culture solution (OD570), the glucose concentration, the propionic acid concentration, and the acetic acid concentration. Fig. 3 is a graph showing the relationship between the culture time and vitamin B ₁₂ in the culture solution.
Concentration, dry cell density, vitamin B ₁₂ per 1g dry cell
It is a graph which shows the relationship with content. In FIG. 1, 1 ... Culture tank, 4 ... Culture solution, 8 ... Magnet stirrer, 19 ... Fresh medium, 24 ... pH electrode, 25 ...
PH controller, 29 ... Micro filter module, 34 ... Liquid extraction line, 35 ... Cell-containing liquid return line.

Claims (1)

[Claims] 1. A method of producing a vitamin B ₁₂ at a high concentration by a microorganism producing vitamin B ₁₂ was cultured in a liquid medium in the culture vessel to obtain a high concentration cells, vitamin B ₁₂ producing bacteria Before the growth is reduced or stopped by its own metabolites other than vitamin B ₁₂ , the culture solution containing the bacterial cells is continuously passed to separate the solution containing microbial cells and the solution containing no microbial cells. , The liquid containing the bacterial cells is returned to the culture tank, the liquid containing no bacterial cells is extracted, and a fresh liquid medium of the same volume as the extracted liquid is supplied to the vitamin B ₁₂ producing bacterial culture liquid to allow overculturing. followed, thereby to obtain a high concentration cells of the vitamin B ₁₂ producing bacteria, characterized by producing a high concentration of vitamin B ₁₂ whereby, vitamin B ₁₂
A method for producing vitamin B ₁₂ by high-concentration culture of a producing bacterium.