JPS594997B2 - Fermentation method Vitamin B↓1↓2 manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides vitamin B1□ production using a fermentation method that can produce and obtain vitamin B1□ with an improved production amount using vitamin B12-producing bacteria belonging to the genus Arthrobacterium, including new bacterial species. Regarding the manufacturing method of B1□.

More specifically, by using a medium containing a specific compound as a carbon source that is easy to obtain and has a stable supply, vitamin B1□ can be produced at low cost and as a feed for culturing operations, and with improved production.
Regarding the fermentation method for producing and obtaining vitamin B1□, in particular, vitamin B1□ belonging to the Arthrobacterium genus.
The producing bacteria are aerobically cultured in a medium containing at least one compound selected from the group consisting of alcohols and ketones having 2 to 3 carbon atoms, and before collecting vitamin B12 from the culture, The concentration of the above compound in
The present invention relates to a fermentation method for producing vitamin B12, which is characterized in that the culture system is maintained under conditions such that the amount of dissolved oxygen in the culture is 30% or less.

Conventionally, vitamin B1□-producing bacteria include Propionibacterium, Bacillus, Corynebacterium, Arthrobacterium, Rhodopsoidmonas, Pseudomonas, Protaminobacter, Streptomyces, Rhodospirinum, and Actinomyces. The existence of vitamin B12-producing bacteria belonging to the genera Selenomonas ruminantium and Nocardia is known.

Among these vitamin B12-producing bacteria, in addition to microorganisms that produce vitamin B12 using carbohydrates as a carbon source, microorganisms that produce vitamin B1□ using hydrocarbons as a carbon source include the genus Corynebacterium, the genus Arthrobacterium, the genus Pseudomonas, and the genus Corynebacterium. The existence of vitamin B1□-producing bacteria belonging to the genus Nocardia is known.

Furthermore, the existence of vitamin B12-producing plants belonging to the genus Pseudomonas and Protaminobacter, which produce vitamin B12 using methanol, which is a C1 alcohol, as a carbon source has been reported.

However, the earth lobe lifter used in the method of the present invention
For the production of vitamin B1□ by conventionally known vitamin B1□-producing bacteria, including vitamin B12-producing bacteria belonging to the genus, a culture medium containing a compound selected from the group consisting of 02 to C3 alcohols and ketones as a carbon source is used. The reports used are completely unknown.

Fermentation method In the production of vitamin B12, carbohydrates are the most widely and commonly used carbon source, but because they are natural sources, they are unstable in terms of price and supply, and industrial fermentation methods are not suitable. It is difficult to say that it is an appropriate material for producing vitamin B1□.

On the other hand, alcohols, which are easily available and inexpensive as petrochemical products, and are provided in stable quantities,
Using ketones and the like as a carbon source is significantly advantageous for industrial implementation.

However, it is extremely difficult to use water-immiscible or water-insoluble hydrocarbons as a carbon source because the residual hydrocarbons make it extremely difficult to isolate vitamin B12-containing bacterial cells from the culture solution. There is a certain flaw.

In addition, when water-soluble methanol is used as a carbon source, the troubles that occur during bacterial cell separation can be avoided, but methanol, which has a low boiling point, evaporates in large amounts during aerobic culture, for example, aerated culture. It has the disadvantage of being inconvenient in operation.

The present inventors conducted research to develop a fermentation method for producing vitamin B12 that can overcome the disadvantages and deficiencies of such conventional methods.

As a result, we succeeded in isolating and collecting a new fungal species, Arthrobafter hyalinus, which belongs to the genus Arthrobacterium and will be described in detail later.

Furthermore, this vitamin B12-producing bacterium belonging to the genus Arthrobacterium can be easily and inexpensively obtained as a petrochemical product, for example, and is miscible with water and is less likely to be lost by volatilization during aerated culture. It has been discovered that the present invention can be advantageously cultured in a medium containing as a carbon source at least one compound selected from the group consisting of alcohols and ketones having 2 to 3 carbon atoms, and that it has the ability to produce vitamin B12 in high yields.

