Antifoaming agent for fermentation, L-amino acid-producing medium and production process of L-amino acids

Disclosed herein are antifoaming agents for fermentation which include, as active ingredient at least one of (a) a reaction product obtained by adding at least one alkylene oxide to a mixture of a fat and/or oil with a trihydric or still higher polyhydric alcohol, or (b) a compound represented by the following general formula (1): ##STR1## wherein n stands for a number of 2-50, R.sup.1, R.sup.2 and R.sup.3 mean individually a hydrogen atom or an acyl group having 2-31 carbon atoms, x denotes an alkylene group having 2-4 carbon atoms, and m1, m2 and m3 are individually a number of 0-200, an L-amino acid-producing medium containing either one of these components, and a production process for L-amino acids making use of this medium. The use of this antifoaming agent permits the quick, sure and durable suppression of bubbling during incubation, and the overall production of the L-amino acid is significantly improved.

BACKGROUND OF THE BACKGROUND 
1. Field of the Invention 
The present invention relates to an antifoaming agent for use in 
fermentation processes, a fermentation medium which incorporates the 
antifoaming agent, a method of producing L-amino acids in the presence of 
the agent, and a method of defoaming using the agent. 
2. Description of the Background Art 
In the fermentative production of useful substances by submerged aerobic 
culture, a great number of bubbles and foam occur causing various 
problems. For example, when a fermenter becomes filled with bubbles the 
culture capacity per unit volume is lowered, and the culture solution can 
overflow. 
Attempts to suppress such bubbling have included the addition of an 
antifoaming agent to the fermentation medium Polyoxyalkylene polyhydric 
alcohol ethers, polyoxyalkylene alkyl ethers, polyoxyalkylene fatty acid 
esters, polyoxyalkylene alkyl ether fatty acid esters, etc. are 
antifoaming agents previously used (Japanese Patent Application Laid-Open 
Nos. 4282/1975, 121482/1975, 135298/1979, 169583/1981 and 35073/1990). 
These antifoaming agents for fermentation baths have not proven very 
satisfactory, however, due to an unacceptable antifoaming effect, an 
adverse affect on fermentative production (inhibition of growth of 
microorganisms, inhibition of formation of products, etc.), a long 
incubation time before developing an antifoaming effect, or the inability 
to retain an antifoaming effect over long periods of time. 
Since the fermentative production of L-amino acids such as L-glutamic acid, 
L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine, 
L-isoleucine, L-histidine, L-proline, L-valine, L-serine, L-ornithine, 
L-citrulline, L-tyrosine, L-tryptophan and L-leucine is commercially 
important, is carried out on an industrial scale by the fermentation of 
microorganisms belonging to Brevibacterium, Corynebacterium, 
Microbacterium, Bacillus, Escherichia or the like and encounters problems 
due to foam and bubbles, it is desirable to provide an antifoaming agent 
that overcomes the above drawbacks. 
Further, conventional L-amino acid fermentation processes are 
unsatisfactory with respect to yield. In order to increase the yield, 
specific surfactants have been added to the fermentation medium so as to 
allow for continuous fermentation while crystallizing out the L-amino acid 
product (Japanese Patent Application Laid-Open No. 288/1977). However, 
this process has proven unsatisfactory with respect to overall yield, 
although improvement as compared with conventionally-known processes is 
obtained. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide an 
antifoaming agent for fermentation which has excellent antifoaming effects 
when added to a fermentative medium while improving the yield of the 
L-amino acid produced, and a process of producing L-amino acids using the 
medium containing the additive. 
The present inventors have found that when a small amount of a product 
obtained by adding an alkylene oxide to a mixture of a fat and/or an oil 
with a polyhydric alcohol, or an acylation product of an (alkylene 
oxide-added) polyglycerol, is added to a fermentative medium, antifoaming 
is achieved quickly and for extended periods, and that the use of this 
antifoaming medium in the fermentation of L-amino acids improves the yield 
of L-amino acids significantly. 
