Note: Descriptions are shown in the official language in which they were submitted.
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1
CLEANING AND/OR DISINFECTING COMPOSITION
Field of the invenrinn
The present invention relates to a cleaning and/or
disinfecting composition comprising a tertiary alkyl amine
and an alkyl betaine as active agents and to the biocidal
use thereof.
BackgrpLnd of th iriy nt-inn
In order to reduce and prevent infection, to minimize the
risk to public health thereof and to prevent product
spoilage, disinfectants are commonly used to kill bacteria
which spread disease.
A known disinfectant is described in WO 95/00613.
However many of the known disinfectants employ
environmentally less acceptable and expensive components
such as amine oxide, EDTA (Ethylene Diamine Tetra Acetic
Acid), and quaternary ammonium compounds.
It is an object of the present invention to yield a cost
effective cleaning and/or disinfecting composition which
does not employ the above environmentally less acceptable
components.
The inventors have found that employing both a tertiary
alkyl amine and an alkyl betaine yielded surprisingly good,
cost effective biocidal activity at a low active agent
concentration, when compared to disinfectants comprising
either only a tertiary alkyl amine or only an alkyl
betaine.
DPfin,_'t,'_on of -hue inv ntinn
According to a first aspect of the present invention there
is provided a cleaning and/or disinfecting composition
comprising a tertiairy alkyl amine and an alkyl betaine.
Another aspect of the present invention is concerned with
the use of a composition according to the present invention
for cleaning surfaces and killing bacteria.
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v
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Preferably the ratio of the alkyl amine to alkyl betaine
lies in the range of 1:3 to 3:1, preferably 1:1 by
weight.
The composition preferably is used at a pH in the range
of 2-10.
15
25
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Detailed description of the invent,'_on
A preferred tertiary alkyl amine is 1.3 propanediamine-n-3-
aminopropyl. A preferred alkyl betaine is alkyl C9-C15
dimethyl amine betaine.
The tertiary alkyl amine and alkyl betaine comprise
preferably 1-15 wt.% of the composition.
The cleaning and/or disinfecting composition may
furthermore comprise an organic acid or soluble salt
thereof, preferably selected from the group consisting of
l0 salycilic acid, acetic acid, sorbic acid, benzoic acid,
lactic acid, citric acid, malonic acid, tartaric acid,
gluconic acid, lactobionic acid, formic acid, malic acid,
parabenzoic acid and peracetic acid.
The addition of an organic acid to the composition effects
surprisingly good results for killing bacteria, especially
gram negative bacteria.
In order to maximize the cleaning effect and to limit the
effects of pH change, the composition may furthermore
comprise a cleaning agent and a buffer.
The invention will now be illustrated by the following
experiments and results, whereby it is noted that:
1) All investigations were carried out using compositions
which comprised, as active agents one or more of the
following:
(A) The alkyl betaine, RN(CH3)2CHzC02; wherein R = 3%
C10, 70% C12, 25% C14, 2% C16,
( B ) The alkyl amine RNH ( CHz ) 3NH ( CHZ ) 3NH2 , where in R =
dodecyl, C12.
(C) amine oxide; RN(CH3)20; R as above
(D) amine oxide; myristyl dimethylamine oxide
(E) alkylamphoacetate; alkyl imidazoline betaine
(F) Coco diethanolamide
(G) polyoxyethylene sorbitan trioleate
(H) Coca-amido betaine, RCONH (CH2) 3N (Me) ZCH2C02, R = as
above;
wherein the desired pH of the active agents was adjusted
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using HC1(aq);
2) The percentages of active agents quoted in all the
experiments refer to the percentage of total
composition;
3) The biocidal effect of the active agents was
investigated by the action thereof on the following
microorganisms:
Staphylococcus aureus ATCC 6538 (Gram positive
bacterium)
Pseudomonas aerug.inosa ATCC 15442 (Gram negative
bacterium)
Saccharomyces cerevisiae ATCC 9763 (Yeast)
Bacillus subtilis PSB 357 tux (Spore culture),
supplied by Dr P. Hill, Univ.
Nottingham; and
4) The first three of these microorganisms were grown in
broth at 30°C for 24 hrs. (bacteria-Tryptone Soya Broth
(Oxoid CM129); yeast - "Malt Extract Broth" (Oxoid CM
57). Cultures were then centrifuged at 4000 r.p.m.
