JPS6291190A - Production of l-aminoacid through fermentation process - Google Patents

Abstract

PURPOSE:A microorganism capable of producing L-aminoacid is subjected to seed culture under oxygen-enriched conditions, then the resultant culture is inoculated in the major culture of the fermentation to improve the yield, production rate and accumulation of the product. CONSTITUTION:A microorganism which is capable of L-aminoacid, such as L-glutamic acid, L-lysine, L-glutamine, L-arginine, L-phenylalanine or L-threonine is subjected to seed culture. At this time, lactic acid in the culture medium is monitored and oxygen is fed into the medium, when lactic acid exceeds a certain level. Thus, high cell body yield and high cell body concentration are achieved. The seed culture is transplanted in the medium for L-aminoacid fermentation and cultivation is effected.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides L-glutamic acid, L-lysine, L-glutamine, and
L-fulginine, L-phenylalanine, L-threonine, L-isoleucine, L-histidine, L-7'loline, L-serine, L-ornithine, L
- Related to a method for producing L-amino acids such as citrulline, L-tyrosine, L-)litphane, and L-leucine. L-glutamic acid, L-lysine, L-glutamine, and
-Arginine, L-phenylalanine, L-threonine, L-inleucine, L-histidine, L-f loline,
L-amino acids such as L-valine, L-serine, L-orthine, L-citrulline, L-tyrosine, L-)litphane, and L-leucine are industrially produced by fermentation methods. Since these microorganisms require all oxygen for growth, supply of oxygen is essential for culturing such microorganisms. Generally, in order to obtain each microbial cell, the conventional method is to supply all the air into the liquid medium as a supplement to the oxygen supply, and to increase the dissolved oxygen concentration in the medium by making the air bubbles finer by aeration and stirring. is being attempted. However, in seed culture, simply aerating air will produce organic acids such as phosphoric acid, co-phosphoric acid, and lactic acid in the medium, and among these organic acids, the production of lactic acid is particularly significant. It was found that the production of these organic acids reduces the concentration of the desired bacterial cells producing each amino acid and the bacterial yield of the main carbon source in the seed culture.

In this way, the conventional amino acid fermentation in the main fermentation medium using a seed culture solution with low bacterial cell concentration and bacterial cell yield has been shown to be effective in achieving the desired yield and production rate of L-amino acids. It had the disadvantage of decreasing both the amount and the amount of accumulation.

[Problems to be solved by the present invention]

The problem to be solved by the present invention is to solve the above-mentioned drawbacks and to develop a method for industrially producing L-amino acids at a lower cost than before.

[Means to solve all problems]

In order to further improve the fermentation yield, production rate, and accumulation m-+ of L-amino acids and to develop an industrially advantageous method for producing L-amino acids by fermentation, we As a result of extensive research, we have found that by supplying a total oxygen concentration higher than that in the air to the food culture medium, organic acids such as malic acid, succinic acid, and lactic acid in the culture medium are reduced.
It was found that the yield, production rate and accumulation amount of amino acids can be increased by using the seed culture solution. The present invention was made as a result of further research based on this knowledge.

That is, the present invention involves inoculating all microorganisms capable of producing L-amino acids into a seed culture medium, and after complete cultivation while supplying oxygen-enriched air to the yeast culture medium, this seed culture is transferred to a main fermentation medium. , relates to a method for producing L-amino acids by a fermentation method characterized by carrying out main fermentation. In addition, as for the method of supplying oxygen, from the time when the lactic acid produced significantly in the culture medium reaches at least 10m97a or more, the oxygen supply method is as follows: @Method of supplying oxygen with increased oxygen concentration to the culture medium (t#GanSho 6O-56696) i may be used.

In the present invention, the amino acid referred to is L-glutamine 1! ,
L-lysine, L-glutamine, L-arginine, L-7
enilalanine, L-threonine, L-inleucine,
L-histidine, L-proline.

L-amino acids such as L-valine, L-serine, L-orthine, L-citrulline, L-tyrosine, L-tryptophan, and L-leucine, and the L-amino acids exemplified here.
The method of the present invention can be used with any L-amino acid other than amino acids, as long as it is produced by fermentation under aerobic conditions.

The L-amino acid producing bacteria used in the present invention may be any microorganism that produces the above-mentioned L-amino acids.