In one aspect of the present invention an antifoaming agent for 
fermentation is provided comprising, as active ingredient(s), either (a) 
at least one reaction product obtained by adding at least one alkylene 
oxide to a mixture of a fat and/or an oil with a trihydric or still higher 
polyhydric alcohol, or (b) at least one compound represented by the 
following general formula (1): 
##STR2## 
wherein n stands for a number of 2-50, R.sup.1, and R.sup.3 mean 
individually a hydrogen atom or an acyl group having 2-31 carbon atoms, X 
denotes an alkylene group having 2-4 carbon atoms, and m1, m2 and m3 are 
individually a number of 0-200, or (c) a mixture of (a) and (b). 
In another aspect of the present invention, there is provided an L-amino 
acid-producing medium comprising water and, optionally a micororganism, 
nutrients and salts, and at least one of (a) the reaction product or (b) 
compound (1), or a mixture of (a) and (b). 
In another aspect of the present invention, there is provided a process for 
the production of an L-amino acid comprising incubating L-amino 
acid-producing microorganisms in a medium containing at least one of (a) 
the reaction product or (b) compound (1), or both, and collecting the 
L-amino acid from the resulting cultured mixture. 
Finally, the present invention provides a method of defoaming wherein at 
least one of (a), the reaction product or (b), a compound of formula (1), 
or both, are added to a medium which would be expected to foam or to a 
medium having foam already present. 
The use of (a) the reaction product or (b) compound (1), or both, permits 
the quick, sure and durable suppression of bubbling during incubation. 
Therefore, the yield of the fermentation product intended per unit 
fermenter is improved. 
In particular, when the above antifoaming agent is added to an L-amino 
acid-producing medium to produce an L-amino acid, the productivity of the 
L-amino acid is remarkably improved. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Component (a) useful in the practice of the present invention is the 
reaction product obtained by adding one or more alkylene oxides to a 
mixture of at least one fat and/or oil with at least one trihydric or 
still higher polyhydric alcohol. Examples of the fat or oil used as a raw 
material herein include vegetable oils such as coconut oil, palm oil, 
olive oil, soybean oil, rapeseed oil, linseed oil and castor oil; animal 
oils such as lard, beef tallow and bone oil; fish oils; and hardened oils 
and partially hydrogenated oils thereof, as well as recovered oils 
obtained in the purification process of these fats and oils. 
Any generally-known alcohol containing three or more --OH groups may be 
used as the trihydric or still higher polyhydric alcohol. Among these, 
however, tri- to hexahydric alcohols having 3-15 carbon atoms, such as 
glycerol, sorbitol, glucose, trimethylolpropane, trimethylolethane, 
1,2,4-butanetriol, 1,2,6-hexanetriol, 1,1,1-trimethylolhexane, 
pentaerythritol, erythrose, tetramethylolcyclohexanol, diglycerol and 
polyglycerol are preferred and may be used singly or in combination. Of 
these, glycerol is particularly preferred. 
Examples of the alkylene oxide useful herein include ethylene oxide, 
propylene oxide and butylene oxide. These alkylene oxides may be added 
either singly or in any combination thereof. It is however preferred that 
two or more of these alkylene oxides should be added in combination. 
Examples of the combination of two or more alkylene oxides include 
ethylene oxide-propylene oxide, ethylene oxide-butylene oxide, and 
ethylene oxide-propylene oxide-butylene oxide. In the combination of 
ethylene oxide and propylene oxide or butylene oxide, it is desirable that 
the number of moles of the added propylene oxide or butylene oxide should 
be more than that of the added ethylene oxide. The addition of the 
alkylene oxides may be conducted either by adding them as a mixture 
(random addition) or by successively adding them (block addition). The 
total number of moles of the alkylene oxides added is preferably 1-100 
moles, more preferably 5-100 moles, most preferably 5-50 moles per mole of 
the mixture of the fat and/or oil with the polyhydric alcohol. The mixing 
proportion of the fat or oil with the polyhydric alcohol is preferably 
1:0.1-1:6, more preferably 1:0.3-1:3 by mole. 