(Sigma model 3K-1) for 10 minutes, and the cell pellets
resuspended on 0.1% peptone water.
p~Dara ~ on of h B s ~ht; 1 i snore c,~) t»rP
This was grown overnight at 30°C in heart infusion broth
(HIB, Difco) with erythromycin(10 mg/ml) to approximately
1x109 cells/ml. This was then used to inoculate heart
infusion agar(HIA) plates {with 10 mg/ml erythromycin)
which were incubated at 30°C for 7-9 days until 80-100%
phase-bright spores were visible under a light microscope.
Spores were then harvested by washing the HIA plates with
sterile distilled water, followed by centrifugation at 4000
g for 20 minutes at 4°C. The spores were washed three times
by centrifugation and resuspensed in sterile distilled
water, before being pasteurized at 70°C for 40 minutes.
Finally, the spores were resuspended in distilled water
containing erythromycin (10 mg/ml) and stored at -80°C.
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To stimulate spore germination, 100 ~.l of the thawed
suspension was heat activated, at 70°C for 30 minutes. 40/~l
of the heat activated spores was added to a solution (30°C)
of NBLi (138 ~1) and sterile distilled water (20 ~,1).
Samples were removed from the 30°C water bath every ten
minutes and the percentage phase dark enumerated.
Investigation into the biocidal effect of (A), (B) and
(A):(8) mixtures on the first three micro-orgaaisms
..
To investigate the effect of the agents on the first
three microorganisms (S.aureus, P.aeruginosa and
S.Ceresiviae) the following suspension test was used
to determine the log reduction of the micro-organisms
in all experiments.
The microorganism suspension (0.1 ml) was added to the test
agent (10 ml) and mixed thoroughly and left at ambient
temperature for five minutes. Following the contact time,
an amount (1 ml) was transferred to Universal Quenching
Agent (9 ml), peptone (1 g), TweenTM80 (5 g), sodium
thiosulphate (1 g) and lecithin (0.7 g) per litre of
deionized water)) to inactivate the disinfectants.
Survivors were counted following serial dilution in 0.1%
peptone water on appropriate agar using 0.1 ml spread
plates. The plates were incubated at 30°C for 48 hours. The
log reduction of the microorganisms was then calculated.
Experiment 1
Investigation into the effect on S.aureus and P.aeruginosa
when treated with (A) and (B) respectively after a five
minute contact time, at a pH between pH=5 and pH=9, the
results of which are graphically shown in figures 1 and 2.
Conclusions from figures 1 and 2:
At pH~s of 5 to 9 (A) was effective against the Gram
positive bacterium, S.aureus. At lower concentrations of
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(A), the lower pH solutions were more effective biocides
against this organism.
(B) was effective against the gram negative bacterium
P.aeruginosa. However (B) was ineffective at low pH except
5 at high concentrations against the gram positive bacterium
S.aureus.
For both (A) and (B), a concentration of 0.01% recorded
over five log reductions in the bacteria (kills) at pH=9.
At pH=7 only 1.5 log reductions were observed and a very
small reduction at pH=5. At the 0.1% level and at all pH
values examined there was greater than log 5 kill with
P.aeruginosa. At 0.01% and pH=9 greater than log 5 kill was
observed whereas at pH of 7 and 5, only a log reduction of
4.5 was recorded.
Experiment 2
Figures 3a, b and c and figures 4a, b and c respectively
show the effect of mixtures of the active agents (A) and
(B), and pH on the log reduction of S.aureus and
P.aeruginosa.
Conclusions from figures 3 (a, b, c) and 4 (a, b, c)
Figure 3a shows that, as the mix changes to more (B), at
pH=5, a higher concentration of total active agent is
required to yield a satisfactory log reduction. As the pH
was raised, the total amount of active agent required to
effect >log 5 reduction, decreases. At pH=9, figure 3c, the
effect of (B) appears to outweigh the activity of (A).
However at pH=7, the 25:75 and the 75:25 (B):(A)
compositions appear to have acted in a synergistic way.
Figures 4a, b and c show that at pH=5 as the amount of (B)
increases, a steady progression of increasing log reduction
is observed which peaks at the 75:25 mixture. This effect
is,reflected in the graphs at pH=7 and pH=9. At a
concentration of 0.001% active, the 75:25 mixture achieves
nearly 3 log reductions and total kill at the b.01°s level
at pH=9.