Furthermore, in addition to the above-mentioned new bacterial species, vitamin B12-producing bacteria belonging to the genus Arthrobacterium can also be produced by using at least one compound selected from the group consisting of alcohols and ketones with 2 to 3 carbon atoms as a carbon source. , we found that similar benefits can be achieved.

Based on the above-mentioned new findings, the present inventors first developed vitamin B12-producing bacteria belonging to the genus Arthrobacterium,
A fermentation method for vitamin B1□, which is characterized by collecting vitamin B1□ from a culture obtained by culturing in a medium containing at least one compound selected from the group consisting of alcohols and ketones having 2 to 3 carbon atoms. Manufacturing method (Special Publication 58-4
No. 9236) was proposed.

As a result of further research, the present inventors have made a sufficient number of the vitamin B1□ producing bacteria exist in the culture, or have obtained vitamin B1□ from the culture in which the producing bacteria are present. At any time before harvesting, the concentration of the above compound selected from the group consisting of alcohols and ketones having 2 to 3 carbon atoms in the culture is below 1 g/l of the culture, and It has been surprisingly discovered that by maintaining the culture under conditions where the amount of oxygen is 3 p- or less, the production amount of vitamin B1□ can be further significantly increased.

Therefore, an object of the present invention is to provide an improved method for producing vitamin B12 by fermentation, which industrially advantageously produces and obtains vitamin B12 using vitamin B12-producing bacteria belonging to the genus Arthrobacterium.

The above objects and many other objects and advantages of the present invention include:
This will become clearer from the description below.

The vitamin B12-producing bacteria belonging to the genus Arthrobacter used in the method of the present invention include known bacteria, such as Arthrobacter simplex (A
TOO6946), Arthrobafter Tsumesens (A
rthrobacter tumescens)
[IFO12960), Arthrobacter globiformis
mis)
US ) C Microtechnical Research Institute Accession No. 3125] is available.

The above-mentioned Arthrobafter hyalinus cures the mycological properties listed below, and
Comparing with the description in the 7th edition of Determinative Bacteriology, no corresponding bacterial species was found, so it was identified as a new bacterial species belonging to the genus Arthrobacterium, and Arthrobafter.
・It was named Hiarius.

Arthrobacterium hyalinus ■, morphological characteristics: knee-cocoon shape; exhibits long rods in the early stage of culture, is curved, and has a size of 0.
Some of them are 8 x 4 to 5 microns and show a V-shape, and in the later stage they change to short rods of 1.2 x 1.5 microns.

Motility: None.

Durham stain; positive or negative.

Sporulation; none.

2.Culture properties Knee broth agar plate culture; jagged edges, convex center, transparent, mixed light.

Meat juice agar shell culture; poor growth, stringy, transparent, mixed light.

Meat juice liquid culture; turbid.

3. Physiological characteristics Growth temperature: 25-35℃ Growth pH: 6-9° Oxygen requirement; facultative anaerobic.

Liquefaction of gelatin: Not performed.

Lidmus milk; turns slightly alkaline and slowly peptonizes.

Indole: Does not occur.

Hydrogen sulfide; occurs.

Nitrate reduction: Not performed.

Catalase; not produced.

Urease; produced.

Anti-acidity; negative.

Decomposition of starch: Not performed.

Fermentability of sugars: Neither acid nor gas is generated from glycerin, propylene glycol, arabinose, xylose, fructose, galactose, glucose, mannitol, sorbitol, lactose, maltose, sucrose, and raffinose.

Asparagine; does not degrade.

Citric acid; does not decompose.

According to the present invention, vitamin B12 can be collected directly or indirectly from the culture obtained by culturing the above-mentioned vitamin B1□-producing bacteria belonging to the genus Arthrobacter in a medium.