Specifically, the IL-glutamic acid producing bacterium Previvucterium tinoro 7catam AT
CC14020b) 4Tagojuku Zolium Furano%
ATCC14067 Plebipucderium lactofermentum ATCC13869 Plebipucderium lactofermentum FERM-P
5012 (AJ 11360) Corycetes thylium acetoacidophyllum ATCC13870 Corycetes tyrium ri>p micum ATCC130322
L-lysine producing bacterium Vk) 49 Thylium futitum F'E
RM-P 1708 (AJ 3419) Corine z41
Liu A, /'14;l Mikumu FERM-P
1709 (AJ 3420) Brevizu Cuderium
Lactofermentum F'1M-P 1712 (
AJ 3425) Buhair 4 polysilylium lactofermentum FEag-p 1711 (AJ 34
24) 3 L-glutamine producing bacteria fv69f4nowA-7rano? A F
ERM-P 5502 (AJ 11576) Corynebecterium acetoacitophyllum ATCC1387
04L-Aldani-producing bacteria FERM-
P 7642 (AJ 12144) Corynebacterium glutamine A F'ERM-P 72
74 (AJ 12093) 5 L-phenylalanine-producing bacterium Kolyashio Thirium cultamicum ATCC2
16706L-Threonine producing bacterium Esselia coli F
'ERM-P 4900 (AJ 11334) Coriopaculataum cultamicum FERM
-P 5835 (AJ 11654) Hive t1 Zlf') Ram Fra fb
FERM-P 4164 (AJ 11172)
”Brevipicterium lactofermentum
FERM-P 4180 (AJ 11178) 7 L
- Isoleucine-producing bacteria y”vb labor-7 lapam FER
M-P 805 (AJ 3271)=y”L439
f4 noum lactoferno 4m F'ERM-P
4192 (AJ 11190) 8 L-histidine-producing bacterium Brekkkushika Hanoum flavum FER
M-P 2316 (AJ 3620) BuV Riricterium lactofermentum FERM-P 156
5 (AJ 3386) Corynebacterium glutamicum ATCC142979L-Zololine-producing bacterium V Tazatherium furanogum FERM
-P 5332 (AJ 11512) Brevipicterium lactofermentum FERM-P 437
0 (AJ 11225) Corynebacterium glutamicum FERM-P 4372 (AJ
11227) 10 L-valine producing bacterium Brevizukterium lactofermentum FER
M-P 1945 (AJ 3446) B) Hexedatherium flavum FERM-P 51
2 (AJ 3276) Corynebacterium lactofermentum FERM-P 1968 (AJ
3455) 11 L-serine producing bacterium Corynebacterium glycinophyllum FER
M-P 1688 (AJ 3414) Dadutherium
Lactofermentum FERM-P 1371 (
AJ 3360) Shiα Shikudozo Pufupe Mekap v'ztsu → UCS F'ERM-P 4532 (
AJ 11262) 12 L-orthine producing bacterium Plebipecterium lactofermentum FEB
I■-P 5936 (AJ 11678) Corynebacterium glutamicum FERM-P 5
644 (AJ 11589) 13 L-citrulline producing bacterium Corynebacterium glutamicum FER
M-P 5643 (AJ 1158B) Brevi (Cterium flavum FE purchase-P 16
45 (AJ 3408) 14 L-tyrosine producing bacterium Corynebacterium ri) Shiomicum FER
M-P 5836 (AJ 11655) Butt1 Kujidaderium furanohiro FERM-P
7914 (AJ 12180) Nog-, vs. Not Chilis FERM-
P 6758 (AJ 11968) 15L-)
Liptophan-producing bacteria Kaoru Bachi← subtilis F'ERM-P
5286 (AJ 11483)
Lactofermentum FERM-P 7127
(AJ 12044) Previdotherium furanohiro
FERM-P 7034 (AJ 1202
2) Cori, ￥Siri2ium Glutamicum
FERM-P 7128 (AJ 12052) 16
L-leucine producing bacterium Prehihycterium lactofermentum FER
M-P 1769 (AJ 3427) Fu1 Hen Town t1
Rium Furanoum F'ERM-P
420 (AJ 3226) Corinne glutamicum FERM-P 1966 (AJ
3453) etc. are used.

As the seed medium for the L-amino acid producing bacteria used in the present invention, any conventionally known seed medium suitable for the growth of monoamino acid producing bacteria can be used.