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Experiment 3
The inventors then carried out research to optimize the
mixture in order to improve the log reductions in both
organisms. This was done by multiplying the log reductions
for each of the organisms and plotting this against the
concentration and percentage composition wherein the
highest point reached on graphs represented the theoretical
mixture best able to deal with both organisms. Such graphs
are shown in figures 5a, b and c, for the three pH values
examined. (Note this was done by the inventors purely for
graphical demonstration, the multiplying of two logs in
this manner is not related to any known physical
phenomenon).
Conclusions from figures 5(a,b,c):
At pH=5, the theoretical optimum compositions against the
bacteria S.aureus and P.aeruginosa were the 25:75 and the
50:50 mix of (B):(A) at 0.1% total active agent in the
mixture composition. The 25:75 mix gave a better log
reduction at 0.01% total active agent in the composition.
At pH=7, the 25:75 or the 75:25 appeared to be suitable and
could be used at the 0.01% level. At pH=9, the 50:50, 75:25
and the 100% (B) compositions gave the best results.
Experiment 4
An examination of the 50:50 (B):(A) mixture was conducted
at a variety of concentrations against S.cerevisiae. A plot
of log reduction is shown in figure 6.
Conclusions from figure 6:
Unlike the bacteria examined, the yeast showed a decrease
in biocidal effect as pH was increased at the lowest
concentrations of active agent used. With this mixture
composition, 0.01% of active agent gave a total kill of the
yeast at all pH values examined.
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Investigation into the comparative effect of disinfecting
compositions, comprising the above active agents (A)-(H) on
the three organisms at a pH of 5.0 and various active agent
concentrations. (Experiments 5-7)
Experiment 5
Effect of active agents on S.aureus
At 0.5% levels, the ability to reduce the levels of
organisms showed the following activity:
(A) > (C) » (B) > (D) > (E) > (G) - (F) - (H) - Control
Experiment 6
Effect of active agents on P.aeruginosa
At 0.5% levels, the ability to reduce the levels of
organisms showed the following activity:
(B) - (C) > (D) » (E) > (G) - (A) - (F) - (H) - Control
Experiment 7
Effect of active agents on S.cerevisiae
At 0,5% levels, the ability to reduce the levels of
organisms showed the following activity:
(B) _ (C) _ (D) > (A) » (E) - (F) > (G) - (H) - Control
Figures 7a, b and c show the comparative effect of the 8
active agents used in this study on the organisms in
experiments 5, 6 and 7, respectively.
Conclusions from figures 7a, b and c:
At a pH of the test (pH=5), (B) is considered to be a
cationic surfactant, and as such it was to be expected that
good activity against gram positive bacteria would be
achieved. However, this was not the case
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The inventors believe that at this pH there may be
a chemical reaction between the surfactant and the techoic
acids of the murein layer of the bacteria. This essentially
makes the surfactant unable to act as a biocide. At higher
concentrations this is circumvented as the layer is swamped
with the surfactants.
Investigation into the effect of (B) sad (A) on spore
germination of S.cerevisiae (Experiment 8):
Background
Spores can be likened to an armour plated bacterium. As
such they are much more difficult to kill than a normal,
vegetative cell. Normal disinfectants and levels used do
not readily kill bacterial spores. After cleaning,
bacterial spores can therefore cause contamination problems
if they germinate and multiply. One strategy for
inactivating bacterial spores is to germinate them into the
more sensitive vegetative form; In Tyndalisation, spores
are heated and cooled repeatedly, the heating cycle
stimulates the spores to germinate into the vegetative
(easy to kill) state. The next heating cycle kills the
germinated bacteria and induces another batch of spores to
germinate and so on.
It has been shown that known disinfectants comprising
quaternary ammonium compounds (QACs) are not effective
sporicides. Apparent sporicidal activity is due to the QACs
sticking to the spores. Upon germination, the locally high
concentration of biocide kills the bacterium. However,
repeated washing or using universal quenching agent allows
recovery, because the biocide is removed.
The term "chemical germination" was coined to summarize the
germination-like changes induced in spares by active
agents.
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Experimental:
Active agents (A) and (B) were mixed with the Bacillus
subtilis spores, in the following composition
concentrations:
(A) 0.5%
(B) 0.5%
(A) : (B) 0.05%
Figure 8 shows the results of this.
Peracetic acid a known sporicide, was used as a
positive control.
Results and conclusions from figure 8:
(B) at 0.5% . Inhibition of germination occurred and was
greater than that of (A) alone.