No particular limitation is imposed on the addition reaction of the 
alkylene oxide, fat and/or oil and polyhydric alcohol. The reaction may be 
conducted under the general conditions of addition reactions of an 
alkylene oxide to an active hydrogen-containing compound. More 
specifically, the reaction may be conducted by adding a catalytic amount 
of an alkaline substance to a mixture of the fat and/or oil with the 
polyhydric alcohol, which materials have been charged in the 
above-described molar ratio, and reacting 1-3 kg/cm.sup.2 of the alkylene 
oxide with the charged mixture at about 100.degree.-200.degree. C. 
Examples of component (b), i.e., the compound of formula (1), useful in the 
practice of the present invention include polyglycerol, mono-, di- and 
triacylated products of polyglycerol, adducts of polyglycerol with a 
polyoxyalkylene, and mono-, di- and triacylated products of adducts of 
polyglycerol with a polyoxyalkylene. Other preferred compounds are those 
where m1, m2 and m3 are from 0 to 100, n is from 2 to 10, R.sup.1, R.sup.2 
and R.sup.3 are acyl groups having 4 to 24 carbon atoms, and x is alkylene 
of 2-4 carbons. 
These compounds (1) can be produced by any method known in the art. An 
example of the production process of polyglycerol includes a process 
wherein glycerol is subjected to dehydrocondensation at an elevated 
temperature of 200.degree.-300.degree. C. in the presence of an alkali 
catalyst. Examples of the alkali catalyst used herein include NaOH, KOH, 
LiOH, Na.sub.2 CO.sub.3, K.sub.2 CO.sub.3, Li.sub.2 CO.sub.3, CaO and MgO. 
The degree of polymerization of polyglycerol may be regulated by changing 
the reaction conditions. However, the resulting polyglycerol is not a 
single component, but a mixture having a certain molecular weight 
distribution. For example, the hydroxyl number of the commercialized 
polyglycerol referred to as hexaglycerol conforms with the calculated 
chemical formula thereof, but the polymer is in reality a mixture composed 
of polyglycerols of different polymerization degrees. 
The polyglycerol obtained by the above-described method is a yellow or 
blackish brown liquid having a high viscosity. As its polymerization 
degree becomes higher, its hue becomes poorer, approaching blackish brown. 
Therefore, before use the colored polyglycerol is subjected to a 
decoloring treatment with an adsorbent like activated carbon or activated 
clay, or to removal of catalyst and decoloring treatment with an 
ion-exchange resin. Diglycerol, tetraglycerol, hexaglycerol and 
decaglycerol have been commercialized. 
The present adduct of polyglycerol with a polyoxyalkylene may be produced 
by any known method including a well-known process wherein after adding an 
alkali catalyst, an alkylene oxide is added to, for example, the 
polyglycerol obtained in the above-described manner under pressure and 
heat. Examples of the alkylene oxide to be added include ethylene oxide, 
propylene oxide and butylene oxide, which have 2, 3 and 4 carbon atoms, 
respectively. These alkylene oxides may be added either singly, or in 
combination, as blocks or at random. They are added in an amount of 
preferably 1-200 moles, more preferably 1-50 moles on the average per mole 
basis of polyglycerol. 
The mono-, di- or triacylated product of polyglycerol, and mono-, di- or 
triacylated product of an adduct of polyglycerol with a polyoxyalkylene 
(both products may be called polyglycerol fatty acid esters) can be 
generally produced by any direct esterification reaction. Various kinds of 
hydrophilic or lipophilic esters may be obtained by suitably combining 
polyglycerols of different polymerization degrees with each other and 
selecting the kind of a fatty acid to be used and the degree of 
esterification. Therefore, any ester having an HLB as measured by a Davis 
method of from 1 to 20, more preferably from 2 to 10 in the case of 
antifoaming agents, and from 10 to 18 in the case of improving the 
productivity of L-amino acids may be prepared and used. 
The esterification reaction is generally conducted at a temperature not 
lower than 200.degree. C. without using any catalyst or may be conducted 
in the presence of an alkali catalyst. Sulfite may be added during the 
reaction as may lipase, etc. Various products are provided by varying the 
degree of purification according to their end applications intended. The 
quality of the polyglycerol fatty acid esters produced depends in large 
part upon the quality of the polyglycerol starting material. This tendency 
becomes more pronounced as the polymerization degree of the polyglycerol 
increases. 