Furthermore, in the present invention, in order to supply an amount of oxygen higher than the conventional oxygen concentration in the air and obtain a high concentration of bacterial cells, the nutrients in the medium can be used even if they have a concentration higher than the conventional medium composition. High concentrations may be used as long as the balance of other nutrients is not lost.

In the present invention, the amount of lactic acid in the seed medium is measured, and from the time when the amount of lactic acid exceeds a predetermined concentration, oxygen at a concentration higher than the oxygen concentration in the air is supplied to the seed medium. Lactic acid may be measured by any known method as long as it is rapid. The amount of lactic acid produced at the time of supplying atmospheric oxygen may be arbitrarily selected depending on the type of amino acid used and the type of producing bacteria. Specifically, oxygen at a concentration higher than that in the air may be supplied into the culture medium when the temperature reaches 10/dt to 200 Tn9/dt, preferably 10 da/dt or more. The oxygen used in the present invention includes liquid oxygen, oxygen produced by the P, S, A (pressure, swing, absorption) method or oxygen enriched membrane method.

The amount of oxygen supplied may be adjusted as appropriate depending on the level of lactic acid in the medium. In this case, even if excess oxygen is supplied, it will have almost no effect on bacterial yield or bacterial cell concentration, but economically the amount of oxygen supplied is better than when the amount of lactic acid in the medium is 200 μ/a or less. Generally, the oxygen concentration is 25~
For a mixed gas of 50% oxygen and air, it is 1/10 to 1/
It is about 2 VVM.

As for the method of supplying more oxygen than air, air and oxygen may be supplied into the culture tank at separate intervals, or they may be supplied after mixing air and oxygen, or ammonia gas and They may be mixed and supplied.

Furthermore, it is also possible to supply oxygen from the surface of the medium.

In the present invention, the supply of oxygen at a concentration equal to or higher than that in the air may be stopped at the time when no production of lactic acid in the culture medium is observed even if the supply of the gas is stopped.

As described above, by carrying out the entire seed culture, a seed culture solution with a high bacterial cell yield and high bacterial cell concentration can be obtained. This seed culture solution is transferred to a conventionally known L-amino acid production medium for main fermentation that is suitable for monoamino acid producing bacteria, and cultured to increase the fermentation yield, fermentation production rate, and accumulation amount of L-amino acids. will improve.

In addition, each amino acid can be isolated from the fermentation liquid according to a conventional method, and no special method is required.

Example 1 Glucose 5g7d1%KH2PO40,11? /
dt.

MgSO4・7H200-051/di, F@S
O4・7H201η/dt.

Mn so 4・4H201rn9/dt, urea 0.
51/di, Bio f7300μI/l, thiamine hydrochloride 3000μ9/l, soybean protein acid hydrolyzed solution as total nitrogen and 100w1/dt gold as a seed medium adjusted to pH 7.5 in a 300dFc 11 volume fermenter. Sterilized. This is Brevibacterium lactofermentum FERM-P 5012 (AJ 1
1360) and cultured at 31.5℃ and rotation f1.1.
-7.0 to 7.000 rpm with ammonia gas.
I kept it at 5. Ventilation is air (04WM) only, and air (0,4vVM) and oxygen (0,I VV).
M) mixed gas was used. In the case of mixed gas with oxygen, the aerated oxygen was controlled so that the production of lactic acid did not exceed 200 m97 dt, and the culture was continued until the consumption of glucose was completed. The seed culture solution obtained in this way was added to the main fermentation medium (Table 1) at 5 vol%.
The cells were transplanted and cultured at 35°C. Glucose concentration is 31/
/dl or less, the glucose concentration decreases from 1 to 3 g.
/d1. 709/d so that it is controlled within the range of
The culture was continued for up to 48 hours while continuously feeding 1 liter of glucose solution.

Table 2 shows the bacterial cell concentration in the seed culture and the L-glutamic acid produced in the main culture.

Table 1 Glucose 8 97dK) I2P
O40,2# Mg 804・7)120 0.05 1
FeSO4' 7H201fn9/litMn SO4
・4H201 Piotin 100 μi/1 Thiamine・HCl 500 μg/No 7.
5 Table 2 Example 2 A seed culture medium with the composition shown in Table 3 (300 tRtt-1) was poured into a jar fermentor with an O1 volume, sterilized at 120°C for 15 minutes, and Brevibacterium flavum FERM-
Inoculated with P 1708 (AJ 3419), 31.5
℃, rotation speed 1+OOOrpm, p with ammonia gas
i(6,s to 7.0).

