Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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REDUCTION OF MALODOR FROM LAUNDRY
FIELD OF THE INVENTION
The invention relates to enzymatic reduction of malodor from laundry.
BACKGROUND
Clothes that have been used for e.g. sports activities or in other ways have
been exposed to sweat from the wearer during use are often difficult to clean
in terms
of the smell of sweat and other body odors (malodor), when subjected to wash
in a
laundry machine.
SUMMARY OF THE INVENTION
We have found that an enzyme having lysostaphin activity is capable of
reducing malodor from laundry. Accordingly, the invention provides a method
~5 comprising contacting laundry with an enzyme having lysostaphin activity.
In a second aspect, there is provided a composition comprising a surfactant
and
an enzyme having lysostaphin activity.
In another aspect, an enzyme is used for reducing malodor in laundry.
2o DETAILED DESCRIPTION
L sY osta~phin
The enzyme Lysostaphin is a glycyl-glycine endopeptidase from the enzyme
class E.C. 3.4.24.75. It hydrolyses the -Gly-Gly- bond in the polyglycine
inter-peptide
link joining staphylococcal cell wall peptidoglycans. Lysostaphin is
commercially
25 available from several suppliers, such as from Sigma-Aldrich, Inc.
An enzyme having lysostaphin activity may be a natural or synthetic variant of
lysostaphin, wherein amino acid substitutions or deletions have been
introduced. It
may also be an amino acid fragment with lysostaphin activity, which is
optionally
fused to one or more other proteins.
Lysostaphin Activity
One unit of lysostaphin activity will reduce the turbidity (absorbance at 620
nm)
of a suspension of Staphylococcus aureus (ATCC 6538) cells by 50%, when the
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initial absorbance is approximately 0.250, after 10 minutes at pH 7.5 and 37
degrees
celcius.
Malodor
During wear, fabrics are contaminated with microorganisms from the human
skin together with sebaceous lipids, sweat and dead skin cells serving as
microbial
substrates. Particularly at mild wash conditions these microorganisms survive
laundering. A consequence of microorganisms surviving household laundering is
malodor generation in the fabrics. We have now found that these microorganisms
are
~o mainly Staphylococcus species.
The malodor of the method of the invention thus comprises all body odors
present in laundry originating from contact with the human skin. In an
embodiment
the malodor may be axillary odors, such as the smell of sweat.
In another embodiment, the malodor may originate from activity of
Staphylococcus species (such as S, aureus, S. epidermis, S. intermedius, S.
saprophyticus and S. hyicus).
In yet another embodiment the malodor originates from fabrics which have been
in contact with the axilla.
2o Evaluation of Malodor
Wet laundry swatches are placed in tinted 200 ml glasses with snap lids. A
trained sensory panel (10 persons) evaluates the odor by sniffing the
headspace
over the wet items and indicating the total odor intensity. The odor intensity
is
indicated on a scale from 0 to 15 where 0 equals 'no malodor' and 15 equals
'very
2s strong malodor'. All evaluations are performed twice (double
determinations). The
swatches are evaluated after approximately 24 hours and after 48 hours
(swatches
are kept in the glasses at all times). An average of all 20 evaluations (10
persons
each evaluate twice) is calculated after both 24 hours and 48 hours. These
average
values are referred to as "Malodor index (24 hours)" and "Malodor index (48
hours)".
Laundry
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The laundry of the method of the invention comprises all kinds of textile
items or
fabrics suitable for being used as clothes or for personal use in other ways
comprising contact with the human skin.
Methods and Uses
By contacting laundry with an enzyme having lysostaphin activity, as defined
in
the method of the invention, the "Malodor index (48 hours)", as defined above,
may
be reduced by at least 10% (preferably 20%, more preferably 30%, most
preferably
40%, and in particular 50%) compared to laundry which has not been contacted
with
1o an enzyme having lysostaphin activity.
The method of the invention may also result in killing or inhibiting growth of
microbial cells in laundry. In an embodiment the microbial cells are bacteria,
such as
Staphylococcus species. In another embodiment, the method of the invention may
result in a reduction in the number of living microbial cells of at least 25%,
preferably
~5 at least 50%, more preferably at least 90%, and most preferably at least
99%.