A preferred material is a polyglycerol condensed-ricinoleic acid ester 
synthesized by dehydrating ricinoleic acid (castor oil fatty acid) under 
heat to precondense it for 3-6 minutes and esterifying polyglycerol with 
the thus-precondensed ricinoleic acid. The reaction conditions are 
generally the same as those described above for any polyglycerol fatty 
acid ester. 
Reaction product (a) or compound (1) or their mixture may be used directly 
as an antifoaming agent for fermentation media and fermentation processes 
or for any other applications where defoaming is desired. However, they 
may also be mixed with known antifoaming agents before or after use. 
Reaction product (a) or compound (1) or their mixture may be added to a 
foaming medium in one to several portions before or after foaming begins. 
For fermentation media the agents can be added at the beginning of 
incubation or during incubation. The amount to be added is preferably 
0.0001-5 wt. %, more preferably 0.001-2.5 wt. % based on the medium. It is 
preferable to add the antifoaming agent according to the present invention 
in an amount of 0.0001-2 wt. %, more preferably 0,001-1.0 wt. % where it 
is used to exert only an antifoaming effect, or in an amount of 0,001-5 
wt. %, more preferably 0.01-5 wt. %, most preferably 0.05-2.5 wt. % where 
it is expected to improve the fermentative productivity of an L-amino acid 
in addition to exerting an antifoaming effect. 
No particular limitation is imposed on fermentation culturing means to 
which the antifoaming agent according to the present invention is applied. 
Examples include aerobic culture, stirring culture, shaking culture and 
the like, all of which product a great number of bubbles. The fermentative 
production of an L-amino acid using the above-described components 
according to the present invention will hereinafter be described. 
Upon production of an L-amino acid, at least one of the reaction products 
(a) or compound (1) or their mixture may be added either to a medium for 
seed culture or to a medium for principal fermentation. As a medium to 
which the at least one reaction product (a) and/or the compound (1) is to 
be added, media generally used in the incubation of L-amino acid-producing 
bacteria containing a carbon source, a nitrogen source, salts and other 
additives can be used. In the present invention, examples of the carbon 
source include carbohydrates such as glucose, dextrose, sucrose, fructose, 
maltose, crude sugar, fruit sugar, glucose, liquid sugar, cane molasses, 
beet sugar, blackstrap molasses, tapioca and starch-saccharified liquor; 
fatty acids such as acetic acid and propionic acid; organic acids such as 
pyruvic acid, citric acid, succinic acid and malic acid; and alcohols such 
as ethyl alcohol and butyl alcohol, all of which may be used either singly 
or in any combination thereof. As the nitrogen source, examples include 
ammonium salts such as ammonium sulfate, ammonium chloride and ammonium 
acetate, urea, aqueous ammonia, corn steep liquor, yeast extract, soybean 
hydrolyzate, peptone, polypeptone, meat extract, and the like. As the 
salts, phosphates, magnesium salts, calcium salts, potassium salts, sodium 
salts, iron salts, manganese salts, zinc salts, copper salts and the like 
may be used. Other metal salts may be further added as needed. 
As described above, a surfactant other than (a) or (b) may be added to the 
fermentative medium to enhance the yield of the L-amino acid. Examples of 
such surfactants include anionic surfactants such as higher (C.sub.6 
-C.sub.25) alcohols, sulfates, alkylbenzenesulfonates, alkyl phosphates 
and dialkyl sulfosuccinates; cationic surfactants such as alkylamines and 
quaternary ammonium salts; nonionic surfactants such as polyoxyethylene 
alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sorbitan fatty 
acid esters, polyoxyethylene fatty acid esters, polyglycerol fatty acid 
esters, alkylglycosides and ester amides; and ampholytic surfactants such 
as imidazoline and betaine. Among these, alkylglycosides and ester amides 
are preferred. These surfactants may be used either singly or in any 
combination thereof, and are preferably added in an amount within the 
range of 0.01-2.5% by weight based on the weight of the medium. 
Further, antibiotics, vitamins and the like may be added to the 
fermentative media as needed. Examples of the antibiotics include 
penicillin, chloramphenicol, erythromycin, streptomycin, kanamycin, 
oleandomycin, kasugamycin, tetracycline, mitomycin, actinomycin and 
cycloserine. Among these, penicillin is preferred. Examples of the 
vitamins include biotin, niacin and thiamin. 
