Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~39358
-- 1 --
ENZYME-CONTAINING DETERGENT COMPOSITION
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an enzyme-
containing detergent composition. More specifically, it
relates to a detergent composition containing alkaline
protozoa APE, which is capable of decomposing proteins
under alkaline conditions.
2. Description of the Prior Art
It is known in the art that enzymes capable of
lo degrading proteins are incorporated into detergent
compositions, whereby proteins or other soils adhered
to materials to be washed are degraded and removed.
Examples of such protein-degradable enzymes convent
tonally used are alkaline pro teases derived from
bacteria belonging to genus Bacillus.
However, alkaline pro teases derived from genus
Bacillus are generally high temperature high activity
type enzymes. That is, most of the alkaline prateases
derived from genus Bacillus exhibit maximum activity
I at a pi of about 9 to 10 and at a relatively high
temperature, especially around 60C, but are inactive or
less active at a relatively low temperature, especially
around a room temperature.
This means that the characteristics of enzymes are
I not fully utilized in such a country as in Japan, where
clothes are generally washed at room temperature.
. ~.~ .
~2393S8
Furthermore, the development of enzymes having a retained
enzymatic activity even at a relatively low temperature
is needed due to the recent increased usage of chemical
fiber clothes having a relatively low heat stability and
also due to the recent energy-saving movement.
SUMMARY OF THE INVENTION
Accordingly, the objects of the present invention
are to eliminate the above-mentioned disadvantages of
- known enzyme-containing detergent compositions and to
provide an alkaline protease-containing detergent
composition capable of washing materials to be washed at
a high washing efficiency even at a low temperature such
as room temperature or less.
Other objects and advantages of the present invent
lion will be apparent from the following description.
In accordance with the present invention, there is provided an enzyme-containing detergent composition
comprising at least one surfactant and alkaline protozoa
having the following physicochemical properties:
I (1) Activity: Hydrolyzes cozen under
alkaline conditions, with an optimum pi range of
-
10 to 11, and has optimum temperature range for activity
of 45C to 50C; and
:
I Enzyme molecule: The protozoa is protein
having a molecular weight of about 22000 as estimated by
a gel filtration method, an isoelectric point of 7.4 as
estimated by an electrofocusing method, an absorption
'?~
peak of 275 to 282 no in an ultraviolet (US) absorption
I, ;,,,
J
.
~L239358
-- 3 --
spectrum, infrared (IT) absorption characteristics as
shown in Figure 5, and a shrine residue in the active
site thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood
from the description set forth below with reference to
the accompanying drawings, in which:
Fig. 1 graphically illustrates the optimum pi
range of the enzyme used in the present invention;
Fig. 2 graphically illustrates the stability,
against phi of the enzyme without substrate used in the
present invention;
Fig. 3 graphically illustrates the active
temperature range and optimum temperature of to enzyme
I used in the present invention;
Figs. PA and 4B graphically illustrate the
stabilities, against temperatures, of the enzyme without
substrate used in the present invention at oh's of 10
- and 8, respectively; and
Fig. 5 illustrates the Fourier Transform
Infrared Spectrum of the enzyme used in the present
invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Alkaline protozoa, APE, which is compounded in
I the detergent compositions according to the present
invention, is a novel enzyme derived from a novel
bacterium belonging to genus Bacillus spy nova NKS-21
(referred to as NKS-21 hereinbelow).
`:
3L~3935~3
-- 4 --
The inventor studied bacteria which can produce
alkaline protozoa having a high activity at a relatively
low temperature. As a result of the classification and
search of numerous strains of bacteria isolated from
natural soil, the novel bacterium NKS-21 which can
produce alkaline protozoa APE was isolated from the
natural soils at Fishiest, Tokyo, Japan. It has been
found from the microbiological properties thereof that a
strain NKS-21 capable of producing alkaline protozoa
APE is a novel aerobic sporangium bacterium belonging
to genus Bacillus. The inventor has applied a patent on
this novel alkaline protozoa and preparation method
thereof (Japanese Unexamined Patent Publication (Cook)
No. 58-134990; published on August 11, 1983). This
bacterium NKS-21 has been deposited since February 3,
1982 in the Fermentation Research Institute (FRY) in
Japan as Bacillus spy FORM BP-93 under the Budapest
Treaty on the International Recognition of the Deposit
of Microorganisms for the Purposes of Patent Procedure.