In the context of the present invention the term "inhibiting growth of
microbial
cells" is intended to mean that the cells are in the non-growing state, i.e.,
that they
are not propagating. The term "microbial cells" denotes bacterial cells (such
as
Staphylococcus species), fungal cells or algae, and the term "microorganism"
2o denotes a fungus (including yeasts) or a bacterium
The present invention covers use of an enzyme for reducing malodor from
laundry items. In an embodiment, the enzyme may have lysostaphin activity. The
invention may also be used for reducing the number of living bacteria in
laundry; for
reducing allergens in laundry; or for sterilizing laundry.
Detergent composition
Lysostaphin may be added to and thus become a component of a detergent
composition.
The detergent composition of the invention may for example be formulated as a
so hand or machine laundry detergent composition including a laundry additive
composition suitable for pre-treatment of stained fabrics and a rinse added
fabric
softener composition, or be formulated as a detergent composition for use in
general
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household hard surface cleaning operations, or be formulated for hand or
machine
dishwashing operations.
In a specific aspect, the invention provides a detergent additive comprising
lysostaphin. The detergent additive as well as the detergent composition may
s comprise one or more other enzymes such as a protease, a lipase, a cutinase,
an
amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase,
a
galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
In general the properties of the chosen enzymes) should be compatible with
the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic
and non
~o enzymatic ingredients, etc.), and the enzymes) should be present in
effective
amounts.
Proteases: Suitable proteases include those of animal, vegetable or microbial
origin. Microbial origin is preferred. Chemically modified or protein
engineered
mutants are included. The protease may be a serine protease or a metallo
protease,
~5 preferably an alkaline microbial protease or a trypsin-like protease.
Examples of
alkaline proteases are subtilisins, especially those derived from Bacillus,
e.g.,
subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and
subtilisin 168
(described in WO 89/06279). Examples of trypsin-like proteases are trypsin
(e.g. of
porcine or bovine origin) and the Fusarium protease described in WO 89/06270
and
2o W O 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO
98/20115, WO 98/20116, and WO 98/34946, especially the variants with
substitutions in one or more of the following positions: 27, 36, 57, 76, 87,
97, 101,
104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
25 Preferred commercially available protease enzymes include AlcalaseT"",
SavinaseT"", PrimaseT"", EverIaseT"", EsperaseT"", and KannaseT"" (Novozymes
A/S),
MaxataseT"", MaxacalT"", MaxapemTM, ProperaseT"", PurafectT"", Purafect
OxPT"",
FN2T"", and FN3TM (Genencor International Inc.).
Li ases: Suitable lipases include those of bacterial or fungal origin.
Chemically
so modified or protein engineered mutants are included. Examples of useful
lipases
include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa
(T.
lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as
described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P.
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pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB
1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO
96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B.
subtilis
(Dartois et al. (1993), Biochemica et Biophysics Acta, 1131, 253-360), 8.
5 stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249,
WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96!00292, WO
95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO
. 97/07202.
~o Preferred commercially available lipase enzymes include LipolaseTM,
Lipolase
UItraTM and LipoprimeTM (Novozymes A/S).
Amylases: Suitable amylases (a and/or ~3) include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are included.
Amylases
include, for example, a-amylases obtained from Bacillus, e.g. a special strain
of B.
licheniformis, described in more detail in GB 1,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO
94/18314, WO 96/23873, and WO 97/43424, especially the variants with
substitutions in one or more of the following positions: 15, 23, 105, 106,
124, 128,
133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391,
408, and
444.
Commercially available amylases are DuramyITM, TermamyITM, FungamyITM and
BANTM (Novozymes A/S), RapidaseTM and PurastarTM (Genencor International
Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin.
Chemically modified or protein engineered mutants are included. Suitable
cellulases
include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium,
Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola
insolens,
Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307,
US
5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having
colour
so care benefits. Examples of such cellulases are cellulases described in EP 0
495 257,
EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are
cellulase variants such as those described in WO 94/07998, EP 0 531 315, US
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5,457,046, US 5,686,593, US 5,763,254, WO 95124471, WO 98/12307 and
PCT/DK98/00299.