(A) at 0.5% . Inhibition of germination occurred and was
lesser than that of (B) alone.
(B):(A) mixture
0.05% . Inhibition of germination occurred, to a
lesser degree than (A) or (B) alone.
The inventors postulate that as the spores germinate the
active agents present on the spore coats kill the
vegetative cells.
Investigation into the possible synergistic effects of the
active agent mixtures (B) : (A) .
(Experiments 9-11)
Solutions of (B):(A), in a ratio of 1:3 at total active
agent concentrations of the solution compositions from
0.002 to 0.01% were prepared. Separate solutions of (B)
and (A) alone were also made up at the same total active
agent concentrations.
The following tables (1-3) give the results of
examples in terms of total composition, and compare the
calculated, expected additive effect with the
experimentally derived effect, carried out at a pH of 9.
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Experiment 9
Effect of a (B) : (A) , (1:3) mixture on S.aureus
The results of this example are shown below in Table 1.
5 Table 1
Total (B)/% (A)/% Expected Actual
active (Additive) effect
agent/% effect log
log reduction
reduction
0.01 0.0025{2)a 0.0075(0.9)a2.9 >5.3
1 0.008 0.002(1.34)0.006(0.01) 1.35 >5.3
0
0.006 0.0015(1) 0.0045(0) 1 3.4
0.004 0.001(1) 0.003(0) 1 1
0.002 0.0005(0) 0.0015(0) 0 0.08
a(x) denotes log reduction of pure surfactant at that
concentration
Experiment 10
Effect of a (B) : (A) , (1:3) mixture on P.aeruginosa
Table 2, below, shows the results of this example.
Table 2
Total (B)/% (A)/% Expected Actual
active (Additive) effect
2 agent/% effect log
5
log reduction
reduction
0.01 0.0025(5.3)a0.0075{0)a 5.3 >5.5
0.008 0.002(4.98)0.006(0) 4.98 >5.5
o(x) denotes log reduction of pure surfactant at that
concentration
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Experiment 11
Effect of a (B) : (A) (1:3) mixture on S. cerevisiae
Table 3, below shows the results of this example.
Table 3
Total (B)/% (A)/% Expected Actual
active (Additive) effect
agent/% effect log
log reduction
reduction
0.01 0.0025(4.5)a0.0075(1.4)a5.9 >5.5
1 0.008 0.002(3.31)0.006(1.22) 4.53 >5.5
0
a(x) denotes log reduction of pure surfactant at that
concentration
15 Conclusions from tables 1-3:
These tables show that a greater effect is achieved than
was to be expected by simply mixing (A) and(B).
20 Investigation to determine the concentration of active
agent (B), (A), and mixtures thereof, in the composition,
required to achieve a log four reduction in S.aureus and
P.aeruginosa respectively. (Experiments 12-15):
25 Experiment 12
Effect of (A) and (B) on S.aureus
Table 4, below shows the results of this example.
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Table 4
pH 5 7 9
(A) 0.1% 0.1% 0.1%
(B) 1% 0.1% 0.01%
Experiment 13
Effect of (A) and (B) on P. aeruginosa
Table 5 below shows the result of this experiment.
Table 5
pH 5 7 9
(A) >1% >1% >1%
(B) 0.01% 0.01% 0.01%
Experiment 14
Effect of mixtures of (A) and (B) on S.aureus
Table 6 below, shows the results of this experiment.
Table 6
(B) : (A) ratio
pH 5 7 9
25:75 0.1% 0.01% 0.01%
50:50 0.1% 0.1% 0.01%
75:25 1% 0.01% 0.01%
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Experiment 15
Effect of mixtures of (A) and (B) on P.aeruginosa
The results of this experiment are shown below in table 7
Table 7
(B) : (A) ratio
pH 5 7 g
25:75 0.1% 0.01% 0.01%
50:50 0.01% 0.01% 0.01%
75:25 0.01% 0.01% <0.01%
Conclusions from tables 4-7:
These tables show that when mixed, the potency of the
disinfecting composition, comprising a (B):(A) mixture is
greater than would be expected from combining (B) and (A).