The microorganisms usable in the cultivation are not
limited to NKS-21, but the microorganisms including
natural or artificial mutants of NKS-21 can also be used
as long as they can produce alkaline protozoa APE.
These microorganisms are included within the scope of
I the present invention.
The novel alkaline protozoa APE obtained from
the cultivation of NKS-21 according to the present
invention will now be explained in detail.
. -it
,
1`:
h
.
~2~9358
-- 5 --
NKS-21 can be aerobically cultured, for example,
in a petunia culture medium having a pi of 8 to 10 by
a shaking culture or aerobic agitating culture after
inoculation of NKS-21 into the culture medium at a
temperature of 15C to 40C for 40 to 150 hours. After
the completion of cultivation, the cultivated microbial
cells are readily separated from the cultured product
and the clear cultivation supernatant solution can be
obtained. The alkaline protozoa APE is readily
precipitated by the addition of an organic solvent such
as ethanol to the supernatant solution. The precipitated
alkaline protozoa APE is subjected to centrifugal
separation and is freeze-dried in vacua. Thus, the
desired enzyme, alkaline protozoa APE, can be
obtained.
The activity of the alkaline protozoa APE thus
obtained is determined as follows.
The cultivated clear solution or the enzyme solution
is appropriately diluted with 0.1 M sodium carbonate
-0.1 M boric acid-potassium chloride buffer solution
(pi 10.0). To 0.5 ml of the resultant solution, 0.5 ml
of I milk-casein solution having a pi of 10.0 is added
and, then, an enzymatic reaction is carried out at 30C
for 10 to 30 minutes. Two milliliters of 0.1 M
Jo I trichloroacetic acid containing 0.2 M acetic cold
- 0.2 M sodium acetate is added to the reaction mixture
to terminate the reaction. The mixture is allowed to
stand for lo minutes or more at 30C and, then, is
. .
~39358
filtered through filter paper. Five milliliters of
0.4 M sodium carbonate and l ml of five times diluted
"phenol reagent" are added to l ml of the filtrate
obtained above, and the mixture is allowed to stand at
30C for 20 minutes to develop color. Thereafter, the
absorbency of the colored mixture is measured at 660 no.
The unit of enzyme is determined according to "Commission
on Biochemical Nomenclature" (Comprehensive Biochemistry
Vow 13, P26-27, 1973), in which the amount of alkaline
lo protozoa which liberates trichloroacetic acid-soluble
substances exhibiting color development at 660 no
corresponding to l mow of Tarzan from lo cozen
substrate having a pi of 10.0 at 30C for l second is
defined as "l Catalina.
The physicochemical properties of the alkaline
protozoa APE (referred to as the present enzyme
hereinbelow) will now be described in detail.
(l) Activity
I; v Hydrolyzes proteins such as cozen, hemoglobin,
Jo albumin, globulin, meat protein, fish protein, and
soybean protein.
(2) Effect of pi on Enzymatic Activity
As shown in Fig. 1, optimum pi is about lo to
about if. The relative activity was determined as
I follows.
Each buffer solution (OHS ml) listed below and
` containing I milk cozen was added to 50 Al of the
present enzyme solution to prepare a test solution.
I"
,
,
-- 7
pi Buffer solution
6-9 0.1 M potassium phosphate - 0.05 M
sodium borate
9-11 0.1 M sodium carbonate - 0.1 M boric
acid - potassium chloride
11-12 0.1 M disodium hydrogen phosphate -
sodium hydroxide
The enzymatic activity of the test solution
was determined in the manner as described above. The
relative activity (%) was calculated, in which the
enzymatic activity at a pi of 10.0 was defined as 100
(3) Effect of pi on Stability without Substrate
As shown in Fig. 2, the present enzyme is
stable in the range of pi of about 7 to about 11.5.
it The stability of the present enzyme was
evaluated as follows.