No specific limitation is imposed on the microorganisms added to the 
fermentative medium according to the present invention. Any one or 
combination of microorganisms may be used so long as they produce an 
L-amino acid. Specific examples thereof include the following 
microorganisms: 
Corynebacterium: 
Corynebacterium glutamicum, Corynebacterium acetoglutamicum, 
Corynebacterium acetoacidophilum; 
Microbacterium: 
Microbacterium amnoneaphilum; 
Brevibacterium: 
Brevibacterium acetoacidophilum, Brevibacterium flavum, Brevibacterium 
lactofermentum, Brevibacterium saccharolyticum, Brevibacterium roseum, 
Brevibacterium divaricatum; 
Arthobacter: 
Arthobacter citreus; 
Bacillus: 
Bacillus subtilis, Bacillus sphaericus. 
Examples of L-amino acids obtained by incubating the above-mentioned 
microorganisms in the invention fermentation bath include L-glutomic acid, 
L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine, 
L-isoleucine, L-histidine, Lproline, L-valine, L-tyrosine, L-tryptophan, 
L-leucine, L-serine, L-ornithine and L-citrulline. 
The conditions under which the medium according to the present invention is 
used to incubate L-amino acid-producing bacteria are the same as those 
used in general amino acid fermentation. Although the incubation 
temperature somewhat varies according to the L-amino acid intended and the 
strain to be used, it may be 20.degree.-40.degree. C. with 
28.degree.-37.degree. C. being particularly preferred. Better results are 
obtained when pH is controlled near neutrality during the incubation. The 
incubation is generally conducted under aerobic conditions such as 
aeration, stirring or shaking culture. The incubation period is generally 
1-7 days. However, incubation may be extended further by continuous 
culture or the like. The isolation of L-amino acids from the respective 
fermented solutions containing the L-amino acids is conducted by ion 
exchange treatment or any other method known in the art.

EXAMPLES 
The present invention will hereinafter be described in more detail by the 
following examples. However, it should be borne in mind that this 
invention is not limited to or by these examples. 
Example 1 
100 ml of media (sterilized at 121.degree. C. for 10 minutes, pH: 7.2) 
having the composition shown in Table 1 were inoculated with 
Brevibacterium flavum and separately placed in fermenters (500-ml 
graduated cylinder) and aerated at a rate of 5 l/min. When bubbles reached 
a marked line at 400 ml, reaction product (a) antifoaming agents shown in 
Table 2 were separately added little by little to the media which were 
then incubated at 30.degree. C. for 2 hours. The amounts of the 
antifoaming agents required to suppress the bubble level at the marked 
line or lower are shown in Table 2 where the antifoaming agent ratios are 
by weight. 
TABLE 1 
______________________________________ 
Glucose 10% FeSO.sub.4.7H.sub.2 O 
10 .mu.g/l 
Meat extract 
0.5% MnSO.sub.4.nH.sub.2 O 
10 ng/l 
Ammonium sulfate 
3% CuSO.sub.4.5H.sub.2 O 
1 mg/l 
K.sub.2 HPO.sub.4 
0.05% Urea 0.5% 
KH.sub.2 PO.sub.4 
0.15% CaCO.sub.3 3% 
MgSO.sub.4.7H.sub.2 O 
0.05% Thiamin hydrochloride 
0.5 mg/l 
______________________________________ 
TABLE 2 
______________________________________ 
Amount of 
anti-foaming 
agent used 
Antifoaming agent (g) 
______________________________________ 
Inventive 
examples: 
1 Beef tallow/glycerol/EO(5)/ 
0.010 
PO(15) = 1/0.3/5/15 
2 Coconut oil/glycerol/EO(10)/ 
0.013 
PO(30) = 1/0.6/10/30 
3 Soybean oil/glycerol/EO(15)/ 
0.016 
PO(25) = 1/1/15/25 
4 Beef tallow/pentaerythritol/ 
0.019 
EO(10)/PO(45) = 1/0.5/10/45 
Comparative 
examples: 
5 Polypropylene glycol 1.250 
6 EO/PO/EO ("Pluronic", product 
1.350 
of Asahi Denka Kogyo K.K. 
