Turbidimetric Microbiological Assay of Amino Acids
Turbidimetric Microbiological Assay of Amino Acids
Turbidimetric Microbiological Assay of Amino Acids
To cite this article: Tomoyuki Ishikura, Tadashi Sakamoto, Ichiya Kawasaki, Toshinao Tsunoda &
Kikuko Narui (1964) Turbidimetric Microbiological Assay of Amino Acids, Agricultural and Biological
Chemistry, 28:10, 700-709, DOI: 10.1080/00021369.1964.10858295
To shorten the time necessary for the determination of L-glutamic acid a turbidimetric micro-
biological assay method with Lactobacillus arabinosus 17-5 has been studied. It was found that the
assay can be completed within 30 hours including the incubation period for the inoculum, the assay
and the time for medium preparation. Turbidimetric assay methods of other amino acids such as
DL-alanine with Leuconostoc citrovorum 8081, L-aspartic acid and L-lysine with Leuc. mesenteroides P-60
have also been studied.
TABLE 1. MEDIUM FOR MAINTANING L. arabinosus TABLE III, BASAL MEDIUM OF L. arabinosus 17-5
17-5 CULTURES
FOR TURBIDIMETRIC METHOD OF L-GLUTAMIC
Baker's yeast extrate* IOml ACID ASSAY (SINGLE STRENGTH II)
Glucose I g Glycine 0.1 g
Na-acetate I g DL-Alanine O. 2 g
Polypeptone I g L-Valine 0.1 g
Agar (powder) 2g L-Leucine 0.1 g
pH 6.0 L-Isoleucine O. I g
* Barker's yeast was heated with 1O·fold volume water for 1 hour DL-Serine O. I g
on boiling wate-r bath and boiled for 5 min on a burner. Solid
L-Threonine O. I g
matter was removed by filtration. Filtrate was employed.
L-Cystine 0, I g
TABLE II. MEDIUM FOR INOCULUM CULTURE
L-Methionine O. I g
OF L. arabinosus 17-5
DL-Asparagine 0.4 g
Baker's yeast extrate lOami L-Proline 0.1 g
Glucose Ig L-Phenylalanine 0.1 g
Na-acetate Ig L-Tyrosine O. I g
Polypeptone I g L-Tryptophan O. I g
Salt A* 2.5ml L-Lysine·HCl 0.2 g
Salt B+ 0.5ml L-Arginine·HCI 0.2 g
Tween 80 0.1 g L-Histidine·HCI 0.1 g
pH 6.0 Adenine·H 2SO, 0.01 g
*- KH 2 PO.. 1 g., K 2 HPO. 1 g.) per 100 m!' Guanine·HCI 0.01 g
MgSO•• 7H,O 2 g., FeSO.,7H,O 0.1 g., MnSO.·4H,O 0.1 g.•
X.Cl 0.1 g., per 100 mi. Uracil 0.01 g
Xanthine 0.01 g
measured by Hitachi Spectrophotometer Type EPB-II
Thiamine· HCI I. mg
with 10 mrn cell. The value obtained by the use of
Riboflavin 2. mg
distilled water was taken as a control. Standard con-
p-Aminobenzoic acid I . mg
centration response curves were prepared each time and
Pyridoxine·HCI I. mg
the amino acid content in samples was calculated by
Pyridoxal· HCl O. 02 mg
interpolation.
Ca-Pantothenate I. mg
The procedures used for turbidimetric assay of other
Niacin I. mg
amino acids were designed on the basis described above.
Biotin 0.01 mg
EXPERIMENTAL RESULTS AND DISCUSSION Folic acid 0.01 mg
1. Assay of L-Glutamic Acid with L. arabinosus KH 2 PO, 0.5 g
17-5. K,HPO, 0.5 g
(A) Assay range MgSO,·7H 2 0 0.2 g
FeSO,· 7H 20 0.01 g
Though the assay range by the titrimetric
MnSO,,4H,O 0.01 g
method with L. arabinosus 17-5 was 1 to 10 micro-
NaCI 0.01 g
grams of L-glutamic acid per tube (2 ml), that
Glucose (anhydrous) 20. g
of the turbidimetric method was more than 10 Na-Acetate (anhydrous) 20. g
micrograms per tube. Turbidimetric response NH,CI 3. g
recorded with the degree of optical density (0. D.) Tween (10 I. g
after 18 hours incubation period is shown in pH 6.0
Fig. 1. These results indicate that the O. D.
values between 10 to 100 microgram per tube which plot of data was more meaningful, a
(2 ml) are readable but values above an O. D. variety of plots with arithmetic dose-arithmetic
of 0.5 are not. response, log dose-arithmetic response and log
To determine which reading was more practical, dose-log response was examined. It was found
optical density or transmission per cent, and that an arithmetic plot of optical density versus.