Each buffer solution (0.5 ml) listed below was
added to 50 I of the present enzyme solution dialyzed
and adequately diluted and the mixture was incubated at
2û 30C for lo minutes.
pi Buffer solution
3-8 Al M citric acid - û.2 M disodium
hydrogen phosphate
8-11 0.1 M sodium carbonate - 0.1 M boric
acid - potassium chloride
11-12 û.15 M disodium hydrogen phosphate -
sodium hydroxide
;
I,
,
93~
After incubating, 9.5 ml of a 0.1 M carbonate
buffer solution containing I cozen and having a pi of
10.0 was added to the mixture. The enzymatic activity
of the mixture was determined in the manner as described
above. The relative activity (%) was calculated, in
which the enzymatic activity at a pi of 10.0 was defined
as 100%.
As shown in Fig. 2, the present enzyme was
most stable at a pi of about 10 to about 11. In this pi
lug region, the original activity of the present enzyme was
completely retained. Furthermore, 80% or more of the
remaining activity was exhibited at a pi of 7 to 11.5.
(4) Effect of Temperature on Enzymatic Activity
As shown in Fig. 3, the present enzyme was
active against cozen at a temperature of 5 to 65C and
at a pi of 10Ø The optimum temperature was 45 to 50C.
(5) Effect of Temperature on Stability without
Substrate
As shown in Figs. PA and 4B, the activity of
the present enzyme is completely retained up to 40C
upon heating without substrate at a pi of 10.0 for
10 minutes, but most of the activity was lost at 50C.
Furthermore, the activity of the present enzyme is
retained up to 45C at a pi of 8 for 10 minutes, but
the deactivation occurred at 50C and the enzyme was
substantially deactivated at 60C.
(6) Stability on Storing
The present enzyme is stable against freezing
,,
~2~3~8
g
and freeze-drying. No substantial decrease in the
activity of the freeze-dried sample was observed when
the sample was allowed to stand at room temperature for
2 weeks. When the freeze-dried sample was stored in a
desiccator at room temperature, the activity of the
present enzyme was completely retained.
(7) Inhibition
The present enzyme was remarkably inhibited by
an active shrine residue inhibitor such as diisopropyl-
fluorophosphoric acid (DIP) and phenylmethanesulfonylfluoride (PMSF). It was also inhibited by benzyloxy-
carbonyl-phenylalanine-chloromethyl kitten (ZPCX).
However, the present enzyme was not inhibited by
tosyl-phenylalanine-chloromethyl kitten (TUCK), which is
it an inhibitor of an animal shrine protozoa, chymotrypsin,
and tosyl-lysine-chloromethyl kitten (TUCK), which is a
trypsin inhibitor.
Furthermore, the activity of the present enzyme
was not adversely affected by the addition of p-chloro-
I mercury bonniest (PCMB) and ethylenediaminetetraacetate
(ETA). In addition, the activity of the present enzymes not adversely affected by the addition of pepstatin.
As a result of the above-mentioned inhibition tests, it
is clear that the present enzyme is a protozoa having a
shrine residue in the active site.
(8) Determination Method of Enzymatic Activity
As mentioned above
(9) Molecular Weight
~L~3~3~3
-- 10 --
The molecular weight of the present enzyme is
about 22000 as estimated by a gel filtration over
Sephadex (Trademark) G-75 at a pi of 10.
(10) Isoelectric Point
The isoelectric point of the present enzyme
obtained from active fractions having a molecular weight
of about 22,000 as estimated above of a gel filtration
over Sephadex trademark) G-75 at a pi of 10 is 7.4 as
estimated by an electrofocusing method.
(11) Ultraviolet US Absorption
The absorption peak is present at 275 to 282 no
in a US absorption spectrum.
(12) Infrared (IT) Absorption Spectrum
As shown in Fig. 5.
it (13) Elemental Analysis
Omitted by the reason that the characteristic
differences in these kind of high molecular weight
substances such as the present enzyme cannot be well
manifested by the comparison of the contents of elements
2û thereof such as carbon, nitrogen or hydrogen.