7 Oleyl alcohol/EO/PO = 1/10/15 
At least 2.5 
8 Stearic acid/PO = 1/15 
At least 2.5 
______________________________________ 
EO: Ethylene oxide; PO: Propylene oxide 
As apparent from Table 2, the antifoaming agents according to the present 
invention exhibit an excellent antifoaming effect in extremely small 
amounts. 
Example 2 
A medium containing 10% (in terms of sugar) of blackstrap molasses, 0.5% of 
urea and 0.3% of corn steep liquor was inoculated with Corynebacterium 
glutamicum and incubated at 30.degree. C. In the prophase of the 
logarithmic growth phase, 0.15% of polyoxyethylene monopalmitate was added 
to the medium and incubation conducted at 30.degree. C. for 30 hours. 
Thereafter, 15-ml portions of the resulting cultured mixtures were 
separately placed in 500-ml graduated cylinders. Air was introduced into 
the cylinders at a rate of 5 l/min. When bubbles reached a marked line at 
400 ml, the antifoaming agents shown in Table 2 were added in amounts of 
0.001 g to the respective medium portions. After aerating for 30 more 
minutes, the height (ml) of bubbles occurred in each graduated cylinder 
was measured. The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Antifoaming agent No. 
Height of bubbles (ml) 
______________________________________ 
1 150 
2 170 
3 160 
4 200 
5 At least 500 
6 480 
7 At least 500 
8 At least 500 
______________________________________ 
In the case of Antifoaming Agents Nos. 5, 7 and 8, bubbles overflowed the 
respective graduated cylinders. 
Example 3 
An L-lysine-producing medium was prepared as follows: 
______________________________________ 
Glucose 10% 
(NH.sub.4).sub.2 SO.sub.4 
4.5% 
Thiamine hydrochloride 200 .mu.g/l 
K.sub.2 HPO.sub.4 0.1% 
Peptone 1% 
Biotin 50 Mg/l 
______________________________________ 
where % is percent by weight. 100 ml-portions of a medium having the above 
composition were placed in 500-ml Sakaguchi flasks, sterilized at 
120.degree. C. for 15 minutes and then inoculated with the 
L-lysine-producing bacteria Brevibacterium SP. Thereafter, the antifoaming 
agents shown in Table 1 were separately added to the medium portions in 
amounts of 0.05% and 1.0% by weight based on each medium portion and 
incubation was further conducted at 30.degree. C. for 30 hours. The 
amounts of L-lysine produced in the respective medium portions were then 
determined. The results are shown in Table 4. 
TABLE 4 
______________________________________ 
Antifoaming 
Amount Amount of L-lysine produced 
agent No. added (%) (g/l) 
______________________________________ 
1 0.05 7.0 
1.0 6.8 
2 0.05 7.2 
1.0 7.1 
3 0.05 6.9 
1.0 7.0 
4 0.05 7.5 
1.0 7.2 
5 0.05 4.5 
1.0 2.1 
6 0.05 5.2 
1.0 3.0 
7 0.05 4.8 
1.0 3.1 
8 0.05 4.0 
1.0 1.9 
______________________________________ 
As apparent from the results in Table 4, the antifoaming agents according 
to the present invention (Nos. 1-4) are excellent antifoaming agents each 
improving the production of L-lysine, a fermentation product. 
Example 4 
A medium composed of the following composition is prepared by weight. 
______________________________________ 
Blackstrap molasses 
4% 
KH.sub.2 PO.sub.4 
0.2% 
MgSO.sub.4 0.05% 
Urea 0.8% 
Biotin 5 .mu.g/l 
Water Balance 
______________________________________ 
and adjusted to pH 7.2 with KOH, and a 30-ml portion thereof was placed in 
a 500-ml Sakaguchi flask and sterilized under heat. This medium portion 
was inoculated with Corynebacterium glutamicum grown on a glucose-peptone 
slant to conduct preliminary incubation at 30.degree. C. for 18 hours. 