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Investigation into the biocidal activity of organic acids
a.n combination with a cleaning/disinfectant composition
comprising one or more of the active agents (A)-(H),
(Experiment 16):
Background
Weak acids are antimicrobiall, however, the extent of their
antimicrobial ability is dependent on several factors which
include the pH of the solution, the pKs of the acids and
their permeabilities or partitioning ability. Lauric acid
is a moderately good anti-microbial material, it is able to
partition itself into microbial membranes, become ionized
and release the proton; the proton disrupts proton-
gradients, the fatty acid anion can disrupt transport
processes and can help permeabilise membranes. However this
effect is based on the protonated material, as such its
ability as an antimicrobial is lost as the pH of the
outside medium is raised to pHs beyond the pK, i.e. as the
acid becomes more ionized. To take advantage of the weak
acid effect low pH solutions should be used. However, low
pH solutions (pH < < 5) can be corrosive on certain
surfaces and are preferably avoided.
Experiment 16
A range of weak acids, see following tables and figures,
was exposed to the three test organisms:
Staphylococcus aureus ATCC 6538
Pseudomonas aeruginosa ATCC 15442
Saccharomyces cerevisiae ATCC 9763,
at a concentration of 0.5% by weight and the effects
recorded according to the suspension test.
Figure 9 shows the results of experiment 16, a plot of the
ratio of the log survivors for the test acid versus a water
control.
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Conclusions from figure 9:
A ratio of log acid . log control greater than the
suggested that the acid acted as a growth promoter, less
than one an inhibitor.
5
Investigation into the effect of disinfectant compositions,
comprising mixtures of the active agents with acids on the
first three organisms. (Experiments
10 17-19)
Figures 10a, b and c show the results of the experiments
17-19 into the effect of mixtures of active agents (A)-(H)
and the acids, salycilic, acetic, sorbic, benzoic, citric,
EDTA on the three microorganisms, at a concentration of
15 0.5% by weight of the active agent, as carried out by the
suspension test.
The pH of the disinfecting compositions was adjusted to
pH=5 with HC1 and NaOH.
Experiment 17
Effect on S.aureus (Figure 10a).
Conclusions from figure 10a:
The most surprising effect observed was the antagonistic
effect of (A) with salicylic acid. In general all the
acid/surfactant mixtures examined were antagonistic.
Experiment 18
Effect on P.aeruginosa (Figure 10b).
Conclusions from figure 10b:
In general there was an improvement in the kill when an
acid was present.
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Experiment 19
Effect on S.cerevisiae {Figure 10c)
Conclusions from figure 10c:
The addition of citric acid at pH=5 gave a large
synergistic killing effect on gram negative bacteria when
mixed with (A).
As was shown above, (A) cannot kill gram negatives by
itself, (see figure 1, experiment 1).
However the addition of citric acid improved its log
reduction from zero to log four reduction, which is
surprising.
Furthermore citric acid at pH=5 at the level used (0.5%)
did not affect the growth of the bacteria,(see figure.8).
Investigation into the biocidal effect of different
(A):acid mixtures concentrations on the microorganisms
(Experiments 20-22)
(In the experiments 20-28, the % of the active agent and
the acid by weight in the composition were in all cases
equal . )
Figures 11a, b and c show the results of the experiments
20-22 into the effect of different concentrations (0.01% -
1.0%) of the (A):acid mixtures against the three micro-
organisms.
Experiment 20
Effect on S.aureus, fig 11a.
Experiment 21
Effect on P.aeruginosa, fig 11b.
Experiment 22
Effect on S.cerevisiae, fig 11c.
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Investigation into the biocidal effect of different
(B):acid mixture concentrations on the microorganisms,
(Experiments 23-25)
Figures 12a, b and c show the results of the experiments
23-25 into the effect of different concentrations (0.01%
1.0%) of the (B):acid mixtures against the three micro
organisms.
Experiment 23
Effect on S.aureus, fig 12a.
Experiment 24
Effect on P.aeruginosa, fig 12b.
Experiment 25
Effect on S.cerevisiae, fig 12c.
Investigation into the biocidal effect of different
(H):acid mixture concentrations on the microorganisms,
(Experiments 26-28)
Figures 13a, b and c show the results of the experiments
26-28 into the effect of different concentrations (0.01% -
1.0%) of the (H):acid mixtures against the three micro-
organisms.
Experiment 26
Effect on S.aureus, fig 13a.
Experiment 27
Effect on P.aeruginosa, fig 13b.
Experiment 28
Effect on S.cerevisiae, fig 13c.