702 Tomoyuki ISHIKURA, Tadashi SAKAMOTo, Ichiya KAWASAKI, Toshinao TSUNODA and Kikuko NARUI
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B Cell concentration, dry matter mg/ml
. 0 L4c":o-'-O--;5oc!O;O==:::;60S0=:~7:'::OO::==;82;o""o ==9""O"';O~-:1-:O~OO FIG. 3. Relation between Turbidity and Cell Con-
Wavelength (mp) centration.
FIG. 2. Wavelength and Optical Density of L. ara-
binosus 17-5 Culture Solution. (C) Relation between turbidity and cell concentration
A: Turbid solution after incubation with L~glutamic acid lOOllg/
tube. B: Almost clear solution after incubation without L-gluta-
In general, the strength of light transmitted
mie acid. through a turbid solution does not always obey
Beer's Jaw. With the instrument employed, if
·dose was more linear and the most satisfactory. the optical density is less than about 0.5, the
(B) Wavelength used for turbidity measurement relation between the bacterial cell concentration
Changes in optical density measured at wave- and the transmitted light follows this law.
lengths between 400 and 1000 mp. are shown in Relation between optical density or per cent
Fig. 2. Absorbance by the media was too high transmission at 625 mp. of the turbid cell suspen-
at shorter wavelengths while on the other hand sion and bacterial cell concentration is shown in
at longer wavelengths, absorbance was too low Fig. 3.
to measure accurately. (D) Change in turbidity during incubation periods
The wavelengths from 600 to 700 mp. gave A previous report 6l suggested that the turbidi-
more accurate values and red light of 625 mp. metric bioassay was less reliabile than the acido-
was selected as the best. The gaps in absorption metric because turbidimetry is a measure of the
curves at 600 m/~ in Fig. 2 were caused by the speed of growth of the test organism. On the
use of different phototubes. Probably the scat- other hand a test in which the change in turbidi-
tering of light by cell suspensions is the cause metric response during incubation periods was
of this difficulty since when the instrument is measured was carried out to make certain of
used as colorimeter, no break of curve takes place. this point (Fig. 4). In the earlier incubation
periods, the speed of growth seemed to depend
Turbidimetric Microbiological Assay of Amino Acids 703:
.2
I was preferable to that of a low inoculum ratio,
(1 : 100) even in the titrimetric method. S )The
effect of pH on the utilization of glutamic acid
by this organism was reported by Sondheimer
et al. 7l and the effect of oleic acid on the cell
permiability of the same organism was described
by Traub. S ) Our preliminary work suggested
,OO':---;:2':c----:ej';:0---I+iC:-)--c:!c:-- - 7 "jl:":-olOc------1 that a younger inoculum such as incubated for
O SO
L-Glutamic acid ltg/tube
seven hours was more effective in preventing
the trouble than an inoculum incubated for the
FIG, 4. Change in Turbidity During Incubation
Period. usual eighteen hours. Growth at an initial pH
6.0 seemed to be better than at pH 6.8 and the
upon the size of inoculum and the response curve addition of Tween 80 to 0.1 % final concentra-
was not linear. Nevertheless at the stage of tion in the media to suspend the bacterial cell
maximum growth (sixteen hours) the response stimulates the growth.
curve became a fine straight line. For at this In regard to the four above-mentioned factors
stage, the substrate concentration must have been and incubation temperature, an experiment de-
a limiting factor for the growth of the organism signed 9 ) with partial fractional design in 2'
and the responses seemed to be independent of factorial in 16 units was carried out. The
the size of inoculum. At later incubation stages factors and experimented levels are shown in
bacterial cells autolysed themselves and a small Table IV. The plan and the results of response
decrease in the optical density was observed. curves in each unit are demonstrated in Fig. 5.
These results indicate that a maximum growth The values in vertical scale were sums of dupli-
response instead of speed of growth plays and cated optical density values.
important part in the measurement by this Since all three or higher order interactions
turbidimetric method and the previous suggestion were assumed negligible, all main effects and
should be altered. In practice, turbidity response two-factors' interaction could be detected by the
should be measured after a 16 to 18 hour in- use of this design. An experiment with 32 units-
cubation period. was considered to be too large to obtain reliable
(E) Size and incubation period of inoculum, addition results. To easily find each main effect, the
of Tween 80 to medium and pH and incubation sums of optical densities against each dose
temperature concentration in eight units of low levels and
As times the response curve of L. arabinosus sums of these in high levels regarding each
17-5 to L-glutamic acid was abnormal and a
7) E. Sondheimer and D.C. 'Nilson, Arch. Biochem. Biophys., 61.-
phenomenon called "lag in response n at lower 313, (1956).