(14) Amino Acid Composition
Amino acid composition of APE calculated
tentatively on the basis of the results by acid
hydrolysis of the enzyme except for cysteine/cystine
and tryptophan, while by per formic acid decomposition
for cysteine/cystine analysis and by alkaline decompo-
session for tryptophan analysis, according to the method
described in the literature methods in En zymology"
, -
I
volume XI, 1967. Academic Press, New York.) were
shown in Table 1, with the compositions of other
alkaline pro teases, whose data were cited from the
literature 1, 2).
As shown in Table 1, there are great
differences between APE and other enzymes cited
in amino acid compositions such as shrine, arginine,
Lawson, valise, ala nine, tryptophan, praline, and so on.
:~39~5~3
- 12 -
Table 1
Cb~parison of Amino Acid Compositions between
APE and Other Alkaline Pro teases
Amino Acid APE subtilisin*l) subtillsm~
Lawson 4 11 9
Histidine 8 6 5
Arginine 8 2 4
Tryptophan 0 3
AsFartic Acadia 11 9
/Asparagine 17 19
Threonine 10 13 19
Shrine 17 37 32
Glutamic Acadia 4 5
guillotine 11 7
Praline 7 14 9
Gleason 30 33 35
Ala nine 26 37 41
Valise 20 30 31
Methionine 4 5 5
Isoleucine 10 13 10
Lawson 14 15 16
Tarzan 12 10 13
Phenylalanine 4 3 4
Sistine 0
_
*l) J. Blot. Chum., 243,5296 (1968)
*2) J. Blot. Chum., 243,2134 (1968)
:~239~58
- 13 -
As is clear from the above-mentioned results, the
present enzyme, which has an optimum pi of 10 to 11,
retains the activity at a relatively low temperature,
but is deactivated at a relatively high temperature
(i.e., 50C or more).
The present enzyme APE can be used with a
detergent builder and thus it can be useful as a
detergent additive, since it is stable at a pi lo 7
to 11.5 and the maximum enzymatic activity is attained
at a pi of 10 to 11 as mentioned above. Especially, the
present enzyme retains its high activity even at a
relatively low temperature (e.g., 20C to 30C), as
compared with conventional enzymes for a detergent.
Consequently, the present enzyme is a most desirable
it type of alkaline protozoa as an additive for a household
detergent in such a country where clothes are washed at
room temperature as Japan.
In the case where the present enzyme APE is
incorporated into a detergent composition, the enzyme
mu APE is generally used in an amount of 5 to 500 natal
(natal = 10 9 Natal), desirably 10 to 100 natal based
on 1 g of the detergent composition, although there is
no limitation in the compounding amount of the enzyme.
; In addition to the enzyme APE, the enzyme-
containing detergent composition according to the
present invention can contain any conventional detergent
ingredient without changing the compounding amount
thereof in conventional detergent compositions. Typical
:,;~....
~239;3~
- 14 -
examples of such detergent ingredients are lo to 50
by weight, preferably lo to 25% by weight, of sun-
fact ant, I to 50% by weight, preferably 10~ to 30~
by weight, of a builder, lo to 50% by weight, preferably
I to 30% by weight, of an alkaline agent or inorganic
electrolyte, and 0.1% to 5% by weight of ingredient(s)
such as an anti-redeposition agent, enzyme(s), a
bleaching agent, an optical (or fluorescent) dye, a
caking preventing agent, and an antioxidant.