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Investigation into the synergistic factor in the active
agent: acid mixtures
The following equation was used to find the synergistic
factor:
Log reductiontotal=log reductiona~ia+log reduction8~ti"e agent+QsF
where aSF (the Synergistic Factor) is positive if
synergistic, negative if antagonistic.
Delineating the effect of the acid and the active agent
from the effect of the mixtures was important in
establishing whether a synergistic effect was present or
not.
The total log reduction can be described by the following
equation:
log reductionobs=log reductiona~t;"e agent+log reductiona~ia+as8
Where
- log reductionobs = total log reduction in organisms
observed,
- log reduCtlOnactive agent = log reduction in organisms due to
the effect of the surfactant alone,
- log reduction8~ia = log reduction in organisms due to the
acid alone,
- aSF, the synergistic factor, is the added log reduction
observed due to the combination of both the acid and
surfactant. This factor can be positive (synergistic) or
negative (antagonistic).
The biocidal activity of the active agent is influenced by
such factors as temperature, concentration, pH, chain
length, permeability and head group charge. The biocidal
activity of the acid is influenced by, for example, time of
contact, permeability, pKa, pH and concentration. Thus the
Synergistic Factor is also influenced by these things since
it is dependent on both.
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Tables 8, 9 and 10 show the QSF factors calculated for the
various active agent:acid mixtures at a concentration of
0.5% (acid and surfactant) at pH=5 at room temperature.
One difficulty encountered in calculating these factors
was, when the sum of the individual active agent and acid
contributions are greater than the control number of
organisms less the lowest recorded survival number
(normally log 2) i.e. if the control number of organisms
was log 7.2, and the log red11Ct10n8~tive agent = 5.5 and the
log reductiona~ia = 1. 8 , then the actual oSF could not be
calculated if it acted synergistically and a maximum value
could only be given if it was antagonistic.
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N
CA 02251941 1998-10-15
WO 97/43368 21 PCT/EP97/02326
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CA 02251941 1998-10-15
WO 97/43368 2 2 PCT/EP97/02326
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CA 02251941 1998-10-15
WO 97/43368 PCT/EP97/02326
23
Conclusions from table 8:
asg factors for S.sureus,(gram positive bacteria).
The values, table-8, show the effects of the various acids
used in combination with the active agents.
(B) gave negative sigma factors especially with polyacids
such as sorbic, citric and EDTA.
With (A) where a sigma factor could be obtained, a strong
antagonistic effect was observed, e.g. salicylate= -1.45.
Conclusions from table 9:
asF factors for P.aeruginosa, (gram negative bacteria).
A difference to the gram positive bacteria was observed
(table 9). The acid:(A) mixtures showed strong synergies,
especially with the chelating acids - NTA gives a
synergistic factor of over 5.8 log units.
Conclusions from table 10:
asg factors for S.cervisise
With (A) the sigma factors were large and negative, as
found in some cases with S.aureus.
Comment on these conclusions from tables 8, 9 and 10:
The inventors postulate that some of the reasons for the
variations of the aSF factors with the different active
agents may be due to reaction of the active agent with the
acid and/or reaction of the acid and/or active agent with
the outer walls of the bacteria inhibiting/aiding the
effect of each other. The large negative effect of (B) with
S.aureus is probably due to a reaction between the amine
end of the surfactant and the techoic acids of the murein
layer, this layer is smaller and less important in the gram
negative bacteria. The large aSF values for the chelating
ligands may be due to them becoming more permeable,
crossing the membrane more easily, and once inside,
dissociating - like a weak acid - but then chelating metal
ions such as calcium within the cell (NTA is smaller
CA 02251941 1998-10-15
WO 97/43368 PCT/EP97/02326
24
molecule and at the lower pH would be more permeable than
EDTA). It is thought that this is a stronger effect than
disruption of proton-motive force and therefore leads to
the greater log reduction observed. At pH 7 EDTA has a
stronger effect than NTA as a preservative, which suggests
that at the higher pH strength of calcium complexation is
the more prominent effect rather than permeability.
Following the above results, further research was carried
out to optimize the composition of the
cleaners/disinfectants according to the present invention.
A first composition according to the present invention was
tested for its biocidal activity at a concentration of 1,0%
w/w.