Separately, 30-ml portions of a medium (pH: 7-8) having the following 
composition: 
______________________________________ 
Blackstrap molasses 
10% 
Urea 1.0% 
KH.sub.2 PO.sub.4 
0.1% 
MgSO.sub.4 0.05% 
Water Balance 
______________________________________ 
were placed in 500-ml Sakaguchi flasks and sterilized under heat. Medium 
samples were then prepared by separately adding to each of these 30-ml 
portions 0.3% by weight of reaction products (a) shown in Table 5, or 0.3% 
by weight of polyoxyethylene sorbitan monostearate and no additive in 
comparative examples. 500 .mu.l portions of the preliminarily cultured 
mixture described above were added to these medium samples having the 
materials in Table 5 added thereto to conduct shaking culture at 
30.degree. C. for 48 hours. The amounts of L-glutamic acid produced in the 
respective media were then determined and the results are shown in Table 
6. 
TABLE 5 
______________________________________ 
Molar Alkylene oxide 
Fat or Alcohol ratio Mole/ 
oil (A) (B) (A/B) Compound 
A + B 
______________________________________ 
Inventive 
9 Coconut oil 
Glycerol 1/1 EO 20 
10 Beef tallow 
Glycerol 1/0.5 EO 50 
11 Palm oil Glycerol 1/0.5 EO/PO 20/5 
(Block) 
12 Palm Pentaery- 
1/2 EO/BO 30/10 
kernel oil 
thritol (Block) 
13 Fish oil Glycerol 1/0.5 EO/PO 40/5 
(Random) 
14 Beef tallow 
Pentaery- 
1/1 EO 20/5 
thritol 
Comp. 
15 Polyoxyethylene sorbitan 
EO 20 
monostearate 
16 Not added -- -- 
______________________________________ 
EO: Ethylene oxide; PO: Propylene oxide; BO: Butylene oxide. 
TABLE 6 
______________________________________ 
Amount of L-glutamic acid (g/l) 
______________________________________ 
9 45.0 
10 38.9 
11 37.9 
12 41.3 
13 37.6 
14 39.5 
15 17.6 
16 1.3 
______________________________________ 
Example 5 
The following media A and B were prepared by weight: 
______________________________________ 
Medium A: 
Blackstrap molasses 
10% (in terms of glucose) 
(NH.sub.4).sub.2 SO.sub.4 
4.5% 
KH.sub.2 PO.sub.4 
0.1% 
Peptone 1% 
Water Balance 
Medium B: 
Glucose 10% 
Biotin 50 .mu.g/l 
Thiamine hydrochloride 
200 .mu.g/l 
(NH.sub.4).sub.2 SO.sub.4 
4.5% 
KH.sub.2 PO.sub.4 
0.1% 
Peptone 1% 
Water Balance 
______________________________________ 
40-ml portions of each of the thus-prepared media were separately poured 
into two 500-ml Sakaguchi flasks and sterilized. The thus-sterilized 
portions were inoculated with L-lysine-producing bacteria, Brevibacterium 
SP and incubation was conducted at 30.degree. C. for 18 hours. 400 
.mu.portions of the resulting cultured mixtures were then subcultured into 
the corresponding media A and B to conduct shaking culture at 30.degree. 
C. for 8 hours. Thereafter, 0.15% by weight of reaction products (a) shown 
in Table 7 were separately added to the subcultured media A and B and 
shaking culture was continued for 24 hours. For the sake of comparison, 
portions of media A and B which contained 0.15% by weight of 
polyoxyethylene sorbitan monopalmitate and no additive therein, were 
incubated in the same manner as described above. The amounts of L-lysine 
in the cultured mixtures were then determined and the results are shown in 
Table 8. 
TABLE 7 
______________________________________ 
Molar Alkylene oxide 
Fat or Alcohol ratio Mole/ 
oil (A) (B) (A/B) Compound 
A + B 
______________________________________ 
Inventive 
17 Beef tallow 
Glycerol 1/0.5 EO 20 
18 Palm oil Glycerol 1/2 EO/PO 25/10 
(Block) 
19 Fish oil Pentaery- 
1/1 EO/PO 15/15 
thritol (Block) 
20 Coconut oil 
Tri- 1/0.5 EO 20 
methylol- 
propane 
21 Beef tallow 
Di- 1/1.0 EO/BO 30/15 
glycerol (Block) 
Comp. 