lo The surfactants usable in the present enzyme-con-
twining detergent compositions are those conventionally
compounded into detergent compositions. Examples of
such surfactants are soaps; aliphatic sulfates such as
linear or branched alkyd or alkenyl sulfates, aside
it sulfates, and alkyd or alkenyl ether sulfates having
linear or branched alkyd or alkenyl groups and having at
least one component of ethylene oxide, propylene oxide,
and battalion oxide added thereto; aliphatic sulfonates
such as alkyd sulfonate, aside sulfonates, dialkylsulfo
, : I succinates, sulfonates of alpha-olefins, vinylidene type
olefins, and internal olefins; aromatic sulfonates such
as linear or branched alkylbenzene sulfonates; alkyd or
alkenyl ether carboxylates or asides having linear or
branched alkyd or alkenyl groups and having at least one
component of ethylene oxide, propylene oxide, and
battalion oxide added thereto; alpha-sulfo fatty acid
salts or esters; amino acid type surfactants; phosphate
type surfactants such as alkyd or alkenyl acid phosphoric
'I '
~935~3
- 15 -
acid esters, alkyd or alkenyl phosphoric acid esters,
and alkyd or alkenyl phosphoric acid ester salts;
sulfonic acid type ampholitic surfactants; button type
ampholitic surfactants; alkyd or alkenyl ethers or
alcohols having linear or branched alkyd or alkenyl
groups and having at least one component of ethylene
oxide, propylene oxide, and battalion oxide added thereto;
polyoxyethylene alkylphenyl ether having linear or
branched alkyd groups and having at least one component
of ethylene oxide, propylene oxide, and battalion oxide
added thereto; higher fatty acid alkanol asides, or
alkyleneoxide addition product thereof; sucrose fatty
acid esters; fatty acid glycerine monstrous; alkyd or
alkenyl amine oxides; and tetraalkyl ammonium salt type
cation type surfactants. Typical examples of counter
ions of the anionic surfactants are sodium ion and
potassium ion. These surfactants can be used alone or
in any mixtures thereof.
Examples of the builders are phosphates such as
I orthophosphates, pyrophosphates, tripolyphosphates,
methaphosphates, hexamethaphosphates, and phytinic
acid phosphoric acid salts such as the salts of
ethane-l,l-diphosphonic acid, ethane-1,2-triphosphonic
acid, ethane-l-hydroxy-1,1-diphosphonic acid and the
derivatives thereof, ethanehydroxy-1,1,2-triphosphonic
acid, ethane-1,2-dicarboxy-1,2-diphosphonic acid,
methanehydroxyphosphonic acid; phosphono carbolic acid
salts such as the salts of 2-phosphonobutane-1,2-
! . r
- 16 ~3935~
dicarboxylic acid l-phosphonobutane-2,3,4-tricarboxylic
acid, and alpha-methyphosphono succinic acid amino acid
salts such as the salts of aspartic acid and glutamic
acid; amino polyacetates such as neutral triacetates,
ethylenediamine tetraacetates, and diethylene thiamine
pentaacetates; polymer electrolytes such as polyacrylic
acid, polyitaconic acid, polymaleic acid, malefic
android copolymer, and carboxymethyl cellulose salts;
non-dissociative polymers such as polyethylene glycol
and polyvinyl alcohol; carboxymethylates of, for example,
diglycolic acid, oxydisuccinic acid, carboxymethyloxy
succinic acid, citric acid, lactic acid, tartaric acid,
sucrose, and lactose; carboxymethylates of punter-
throttle carboxymethylates of gluconic acid; organic
acid salts such as the salts of Bunsen polycarboxylic
acid, oxalic acid, mafia acid, oxydisuccinic acid, and
gluconic acid; and alumina silicates such as zealots.
Examples of alkaline agents or inorganic electrolytes
are the alkali metal salts of inorganic acid such as
carbonates, sesquicarbonates, sulfates, halides, and
methasilicates. Furthermore, triethanolamine, diethanol-
amine, monoethanolamine, triisopropanolamine, and
similar amine can also be used as an organic alkaline
agent.
Other ingredients such as anti-redeposition agents
(e.g., polyethylene glycol, polyvinyl alcohol, polyvinyl
pyrrolidone, and carboxymethyl cellulose), bleaching
agents (e.g., sodium per carbonate and sodium perorate),
~:~
- 171Z3~8
optical dyes (e.g., bis(triazinylamino)stilbene dip
sulfonate derivatives, and cumarin derivatives),
caking preventing agents (e.g., p-toluenesulfonate
salt, and talc), and antioxidant (e.g., tert-butyl-
hydroxytoluene, and 1,1'-bis-(4-hydroxyphenyl)
cyclohexane) can be optionally used in the present
enzyme-containing detergent composition.
The alkaline protozoa APE can be compounded
into the enzyme-containing detergent composition
according to the present invention in any conventional
method. However, the compounding in the form of fine
powder particles is not desirable from the viewpoints
of safety and sanitation for detergent users and workers
in detergent industries because of dusting during the
handling of detergent compositions. Accordingly, the
alkaline protozoa is preferably used in the form of a
solution or is preferably granulated in such a form
that dust is suppressed. The granulator for this
granulation can be any of the known types of mixing
granulators, drum granulators, pan granulators,
extrusion granulators, fluidizing granulators,
centrifugal fluidizing granulators or modifications
of these granulators. However, it should be noted that
the granulation or formation of the alkaline protozoa
APE compounded into the present enzyme-containing
detergent composition is not limited to the methods
described above.
EXAMPLE
,..'
.
~3~58
- 18 -
The present invention will be further illustrated
by, but is by no means limited to, the following
examples, in which all percentages are expressed on
a weight basis unless otherwise specified.
Example 1
The alkaline protozoa APE and, as a comparative
example, a commercially available alkaline protozoa for
a detergent were evaluated by compounding the same into
various detergent compositions. The laundering test of
blood soiled fabrics was carried out as follows:
A. Preparation of Whole Blood Soiled Cotton
Fabrics
Sample fabrics were dipped, under the following
conditions, in freshly collected bovine whole blood
containing 1 volume, per 9 volume of the blood, of a 3.8%
solution of sodium citrate for preventing coagulation.
The soiled sample fabrics were squeezed at 70% by means
of a mangle to obtain uniformly soiled fabrics.
Thereafter, the sample fabrics were air dried in a room
out of direct sunlight and, then, were stored at a
temperature of 0C to 5C in a cold dark place. After
the preparation, sample fabrics were cut into pieces of
a size of 10 cm x 5 cm.
Sample fabrics: 10 cm x 15 cm
Soiling liquid: 100 ml per 10 sample
fabrics
Soiling temperature: 10 + 2C
.
,,
:12~ it
-- 19 --
Soiling time: 5 minutes (agitation
direction was changed in
30 seconds each
B. Washing Procedure
The sample fabrics were washed and rinsed
(three times) under the following conditions in a
Terg-O-Tometer.
Washing Conditions
Soiled fabrics: 5 cm x 10 cm
Detergent: 0.133% (0.7 nKat/ml enzyme)
Bath ratio: 1:85
Agitation: 105 rum
Washing time: 10 min.
Rinsing Conditions
Soiled fabrics: 5 cm x 10 cm
Bath ratio: 1:85
Agitation: 105 rum
Rinsing time: 3 min.
After rinsing, the sample fabrics were air
dried in a room without being subjected to direct
sunlight.
C. Detergent and Enzyme Used
The following six phosphate and non-phosphate
detergents were used as the detergent.
3~5~3
- 20 -
Composition of Detergent
. phosphate Non-phosphate
Composltlondetergentdetergent
Surfactant 15% 15%
Sodium tripolyphosphate 17
- Zealot - 17
Sodium silicate 5 5
Sodium carbonate 3 3
Carboxymethylcellulose
Water 8 8
Sodium sulfate balance balance
The pi of each detergent measured in a 0.133%
concentration at 25C was as follows:
OH of Detergent
pi of Surfactant Phosphate Non-pho plate
LAS type detergent 1 10.18 10.18
SDS type detergent 2 9.76 9.86
ASS type detergent 9.95 10.17
.
*l) LAS: Sodium line æ alkylbenzenesulfonate
*2) SDS: Sodium dodecylsulfate
*3) AS: Alpha-olefin sulfonate
123~
- 21 -
The enzyme used was a commercially available
alkaline protozoa for a detergent composition or the
alkaline protozoa APE. The results are shown in
Table 2.