CA 02251941 1998-10-15
WO 97/43368 PCT/EP97/02326
Composition:
Formulation 1 - (disinfectant) ~ as supplied as 100
Raw material:
5
(2) sodium hydroxide (50%) 10.00 5.00
(3) 1.3 propanediamine-n-3-
aminopropyl (30%) 5.50 1.65
(4) alkyl (C9-15) dimethylamine
10 betaine (30%) 5.00 1.50
(5) citric acid anh 8.40 8.40
(6) sodium carbonate (light) 1.00 1.00
(7) sodium tetra borate 10 h2o 1.00 1.00
15 (1) water (demineralized) up to 100.00 100.00
To yield formulation 1, the raw materials were mixed in the
order given in brackets.
Specification of formulation 1:
Appearance: clear not viscous colourless liquid
Relative density (20°C)
Viscosity: 1.085
pH (1% solution): 9.5 - 10.0 (demi water)
The efficacy of the composition (formulation 1)
was evaluated in a test solution representing clean
conditions. The test was performed according to the
Quantitative European Suspension Test (E.S.T) method, as
described under 3), above for assessing bactericidal and
fungicidal activity, as follows:
Suspensions of the microorganisms were added to a
solution containing formulation 1. After a period of
exposure (5 minutes) at a temperature (20°C), the fraction
CA 02251941 1998-10-15
WO 97/43368 PCT/EP97/02326
26
of surviving organisms was determined. One variant was
tested:
a. with 0.03% bovine serum albumin (B.S.A.)in the test
solution representing clean conditions.
Test organisms
The effect was assessed on both Gram negative and
Gram positive bacteria and a yeast. The test strains used
were:
- proteus mirabilis ATCC 14153;
- pseudomonas aeruginosa ATCC 15442;
- saccharomyces cerevisiae ATCC 9763;
- staphylococcus aureus ATCC 6538;
- streptococcus faecium DVG 8582.
Calculation of the microbiocidal effect:
The microbicidal effect due to the action of the
disinfectant in 5 minutes at 20°C (MEZ°5) is expressed by
the formula:
ME2°5 = log (N~) - log (No) ,
Where,
- N~ is the number of colony forming units per ml of the
test mixture without disinfectant,
- ND is the number of colony forming units per ml of the
test mixture after the action of the disinfectant.
It is generally preferable that a disinfectant
preparation in the lowest use dilution induces a
microbicidal effect (MEszo) of at least 5 logarithmic
reductions for each of the test organisms.
The results of the investigation, carried out at
a pH of 9, are summarized in table 11, below.
Tested under clean conditions all the test
organisms showed a reduction of at least 5 logarithmic
cycli after 5 minutes exposure to the test composition at a
concentration of 1,0% w/w.
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27
Table 11 Microbicidal effect (ME2°5) of formulation 1:
test concentration: 1.0% w%w
bovine serum albumine: 0.03%
time: 5 minutes
temperature: 20°C
Test organism log N~ log ND MEz5
(cfu'/ml) (cfu/ml)
Proteus mirabilis ATCC 14153 6,7 < 1 > 5,'7
Pseudomonas aeruginosa ATCC 6 , 6 < 1 > 5
15442 . 6
Saccharomyces cerevisiae ATCC 6,1 < 1 > 5.1
9763
Staphylococcus aureus ATCC '7,0 < 1 > 6.0
6538
Streptococcus faecium DVG 8582(,5 < 1 > 5.5
cfu: colony forming units
Three further suspension tests were carried out to further
determine the biocidal activity of formulation 1, on one or
more of the following microorganisms.
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28
Key microorganism id number
Sa Staphylococcus aureus ATCC 6538
Sc Saccharomyces cerevisiae ATCC 9763
Ps Pseudomonas aeruginosa ATCC 15442
Ef Enterococcus faecium DVG 8582
Pm Proteus mirabilis ATCC 14153
List Listeria monocytogenes Type 4 b
Sal Salmonella choleraesuis ATCC 13311 {previously
S.typhimurium)
Suspension est 1
Conditions
Temperature 2 0 t 3. ~ C
.Contact time 5 min * 5 ec
_Soi3ing 0:03% BSA
water 17Gh
=Inactivation standard in buffer + horse serum
Concentration 1.-0%
Results of suspension test 1
2 MICROORGANISM Sa Ef Ps Sc Pm
5 LDR LDR LDR LDR LDR
Blanks in log 8.5 7.5 8.9 7.0 9.0
Formulation 1 >7.5 >6.5 >7.9 >6.0 >S.0
LDR = Log reduction
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29
Suspension test 2
Conditions
Temperature 20 t 1C
Contact time 5 min t 5 sec
Soiling 0.03% BSA
Water 17Gh
Inactivation standard in buffer + horse serum
Concentration 0.5%
Results of suspension test 2
MICROORGANISMS Sa Ef Ps Sc Pm Sal List
LDR LDR LDR LDR LDR LDR LDR
Blanks in log 8.9 7.4 8.9 7.5 9.0 9.1 8.0
Formulation 1 >7.8 >6.9 6.0 >6.0 >8.1 >8.0 >7.9
LDR = Log reduction
Suspension test 3
Conditions
Temperature 20 ~ 1C
Contact time 5 min t .~5, see
Soiling 0.03% BSA
\..