According to the present invention, a medium containing a carbon source, a nitrogen source, inorganic salts, an antifoaming agent, etc. can be used as the medium.

In the method of the present invention, the medium contains at least one compound selected from the group consisting of alcohols and ketones having 2 to 3 carbon atoms as a carbon source.

Preferred examples of such carbon sources include ethyl alcohol, n-propertool, i-propertool, ethylene glycol, propylene glycol, glycerin, acetone, and the like.

In the present invention, other carbon sources such as normal paraffin, sugars, fats and oils, etc. can be used in combination with these.

Such combined use is often expected to further improve production.

Nitrogen sources to be included in the medium include, for example, corn steep liquor, yeast extract, peptone, fish cakes, vegetable protein, soybean meal, Sungross (dried powder of two-malt whiskey waste liquor manufactured by Sungross), ammonium salts, nitrates, and urea. etc. can be mentioned.

Furthermore, examples of inorganic salts include cobalt salts, phosphates, magnesium salts, manganese salts, zinc salts, calcium salts, molybdenum salts, and copper salts.

The absence of manganese salts in the medium is essential for vitamin B1.
If it is convenient for the production of No. 2, the addition of manganese salts can be omitted.

Furthermore, a vitamin B1□ precursor such as 5,6-dimethylbenzimidazole can be added to the medium as a vitamin B12 production promoting substance.

The medium composition can be changed as appropriate, and desired components can be appropriately added during the culture to perform the culture.

For example, the carbon source may be used at a concentration of about 20 g/l or less in the culture so as not to reduce the growth rate.
For example, sequential additions can be made to range from about 5 to about 159 per liter culture.

At this time, sufficient dissolved oxygen is supplied under sufficiently aerobic conditions, such as by aeration, stirring, shaking, etc., so that the dissolved oxygen concentration in the culture does not decrease and the growth rate decreases. can be cultured under the following conditions.

Generally, a culture temperature of about 20 to about 40°C is used, and a pH of about 4 to 9.5 can be adopted.

The number of days for culturing is usually about 2 to 8 days, and can be changed as appropriate depending on changes in other culture conditions.

In the method of the present invention, before collecting vitamin B1 The concentration of the compound selected from the group consisting of the above 2 to 3 alcohols and ketones in the substance is 1
g/l culture and the amount of dissolved oxygen in the culture is 3W
The culture is maintained under conditions that are below the IA.

The above-mentioned low compound concentration and low dissolved oxygen content conditions may be provided by any means, but for example, the above-mentioned vitamin B
1□ When the growth of the producing bacteria has progressed to the desired degree and the number of the producing bacteria in the culture has increased to the desired degree, if the supply of the carbon source is reduced or stopped, the above compound (the concentration of the carbon source decreases,
The growth of the producing bacteria can be reduced or stopped to bring about the above-mentioned low compound concentration conditions.

Furthermore, if the supplementation of the amount of dissolved oxygen is reduced or stopped, for example by reducing the oxygen supply or reducing the agitation efficiency for aeration, the above-mentioned low dissolved oxygen concentration condition will occur in the culture. be able to.

The period for which the culture is maintained under the above conditions can be appropriately selected, but for example, about 1 hour or longer, preferably about 1 to 40 hours, more preferably about 5 to about 20 hours, to achieve the desired The effect of increasing vitamin B1□ production can be obtained.

In the present invention, the above-mentioned maintenance is preferably carried out under conditions other than the above-mentioned carbon concentration and dissolved oxygen concentration that satisfy the culture conditions.

According to the method of the present invention, as described above, a vitamin B12-producing bacterium belonging to the genus Arthrobacterium is grown with a carbon number of 2 to 3.
aerobically in a medium containing at least one compound selected from the group consisting of alcohols and ketones, and before collecting vitamin B12 from the culture, the concentration of said compound in the culture is 1 g/g/ The amount of vitamin B12 produced can be further significantly increased by maintaining the culture under conditions in which the amount of oxygen dissolved in the culture is less than 1 culture and the amount of dissolved oxygen in the culture is less than 3 folds.