When ventilation is only air (1/4 V'VM) and when air (0,20YVM) and oxygen (0,05VVM)
+7) I did a mixed caste. In the case of mixed gas with oxygen, the amount of oxygen aerated was controlled so that the production of lactic acid did not exceed 50 q/dti, and the cultivation was continued until the consumption of glucose was completed. In this way, the result is 3
After the glucose concentration drops below 1 i/dt, the glucose concentration decreases from 1 to
6011 to be controlled within the range of 397dt
The culture was continued for up to 96 hours while continuously feeding a mixture of 7 dl of glucose and 317 dt of ammonium sulfate. The bacterial cell concentration in the seed culture and the amount of L-IJ gin produced in the main culture were as shown in Table 4.

Table 3 Medium composition Table 4 Example 3 Glucose 6.9/dt, (NH4)2So40.
2 N/#.

Urea 0.2 g/dt, KH2PO40,15F/
dt, MgSO4・7H200,04#/dt
, FeSO4'7H201■/di, Thiamine HCl 10μ! i/di, biotin 0.3μI
/dt, 30Tn with soybean protein acid hydrolyzate as total nitrogen
The seed medium containing 9/dtf was placed in a 300 dt-11 capacity fermenter containing a medium adjusted to pH 7.0 and sterilized.

In this, Brevibacterium flavum FERM-P 5
502 (AJ11576) was inoculated, the temperature was maintained at 31.5°C, and the pH was maintained at 7.0 to 7.5 with ammonia gas at a rotational speed of 1.00 Orpm. Ventilation is air (
1/2 VVM ) and air (0,4WM
) and oxygen (0,1VVM) (7). In the case of a mixed gas with oxygen, the production of lactic acid is 501n9
The culture was continued with i't-control of the aeration of oxygen so as not to exceed 7 dt until glucose consumption was completed. In this way, after the yield falls below 3 fj/dl, the culture is continued for up to 72 hours while continuously feeding 7011/dt of glucose solution so that the glucose concentration is controlled within the range of 1 to 3 fj/dt. continued. As a result, the bacterial cell concentration in the seed culture and the amount of L-glutamic acid produced in the main culture were as shown in Table 6.

Table 5 Glucose 11 1/dtKH2P
O40, 25# 1i1fgSo4・7H200,04#FsSO4・7
H201rn9/dl Ammonium sulfate 1.5 11/dtNa
2So41.5 z Thiamine/HO235μme 9t Biotin 0.35 #th
6 Table Example 4 Glucose 7 g/dl, KH2PO40,1
g/dl.

MgSO4・7H200,041/a, FeS
O4”7H201′m9/di.

Mn5Oa ・4H201mg7 dl, urea 0.31
/dt, 1007 as total nitrogen for soybean protein acid hydrolyzate
119/#, Thiamine-HCL 30 pg/d
i, Seed medium containing Biofy 20 pg/dl'ft'
ip [(adjusted to 7.0 and sterilized in the 300 de11 capacity fermenter. Brevibacterium.
Flavum FERM-P 7642 (AJ12144
) was inoculated and maintained at 31.5°C.

Aeration was performed with only air (1/4 VVM) or with a mixed gas of air (0.2 VVM) and oxygen (0.05 VVM). In the case of mixed gas with oxygen, the production of lactic acid is 5
0m9/dl'! (The amount of oxygen aerated was controlled so as not to exceed the amount of oxygen, and each culture was continued until the consumption of glucose was completed.
The temperature was maintained at 6.5 to 7.0.

After the glucose concentration became 51/dt or less, the culture was continued for up to 72 hours while continuously feeding the glucose solution at 709/di so that the glucose concentration was controlled within the range of 3 to 5117 dl. As a result, the bacterial cell concentration in the seed culture and the amount of arginine produced in the main culture were as shown in Table 8.

Table 7 Glucose 14 97dlKL (2P
O40,11 MgSO4・7)120 0.04 11
1'@So4・7H201m9/diMn So a
'4H201 Ammonium sulfate 3.0 El/dl Thiamine・HC45μi/dl Biotin 5μ&7dl-7,0

Claims (1)

[Claims] After inoculating a microorganism capable of producing L-amino acids into a seed medium and culturing while supplying oxygen-enriched air to the seed medium, this seed medium is transferred to the main fermentation medium and the main fermentation is carried out. A method for producing L-amino acids by a fermentation method, characterized in that: