sulfate was found more active in neutral and alkaline pH regions. The ... continuously maintained in our laboratory. Protamine. Clupeine sulfate. (from herring.
Bulletin of the Japanese
Effects
Society of Scientific Fisheries
51(5), 811-815 (1985)
of pH, Temperature, Metal Ions and Organic Matters on the Bactericidal Action of Clupeine Sulfate*1
Nazrul
MD.
ISLAM,*2 Terushige
MOTOHlRO,*3
and Takao
ITAKURA*3
(Accepted October 6, 1984)
Various were studied.
factors influencing Clupeine sulfate
the bactericidal action was found more active
ionic strength of the buffer greatly influenced inhibited the bactericidal action of clupeine bactericidal
action.
Although
the
bactericidal action of clupeine, that of molarity of the salts. reduced the bactericidal ions. Organic substances protamine
Few
studies
under
are
the bactericidal action, 0.5M phosphate sulfate. Temperature had only a little
increased
salt concentration
the ionic strength The ionic strengths
under
on the
different
bactericidal
nutritional
included
and
were
and others
are the works
regarding
Protamine
which
influence
antibacterial
the
some
of
cells were reversed by nucleic acids and ribonucleic acids. A recent report also described the factors
either
influencing the bacteriolytic on B. subtilis cells.7)
Corp.
action
and
of protamine
sterilized
Although a number of papers dealt with the study of factors influencing the antibacterial action
after
of protamine, they were neither specific sistent and were rather contradictory
nor con in some
Media
cases.
effects
reports
the
against
organisms
Bacillus
were included
species,15)
only
protamine
the
buffer
study
of
conditions.
Antibacterial Department
*3
Laboratory of Food Shilmoarata,
filter
it
units
buffer (Millipore
otherwise
were
used
as
basal
used
action broth
meat
Penassy
(Difco);
extract
Broth
Nutrient (Difco)
Initial
*2
Ltd.
dissolving
in
the
immediately
were of
and
of
and
0.2%
MgSO4•E
Medium
NO.
Tryptose
routinely
the
for
different
consisting
Antibiotic
agar
medium
under
used
0.5
Blood for
3
Agar
growth
and
strains.
and Methods
Strains Bacillus subtilis ruber IFO 3026 was the principal subject matter of this study. The other organisms *1
was
Nutrient
preservation
Materials
obtained
phosphate
stated
bactericidal
0.3%
7H2O;
Base
in this study.
by
0.01M
solutions
was
preparation.
% peptone,
these
not
Osaka laboratory.
roe)
prepared or
B. were
Industries
Millex-GS If
Phosphate
of
pH, temperature, metal ions and organic matters on bactericidal action of clupeine sulfate. In view of the marked bactericidal action of pro
with
U.S.A.).
our
herring
water
and
strains
Fermentation,
Chemical was
distilled
12583 the
in
(from
Pure
solution
the
of
maintained
sulfate
in
All
Institute
Wako
Clupeine
text
paper
from
Clupeine from
tamines
the
of Na and K action of the
IFO
12200.
continuously
protamine.3-5) BLOOM et al.6) reported that the antibacterial action of protamine on B. subtilis
present
reduced
important than ions drastically
coagulans
IFO
obtained
factors
action
B.
licheniformis
and
The
largely
ions was far more 0.15-0.20 of metal
study.
environmental conditions. Among the available reports, however, the studies carried out by NEGRONI and FTSCHER1,2) are the most elaborate, can
in the media
of the metal of more than
buffer almost effect on the
action. Mg and Ca ions were more inhibitory than that in the media had very little or no effect on the bactericidal
available
action of protamine
of clupeine sulfate on Bacillus organisms in neutral and alkaline pH regions. The
Characteristics of Fisheries
Kagoshima
of Fish Technology,
Quality 890,
Control Japan(元
Suspensions
Initial
of Bacteria
from
suspensions 18h
Penassy
of
broth
Broths.
bacteria
culture The
culture
either was
were in
prepared nutrient
centrifuged
Protamines-II. Bangladesh and
Agricultural
Analysis, Faculty of
広 輝 重 ・板 倉 隆 夫:鹿
University, Fisheries,
Mymensingh, Kagoshima
児 島 大 学 水 産 学 部).
Bangladesh. University, 4-50-20
or and
812
ISLAM, MOTOHIRO, and
Table
1.
Effect
of pH on bactericidal
ITAKURA
action
of clupeine
sulfate
%survivors Test
Clupeine
organism
pH*
,μg/ml
B.subtilis ruber B. coagulans B. licheniformis
200 100 200
Initia cell number: lIecubation:
*
1.
Effect
of pH
pellet
obtained
phosphate 340nm.
The
Action basic
study
10.0
11.0
0.03 -
0.07 -
solution
incubated
at
37•Ž
a thermostat
period. estimated
After by
sulfate
on
B.
subtilis
ruber.
made
tubes the
14mm•~
series
was
adequately
stated
water-bath incubation
count
then in
desired
viable plate
quantified were
otherwise for
com diluted
of
samples
Results
and Discussion
was
distributed of
0.1ml
The
in
protamine was
of
of
not
involved
of
from
standard
was
method
medium series
and
(if
forming units and the percent of surviving or ganisms were calculated. All experiments were accompanied with control samples without pro tamine. Any modification of the above technique, when made, are discussed in the text.
sterilized quantified
Sulfate
the
0.1ml
suspension.
in
count
action
aliquots
adding
bacterial
in
of
in
Each
protamine
were
9.0 0.03 -
2.0•~107cells/ml
Clupeine
basal
by
of clupeine
experiment.
bactericidal
aliquots
160mm. pleted
of
0.1 0.0 1.1
200ƒÊg/ml
plate
each
of
follows:-the 0.8ml
action
photometrically
principle of
number:
standard with
Bactericidal
bactericidal
resuspended
and
A
on
sulfate:
was
buffer
simultaneously
text)
8.0
0.1 0.0 3.3
control cell
Clupeine
in
7.0
2.0 3.7 3.5
clupeine•¬ :
as
6.0
39℃
Initial
the
6.8 7.9 28.5
M/100 acetacebueffer (pH 4.0and 5.0);M/100 phogphate buffer(pH 6,0,7.0 and 8.0)and M/100 sodium bicarbonatebuffer(pH 9.0, 10.0 and 11.0).
•› ---•›:
at
5.0
2.0×107cells/m 2h at
Fig.
the
4.0 20.3 15.0 -
the
length
organisms as
colony
Effect of pH The relationship between pH and bactericidal action of clupeine is presented in Table 1. Bacteri cidal action of clupeine was found to be much higher at neutral and alkaline pH. This result contradicted with a previous report suggesting the higher antibacterial action of protamine in slightly acidic and neutral media.4) Another interesting observation found in, the present study was that in acetate buffer (pH 4.0 and .5.0) bacterial ino-
Factors
Affecting Bactericidal
Action of Clupeine
813
Table 2. Effect of ionic strength of buffer on bacteri cidal action of clupeine sulfate
at the same assumed that
Molarity
0.01
0.03
0.05
0.1
*% survivors
0.14
0.28
0.69
1.08
phosphate molecules prevented protamine to reach the reactive cell surface. The same observation on E. coli K-12 has been reported for another arginine
Initial Clupeine
0.5
21.87
62.50
rich basic protein
B. subtitisruber
clupeine/ml
sulfate:200μg
in phosphate
37℃
2 h at
Table 3.
Effect
of temperature
of clupeine Temp.
5
%survivors Initial
on bactericidal
action
sulfate
℃
10
1.72
0.80 2.5×106
cell number:
20
30
40
50
0.25
0.25
0.20
0.12
cells/ml
Test organism: B. subtilis ruber Clupcine sulfate: Incubation time:
200ƒÊg/ml 90min
in M/100 phosphate buffer(pH7.0)
Table 4. Bactericidal action of clupeine sulfate at different temperatures as a function of time Counts
of survivor
Temp.
Time (min) 0
5
15
30
30
2.0×106
3.3×103
2.9×103
1.9×10
3 40
2.0×106
2.2×103
1.8×103
9.0×10
250
2.0×106
1.3×103
1.1×103
8.0×102
Test organism: Clupeine
sulfate:
B. subtilis 200μg/ml
ruber in M/100
phosphate
histone.8)
buffer (pH
7.0) Incubation:
it can be of anionic
1.6×106cells/ml
cell numbers:
Test organism:
0.3
pH. From this result increased concentration
buffer(pH
7.0)
culum in the control tubes were partially killed but at pH 4.0 where most of the organisms killed in the control tubes, a significant number of organisms survived in the protamine tubes. This result suggests that addition of protamine in the acidic media somwhow protected some of the organisms from the lethanl effect of lower pH (Fig. 1). Considering the overall effect of pH on the bactericial action of clupeine, it is reasonably suggested that the polycationic nature of protamine due to their high arginine content may partially be altered in the acidic medium, consequently result ing in the decreased ability to combine with the bacterial cell surface. On the other hand, it is quite natural to think that cationic nature of pro tamines is not subject to be changed considerably in the alkaline media because isoelectric point of protamine is reported to be about 12.0.16) Bactericidal action of protamine also varied significantly at different ionic strength of buffer (Table 2). In 0.01M phosphate buffer at pH 7.0 the percent survival of organisms was only 0.14 but this figure increased upto 62.5 in 0.5M buffer
Effect of Temperature The data presented in Table 3, indicated that although bactericidal effect of clupeine sulfate is slightly higher at higher temperatures, it is not likely that temperature can influence the bacteri cidal action significantly. Killing at different temperatures as a function of time are given in Table 4. Killing took place at slightly rapid rate at higher temperature, but at all the temperatures studied most of the organisms were killed within 5 minutes and after that reduction in number was quite insignificant. The technique employed in this study was not sufficiently accurate to establish actual rate of killing in the early course of the reaction, therefore, quantitative relationship be tween rate of killing and temperature was not es tablished. Effect of Metal Ions Influence of various metal ions with different ionic strengths on bactericidal action of clupeine sulfate are given in Table 5. Salt concentration of the medium greatly influenced the bactericidal action and the ionic strength of the metal ions was far more important than that of molarity of the salts. Although the effect of molarity on bacteri cidal action varied depending on the salt used, ionic strength higher than 0.15-0.20 in general led to increased survivability of Bacillus subtilis cells. Mg and Ca ions were more inhibitory than that of Na and K ions. Previous studies have also supported the present finding, which suggested that presence of Al, Mg and Ca ions decreased the efficiency of antibacterial action of protamine.9) Considering the multilayered cell wall of Gram positive organism which is composed of peptido glycan and teichoic acid and stebilized as poly anionic polysaccaride,10,11) it is reasonable to assume that the metal ions may compete with polycationic protamine for binding with reactive cell surface. Recent evidence has suggested that polyanionic polysaccarides from bacterial cell wall can antagonize the bactericidal and bacteriolytic action of protamine.7) Effect of Media Containing Organic Matters Effect of some common bacterial culture media
814
ISLAM, MOTOHIRo
Table 5.
and
ITAKURA
Effect of metal ions on bactericidal
action of clupeine sulfate
% survivors Metali ions
Molarity
0.005
0.01
0.05
0.1
0.15
0.2
0.3
0.6
-
-
0.09
0.09
0.12
0.96
5.00
31.40
0.19
1.21
6.28
34.20
NaCl KCl
-
-
0.13
0.15
MgSO
0.07
0.19
2.20
35.50
-
80.20
-
-
MgCl2 CaCl2
0.16 0.25
0.86 1.90
4.05 5.58
37.50 30.30
-
84.00 70.50
-
-
Initial Test
2.0×106
cell number: organism:
Clupeine
B. subtils
sulfate:
Incubation
cells/ml
ruber
200μg/ml
time:
in M/100
60min
Table 6.
at
phosphate
buffer
(pH
7.0)
37℃.
Effect of different culture media
on bactericidal action of clupeine
sulfate
% survivors
Glucose (0.1 M)
Peptone (1.0%)
0.60 Initial
Meat extract (0.5%)
Penassy broth
Tryptic soy broth
Heart infusion broth
0.08
0.0
0.03
0.06
0.93
cell
number:
2.0•~108
Test organism: Incubation
2h
at
action
37•Ž
of protamine
are presented
in Table 6. From these results it can easily be understood that the presence of these organic matters in the media had very little or no influence on strong bactericidal
action of protamine.
More
over in some of the media bactericidal action was even stronger than that of in plain phosphate buffer. Only in peptone the bactericidal action was slightly insignificant
decreased but that seemed to be quite and ignoreable. The earlier reports
of ineffectiveness of protamines in broth media3,12) was greatly contradicted with the results of the present study. in preparation
This can be explained by differences of protamines, organisms involved
and the experimental condition sidering the biochemical nature not
unlikely
that
it may
employed. Con of protamine it is
combine
with
organic
substances like nucleic acids, some proteins anionic polysaccarides and this phenomenon antagonize the bactericidal action. Some vious evidences supports this view.6-8) Throughout the present study phosphate was used desireable though
as the basal interference
previously
like
cationic
and can pre buffer
medium to avoid the un by other materials. Al
a number
made on antibacterial mechanism by which cular organisms are only some
0.09
cells/ml
B. subtilisruber
time:
on bactericidal
Phosphate buffer
of studies
have been
action of protamine, the protamine kill some parti
is not yet clearly known. There speculations that protamine acts bactericidal
detergents
and
poly
peptide antibiotics, which adsorb face, alter the osmotic integrity,
on the cell sur and disturb re
spiration.13,14) It is almost clear from the above study that bactericidal action of protamine is more or less substrate dependent, and especially the pH and ionic strength of the media are the most important. References
1) P. NEGRONIand I. FISCHER: Rev. Soc. Argent. Biol., 20, 307-311 (1944). 2) P. NEGRONIand I. FISCHER: Rev. Soc. Argent. Biol., 20, 487-495 (1944). 3) B. F. MILLER,R. ABRAMS, A. DORFMAN, and M. KLEIN: Science, 96, 428-429 (1942). 4) R. WOLF and J. BRIGNON: Bull. Soc. Chem. Biol., 36, 1125-1132 (1954). 5) T. D. BROCK:Can. J. Microbiol., 4, 56-63 (1958). 6) W. L. BLOOM,M. G. WINTERS, and D. W. WATSON: J. Bacteriol., 62, 7-13 (1951). 7) S. ANTOHIand A. POPESCO: Z. Naturfosch., 34C, 1144-1150 (1979). 8) J. G. HIRSCH: J. Exp. Med., 108, 925-944 (1958). 9) L. MASSARTand P. VAN DEN DAELE: Arch. Intern. Phermacodynamic., 76, 424 (1948). 10) K. H. SCHLEIFER and O. KANDLER: Bacteriol . Rev., 36, 407-477 (1972). 11) J. W. COSTERON, J. M. INGRAM, and K. J. CHENG: Bacteriol. Rev., 38, 87-110 (1974). 12) O. R. BRAEKKANand G. J. BOGE: Reports on Technological Research Concerning Norwegian
Factors
Affecting
Bactericidal
Fish Industry, 4, 1-21 (1964). 13) T. J. FRANKLINand G. A. SNOW: in "Bio chemistry of Antimicrobial action", Chapman and Hall, London, 1971, pp. 53-55. 14) H. FISCHERand L. WAGNER:Naturwisenschaften,
Action
15) 16)
of Clupeine
815
41, 533-545 (1954). N. M. ISLAM, T. ITAKURA, and T. MOTOHIRO: Bull. Japan. Soc. Sci. Fish., 40, 1705-1708 (1984). K. FELIX: Adv. Protein Chem., 15, 1-56 (1960).