8) A, Traub, ibid., 62. 222 (1956).
dose concentrations was observed. To prevent 9) rv1. t-.-Jasuyama, "Jikken Keikaku Ho (Experimental Designs), ",
this trouble, use of a large inoculum ratio (1 : 5) Iwanami Shoten, 1956.
704 Tomoyuki ISHIKURA, Tadashi SAKAMOTO, Iehiya KAWASAKI, Toshinao TSUNODA and Kikuko NARUI
Inoculum size 1 :5
1:100
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L-Glutamic acid J.!g/tube
50 1000 50 100
FIG. 5. The Plan Designed with Factorial Replication in 2 5 Factorial in 16 Units and Each
Response Curve
factor were plotted against doses, which is shown of optical densities of response for each concentra-
in Fig. 6. Care had to be taken that only the tion of dose in four units of low levels and sums
height difference between two curves could be of those in four units of high levels in regard
the subject of this discussion whereas the height to each factor were plotted against doses. Results
of curves in itself could not. It was found that are shown in Fig. 7. In a group of 1: 100
advantageous conditions were seven hours in- inoculum, the advantage of seven hours incuba-
cubation period of inoculum, 1: 5 ratio of tion, pH 6.0, addition of Tween 80 and 37°C
inoculum, pH 6.0 of basal medium, addition of incubation against different level of each factor
Tween 80 to medium and 37°C incubation were apparent. In the group of 1: 5 inoculum,
temperature, against another level of each factor. however, there were no significant differences
Furthermore, in order to obtain more detailed in response between high and low levels of each
information about these effects, results of an factor except that an incubation temperature
experiment of 16 units were divided into two of 30°C was better than 37°C. The analysis of
groups with respect to two conditions (l: 5 and variance from the data of these experiments
1: 100) of inoculum ratio. In each group, sums proved these main effects and their interactions
Turbidimetric Microbiological Assay of Amino Acids 705
0
pH
Tween SO Temperature
S
6.0 0.1%
2
6.S None
30'C
E:;' 0
bIJ
..9
I
~~ Inoe. inc. Tween 80 Temp.
'" period 7 hrs
0.1%
.~ 3
None
,6 IS hrs
:; 2
g
..s
with the factor of inoculum ratio to be significant. From these results, it can be concluded that
The effect of temperature on nutritional require- routine assays should be carried out under
ment of L. arabinosus was already described by conditions as follows; seven hour incubated
Borek et al. IO ) inoculum, pH 6.0 of media and 0.1 % addition
10) E. Borek and H. Waelsch, J. BioI. Chern., 190, 191 (1951). of Tween 80, since these conditions were ap-
706 Tomoyuki ISHlKURA, Tadashi SAKAMOTO, Ichiya KAWASAKI Toshinao TSUNODA and Kikuko NARUI
.5 .5
L -Glu.
.4
.4
h .3
.3 bD
h ..s
I
~
I .2 IJ-Glu .
.2
.0 OL.---c:2:':oO--4-;';O:---~60;;----c:S'c-O---o1.".Or"...)
c-1,-_G"'Ic-u-.fJ-g-,J/tube-
o 40 St) 120 160 21ln D-Glu. fJg/tube-
. 00;,----"I20"..---4""0--...."6'::-0---:S,,,,0,,----,1..,07"0----...J FIG. 9. Activity of D-Glutamic Acid .
L-Glutamic acid Ilg/tube
I
cause color formation and the color influences
.2
turbidity measurements. The results of heating
carried out under a pressure of 20lbs/inch 2 for
15 minutes showed that the reading of optical .1 ~-PCA
time is most desirable. It was found that a pres- 0 .5 1.0 1.5 2.0 2.'5 L-PCA rng/tube-
sure of 10 Ibs/inch 2 for ten minutes was sufficient. FIG. 10. Activity of L-Pyrrolidone Carboxylic Acid.
"
~
~
.5 .5
.4 .4
E-o
E-o
~ .3 .3
"I .9"
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FIG. 14. Standard Response Curve for DL-Alanine FIG. 16. Standard Response Curve for L-Lysine
with Leuc. citrol'orum 8081. Hydrochloride with Leuc. mesenteroides P-60.
Turbidimetric Microbiological Assay of Amino Acids 709
Acknowledgements. The authors are indebted vice-Dicector of the Central Research Laboratory
to Mr. M. Suzuki, President of Sanraku-Ocean of Sanraku Co. for their constant guidance
Co., Ltd. and Mr. T. Domen, President of through the course of this work and Miss S. Imai,
Ajinomoto Co., Inc. for their encouragement Miss T. Kanno and Miss T. Furuya for their
and to Dr. Y. Takeda, Director and Dr. A. Ozaki, assistance in carrying out the experiments.