Water l7~Gh
:Inactivatior. standard in. buffer + horse>seruni
Concentration 0.5%, 0::4%, 0..3%
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WO 97/43368 PCT/EP97/02326
Results of suspension test 3
MICROORGANISM Sa Ef Ps Sc Pm Sal List
LDR LDR LDR LDR LDR LDR LDR
Blanks in log 8.9 7.4 8.9 7.5 9.0 9.1 8.0
Formulation 1 >7.8 >6.9 6.0 >6.0 >8.1 >8.0 >7.9
5 o.s%
Formulation 1 >7.8 >6.9 >3.6 >6.0 5.4 >8.0 >7.9
0.4%
Formulation 1 >7.8 >6.9 1.9 >6.0 3.1 >8.0 >7.9
0.3%
10
LDR
=
Log
re
uction
A
second
formulation
of
the
composition
according
to
the
present
invention
was
also
subjected
to
two
suspension
tests.
Formulation
2
(detergent
%
as
supplied
as
7.5
sanitizes)
100%
Raw material:
(2) sodium hydroxide (50%) 10.00 5.00
20 1.3 propanediamine-n-3-
(3)
aminopropyl (30%) 6.50 1.95
(4) alkyl (C9-15) dimethylamine
betaine (30%) 5.00 1.50
(5) citric acid anh 8.40 8.40
25 fatty acid alcohol (C9-11)
(6)
ethoxylate 5 EO 4.00 4.00
(7) sodium carbonate (light) 1.00 1.00
(8) sodium tetra borate 10 h2o 1.00 1.00
30 water (demineralized) up to 100.00 100.00
(1)
Production method:
The raw materials were mixed in the in order
given in brackets.
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31
Specification of formulation 2:
Appearance: clear non viscous colourless liquid
Relative density (20°C): 1.086
pH (1% solution): 9.5-10.0 (demi water)
Suspension
test 1 to
determine
the microbiocidal
efficiency formulation
of 2.
Conditions
Temperature 20 t 1C
Contact time 5 min 5 sec
Soiling 1% BSA
Water 17Gh
Inactivation standard in buffer + horse serum
Concentration 0.5%, 1%
Results of suspension test 1
MICROORGANISM Sa Ef Ps Sc Pm Sal List
LDR LDR LDR LDR LDR LDR LDR
2 Blanks in log 8.9 7.4 8.9 7.5 9.0 9.1 8.0
0
Formulation >5.4 >1.7 1.9 >0.5 >9.0 >3.6 >7.0
2,
1% BSA, 0.5%
conc.
Formulation >7.7 >6.7 7.8 5.6 7.3
2,
2 1% BSA, 1% conc.
5
LDR = Log reduction
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WO 97!43368 PCT/EP97I02326
32
Suspension test 2 Conditions
Temperature 20 t 1C
Contact time 5 min t 5 sec
Soiling 1% BSA
Water 17Gh
Inactivation standard in buffer + horse serum
Concentration 0.5%, 0.40, 0.3%
Results of suspension test 2
1 MICROOGANISM Sa Ef Ps Sc Pm Sal List
0
LDR LDR LDR LDR LDR LDR LDR
Blanks in log 8.9 7.9 9.1 7.0 >8.19 9.0 8.9
.0
Formulation >7.8 >6.9 5.1 >6.0 >8.1 >B.0 >7.9
2
0.5%
Formulation >7.8 >6.9 4.8 >6.0 5.4 >8.0 >7.9
2
0.4%
Formulation >7.8 >6.9 2.0 >6.0 3.1 >8.0 >7.9
2
0.3%
LDR = Log reduction
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33
REFERENCE
1. Jones, M.V., Food Preservative Interactions, PCW
851201