22 Polyoxyethylene sorbitan 
EO 20 
monopalmitate 
23 Not added -- -- 
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TABLE 8 
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Amount of L-lysine (g/l) 
Medium A 
Medium B 
______________________________________ 
17 7.2 6.5 
18 6.9 6.2 
19 6.5 5.9 
20 7.8 7.0 
21 6.7 6.0 
22 3.5 3.2 
23 1.6 1.4 
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Example 6 
A medium composed of the following composition by weight: 
______________________________________ 
Blackstrap molasses 
4% 
KH.sub.2 PO.sub.4 
0.1% 
MgSO.sub.4 0.05% 
Urea 0.8% 
Biotin 5 .mu.g/l 
Water Balance 
______________________________________ 
was adjusted to pH 7.2 with KOH, and a 30-ml portion thereof was placed in 
a 500-ml Sakaguchi flask and sterilized under heat. This portion was 
inoculated with Corynebacterium glutamicum grown on a glucose-peptone 
slant to conduct incubation at 30.degree. C. for 18 hours. 
Separately, 30-ml portions of a medium (pH: 7-8) composed of the following 
composition: 
______________________________________ 
Blackstrap molasses 
10% (in terms of sugar) 
Urea 1.0% 
K.sub.2 HPO.sub.4 
0.1% 
MgSO.sub.4 0.05% 
Water Balance 
______________________________________ 
were placed in four 500-ml Sakaguchi flasks and sterilized under heat to 
prepare four media A, B, C and D. Then, 300 .mu.l portions of the cultured 
mixture obtained above were separately subcultured into media A-D. 
Further, 0.2% of polyglycerol monolaurate and 0.2% of polyoxyethylene 
sorbitan monostearate were added to the medium A, 0.3% of polyglycerol 
monolaurate to the medium B, and 0.3% of polyoxyethylene sorbitan 
monostearate to the medium C. Medium D had no additive. 
The media A-D were separately subjected to shaking culture at 30.degree. C. 
for 48 hours to determine the amounts of L-glutamine in the respective 
media. The results are shown in Table 9. 
TABLE 9 
______________________________________ 
Medium Amount of L-glutamine (g/l) 
______________________________________ 
Inventive: 
A 39.5 
B 34.0 
Comparative: 
C 18.0 
D 1.2 
______________________________________ 
Example 7 
The following media A and B were prepared by weight: 
______________________________________ 
Medium A: 
Blackstrap molasses 
10% (in terms of glucose) 
(NH.sub.4).sub.2 SO.sub.4 
4.5% 
KH.sub.2 PO.sub.4 
0.1% 
Peptone 1% 
Water Balance 
Medium B: 
Glucose 10% 
Biotin 50 .mu.g/l 
Thiamine hydrochloride 
200 mg/l 
(NH.sub.4).sub.2 SO.sub.4 
4.5% 
KH.sub.2 PO.sub.4 
0.1% 
Peptone 1% 
Water Balance 
______________________________________ 
40-ml portions of the thus-prepared media were separately poured into two 
500-ml Sakaguchi flasks and sterilized. The thus-sterilized medium 
portions were inoculated with L-lysine-producing bacteria, Brevibacterium 
SP to conduct incubation at 30.degree. C. for 18 hours. 400 .mu.l of the 
resulting cultured mixtures were subcultured into their corresponding 
media A and B to conduct shaking culture at 30.degree. C. for 8 hours. 
Thereafter, 0.15% of polyglycerol stearate was added to subcultured media 
A and B to continue the shaking culture further for 24 hours. For the sake 
of comparison, portions of media A and B, which contained no polyglycerol 
stearate therein, were incubated in the same manner as described above. 
The amounts of L-lysine produced in the respective cultured mixtures thus 
obtained are shown in Table 10. 
TABLE 10 
______________________________________ 
Agent for improving 
Amount of L-lysine 
Medium the yield of amino acid 
(g/l) 
______________________________________ 
A Added 7.2 
Not added 1.8 
B Added 6.0 
Not added 1.9 
______________________________________