The detergent efficiency of protein soil was
determined as follows:
eat sample liquids were obtained by
thermally extracting soiled fabrics before and after
washing with 100 ml of a 0.1 N aqueous Noah solution at
it a temperature of 90 + 2C for 120 minutes. The test
sample liquid was chemically colored with a copper-Folin
reagent and the absorbency thereof was measured at a
wavelength of 750 no. The detergent efficiency (D) was
calculated from the following equation:
D (%1 = Do DOW x 100
wherein Do = Absorbency of extract from standard cotton
fabrics (1 g)
I: Do = Absorbency of extract from soiled fabrics
(1 g) before washing
Do = Absorbency of extract from soiled fabrics
. (1 g) after washing
':
,:
~39;~
-- 22 --
' O D 0 0 0
o co ox Jo a o
_ 'I
'I
It o co o a ox o o o I`
H lo D a O --i I 0 I I
co ox ox ox Jo co co a a
I
I I a o o co 0,
l D O o ox
n o us
'I I
'
. a) a
,
~X39~S8
- 23 -
Example 2
The washing test procedure of Example l was
repeated, except that the protozoa activity in the
washing solution and the washing temperature were
changed in the case of the LAS type non-phosphate
detergent containing the alkaline protozoa APE.
The results are shown in Table 3.
Table detergent Efficiency !%)
.. . . .. .. _ _
Protozoa Activity Washing Temperature
in Washing Solution
nKat/ml) 10C 25C 4ûC
0 68.8 72.0 74.8
0.7 78.2 87.8 89.4
2.1 80.5 88.6 92.8
3.5 80.8 89.4 92.8
7 82.0 89.8 94.3
2û Example 3
The laundering tests of soiled cotton collar
fabrics were carried out by using various detergents
containing the alkaline protozoa APE or a common-
Shelley available alkaline protozoa for a detergent
composition as follows:
A. Preparation of Soiled Collar Fabrics
According to a method described in Motor
: Managua and Ikuko Okamoto "Sweeney Sweeney Shea Kagaku",
239~8
- 24 -
_ , 106-115 (1978), two hundred pieces of soiled collar
fabrics made of standard cotton fabrics for detergency
test and having a size of 7 cm x 37 cm were cut into
small pieces having a size of 1 cm x 5 cm. The collar
fabrics used were worn for a week prior to being cut as
above. Any ten small pieces were sewn to form test
fabrics having a size of 5 cm x 10 cm and were stored at
a temperature of 0C to 5C in a cold dark place.
B. Washing Procedure
The sample fabrics were washed and rinsed
(three times) under the following conditions in a
Terg-O-Tometer.
Washing Conditions
Soiled fabrics: 5 cm x 10 cm
Detergent:- 0.133% (0.7 nKat/ml
enzyme)
Bath ratio: 1:85
Agitation: 105 rum
Washing time: 10 min.
Rinsing Conditions
Soiled fabrics: 5 cm x 10 cm
Bath ratio: 1:85
Agitation: 105 rum
Rinsing time: 3 min.
After rinsing, the sample fabrics were
air dried in a room out of direct sunlight.
C. Detergent and Enzyme Used
The following four phosphate and non-
-
,;.. I.. ,~.. , ... - -
,: ;
I
-- 25 --
phosphate detergents were used as the detergent.
Position of Detergent
.
Cc~npositionPhosphate N~n-phosphate
detergent detergent
Surfactant 15% 15%
Sodium tripolyphosphate 17
Zealot I - 17
Sodium silicate 5 5
Sodium carbonate 3 3
Carbo~r~ethylcellulose
Water 8 8
Sodium sulfate balance balance
'
; The pi of each detergent measured in a
15 0.133% concentration at 25C was as follows:
pi of Detergent
.
:
Type of Surfactant Phosphate Non-phosphate
Detergent Detergent
LO type detergent 1 10.18 10.18
AS type detergent 2 9.95 10.17
*l) ITS: Sodium linear aLlcylbenzenesulfonate
*2) Alps: Alpha-olefin sulfonate
..
3513
- 26 -
The enzyme used was a commercially
available alkaline protozoa for a detergent composition
or the alkaline protozoa APE. The results are shown
in Table 4 below.
,,, ., . . ` `: -
I 8
_ 27 --
'1 0 N 1~1 H Jo 1` 0 r
I ox ox- 1` 00 CO I` I` CO I 0 a ox a
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