Although the reason why the production amount of vitamin B1□ is increased by the method of the present invention is not exactly clear.

It is thought that the microbial cells perform their functions in a state where the concentrations of both the carbon source and oxygen are extremely low, resulting in a state suitable for producing vitamin B1□ more selectively.

When carrying out the method of the present invention, vitamin B12 from the culture
can be collected in the same manner as in conventional methods.

Since vitamin B12 mainly accumulates within bacterial cells, for example, the culture is first centrifuged to obtain bacterial cells.

When separating cyanoid vitamin B1□ from a culture, it can be extracted by adding cyanide ions to the bacterial cells, adjusting the pH to 5 with an acid such as sulfuric acid, and boiling.

When separating coenzyme-type vitamin B12 (5,6 dimethylbenzimidazole cobamide coenzyme) and hydroxy-type vitamin B1□ from the culture, the bacterial cells are infused in the dark with methanol, ethanol, acetone, pyridine, etc. according to the conventional method. It can be extracted using a solvent of

Purification of vitamin B1□ extracted from the culture can be carried out by appropriately combining phenol extraction means, adsorption means using activated carbon, column chromatography using ion exchange resin, cellulose, etc.

The present invention will be explained in more detail with reference to Examples below.

Example 1, Comparative Example 1, and Comparative Example 2 Arthrobacter hyalinus [Feikoken Bacteria No. 3125
] i-propatool 10 m11 peptone 3g, yeast extract B, NH4N033. ! il, Na2HPO4
+ 12H201,5El, KH2PO,0,4&1
Mg504-7H200,5,9, FeSO4・7H2
010F71'P, ZnSO4・7 H2O10"i,
C.

(N03)25'#, CuSO4・5H2050μs,
Four 500-capacity Erlenmeyer flasks containing 100-mL of sterilized medium containing Mo0310 μEl and 0a0035 g were inoculated and cultured with shaking at 30° C. for 2 days, and the culture solution was used as a seed seed.

i-proptool 10m, peptone 3g, sun gloss 5
g, NH4N0312g, Na2HPO4m 12H
2O6g, KH2PO41.6g, Mg504-7H2
0:l, FeSO4・7H7H2O40, Zn S 0
4 ・7 H2040”?, Co(NO3) 25mg,
CuSO4・5H20200μ91MoO310pg,
0a0035. ! i', antifoaming agent 0.5mA! A culture medium 41 consisting of 11 ion-exchanged water was prepared in a fermentation tank 101, and after sterilization, 400 yd of the above seed mother was inoculated, and the culture medium was incubated at 32°C for 7 hours.
The cells were cultured for 36 hours while stirring at 00 rl) I and aerating sterile air at 2.0 d/min.

During that time, the i-propatool in the culture solution was 3. omi/la
Add i-proper tool 1 without exceeding W.
30rnl was supplied per culture solution.

When i-propertool was no longer detected in the culture solution, the air supply was stopped and the stirring speed was reduced to 3 oorpm.

The concentration of vitamin B12 in the culture solution just before decreasing is 1
,010μ9/l.

Immediately after that, the dissolved oxygen concentration decreased to 0.7 ppII,
The temperature was maintained at the same temperature for 5 days.

During this period, the concentration of vitamin B12 in the culture solution increased and reached the maximum at 12 o'clock after the air supply was stopped, and the concentration was 1.4 hours.
It was 50μfl/l.

On the other hand, without stopping the air supply, the stirring speed was increased to 70%.
When a total of 48 hours have passed while keeping the value at 0 rial (
Comparative Example 1) Vitamin B12 production aU-i 1.13
It was 0 pEl/1.

Note that the dissolved oxygen concentration in this case was 5.5pIIIl.

Also, as mentioned above, while supplying i-property tools, 3
After culturing for 6 hours, when i-propatool was no longer detected in the culture solution, 2.5 g of i-propatool was added per 11 cultures, and the original aeration and stirring conditions were continued for a total of 48 The production concentration of vitamin R12 over time (Comparative Example 2) was 1,060 μfi//l.

The dissolved oxygen concentration at this time was 2.0 pTMIl.

As shown in Comparative Examples 1 and 2, it can be seen that the improvement effect of the present invention cannot be achieved even if either of the compound concentration conditions and dissolved oxygen amount conditions specified in the present invention are not satisfied.

Example 2 Table 1 shows the vitamin B12 production rate and bacterial cell concentration obtained in the same manner as in Example 2 except that various carbon sources were used instead of i-propertool in Example 1.

Example 3 Earthlobafter Simplex (ATOO6946
) to n-Proper Tools 1o7727! , peptone 3g,
Yeast extract 1g, NH4N033g, Na2HP0
・12 H2O1.5g, KH2PO40.4.9, M
gSO4・7H200, 5y1FeSO4・7H201
0"11ZnS04・7H2010'?, co(N03
)25mfj1CuSO4・5H2050μy1Mo0
310μL Sterilized medium 1 containing Oa0035g
The seeds were inoculated into four 500-capacity Erlenmeyer flasks containing 00rrll and cultured at 30° C. for 5 days.

n-propatool 10 yd, peptone 3 g, sun gloss 5 g, NH4No36. ! i', NaHPO+ ・12
H20:l, KH2PO40.8g, Mg5O, 7H
,,01g,FeSO4・7H2020～,ZnSO4
・7H2020■, Co (N 03) 251119゜0uSO, ・5H20100p&, Mo0320 μf
i.

0a0035. ! i', 0.5 rnl of antifoaming agent, and 11 rnl of ion-exchanged pure water were prepared in a fermentation tank, and a sterilizing layer was inoculated with 400 rnl of the aforementioned seed mother.
The cells were cultured for 7 days with stirring of rpII and aeration of sterile air at 2.0 l/min.

During that time, do not add n-propatool to the culture medium so that the concentration does not exceed 3.0 ml/l.
10rnl was supplied per culture solution.

When the concentration of n-propertool in the culture solution was no longer detectable, the air supply was stopped and the stirring speed was reduced to 25 Å.

Immediately after that, the dissolved oxygen concentration decreased to 1.3 degrees, and the temperature was maintained at the same temperature.

During this period, the concentration of vitamin B12 in the culture medium increases and reaches its maximum 18 hours after the air supply is stopped, and the concentration is 2.
It was 80 μg/l.

In contrast, the stirring speed was increased to 5o without stopping the air supply.
The concentration of vitamin B12 produced after a total of 8 days while maintained at ORPM was 197 μg/l.

In the above examples, the determination of vitamin B1□ in the culture solution was as follows:
Potassium cyanide was added to the diluted culture solution to adjust the pH to 5, and then boiled for 15 minutes to convert it to dianocobalamin, followed by a microbial quantitative method using Lactobacillus leichmani ATO07830.

Claims (1)

[Claims] 1. A vitamin B1□-producing bacterium belonging to the genus Arthrobacterium is aerobically cultured in a medium containing at least one compound selected from the group consisting of alcohols and ketones having 2 to 3 carbon atoms. , before collecting vitamin B12 from the culture, the culture is grown under conditions where the concentration of the above compound in the culture is less than 19/l culture and the amount of dissolved oxygen in the culture is less than apr111. A fermentation method for producing vitamin B1□, which is characterized by its retention. 2 The vitamin B1□ producing bacterium is Arthrobacter hyalinu.
s). 3 The vitamin B12-producing bacteria are ground 0/<lid.
Arthrobacter sympl
The manufacturing method according to claim 1, which is (ex). 4. The method according to claim 1, wherein the compound is selected from the group consisting of ethyl alcohol, n-propanol, 1so-7'' lovanol, ethylene glycol, propylene glycol, glycerin and acetone.