Commercially available cellulases include CelluzymeT"", and CarezymeT""
(Novozymes A/S), ClazinaseT"", and Puradax HAT"" (Genencor International
Inc.),
and KAC-500(B)T"" (Kao Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant,
bacterial or fungal origin. Chemically modified or protein engineered mutants
are
included. Examples of useful peroxidases include peroxidases from Coprinus,
e.g.
from C. cinereus, and variants thereof as those described in WO 93/24618, WO
~0 95/10602, and WO 98/15257.
The detergent enzymes) may be included in a detergent composition by adding
separate additives containing one or more enzymes, or by adding a combined
additive comprising all of these enzymes. A detergent additive of the
invention, i.e. a
separate additive or a combined additive, can be formulated e.g. as a
granulate, a
~5 liquid, a slurry, etc. Preferred detergent additive formulations are
granulates, in
particular non-dusting granulates, liquids, in particular stabilized liquids,
or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991
and 4,661,452 and may optionally be coated by methods known in the art.
Examples
of waxy coating materials are polyethylene oxide) products
(polyethyleneglycol,
2o PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols
having
from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the
alcohol
contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene
oxide
units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of
fatty acids.
Examples of film-forming coating materials suitable for application by fluid
bed
25 techniques are given in GB 1483591. Liquid enzyme preparations may, for
instance,
be stabilized by adding a polyol such as propylene glycol, a sugar or sugar
alcohol,
lactic acid or boric acid according to established methods. Protected enzymes
may
be prepared according to the method disclosed in EP 238,216.
The detergent composition of the invention may be in any convenient form,
e.g.,
so a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid
detergent may be
aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or
non-
aqueous.
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The detergent composition comprises one or more surfactants, which may be
non-ionic including semi-polar and/or anionic and/or cationic and/or
zwitterionic. The
surfactants are typically present at a level of from 0.1 % to 60% by weight.
When included therein the detergent will usually contain from about 1 % to
about
40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha
olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate,
secondary
alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or
alkenylsuccinic acid or
soap.
When included therein the detergent will usually contain from about 0.2% to
1o about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol
ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty
acid
monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid
amide,
or N-acyl N-alkyl derivatives of glucosamine ("glucamides").
The detergent may contain 0-65 % of a detergent builder or complexing agent
~ s such as zeolite, diphosphate, triphosphate, phosphonate, carbonate,
citrate,
nitrilotriacetic acid, ethylenediaminetetraacetic acid,
diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates
(e.g. SKS-6
from Hoechst).
The detergent may comprise one or more polymers. Examples are
2o carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),
polyvinyl
alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates
such as
polyacrylates, maleiclacrylic acid copolymers and lauryl methacrylate/acrylic
acid
copolymers.
The detergent may contain a bleaching system which may comprise a H202
2s source such as perborate or percarbonate which may be combined with a
peracid
forming bleach activator such as tetraacetylethylenediamine or
nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise
peroxyacids of e.g. the amide, imide, or sulfone type.
The enzymes) of the detergent composition of the invention may be stabilized
so using conventional stabilizing agents, e.g., a polyol such as propylene
glycol or
glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid
derivative,
e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4
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formylphenyl boronic acid, and the composition may be formulated as described
in
e.g. WO 92/19709 and WO 92/19708.
The detergent may also contain other conventional detergent ingredients such
as e.g. fabric conditioners including clays, foam boosters, suds suppressors,
anti-
corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes,
bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or
perfumes.
It is at present contemplated that in the detergent compositions any enzyme,
in
particular lysostaphin, may be added in an amount corresponding to 0.01-100 mg
of
enzyme protein per liter of wash liquor, preferably 0.05-10 mg of enzyme
protein per
~o liter of wash liquor, more preferably 0.1-5 mg of enzyme protein per liter
of wash
liquor, and most preferably 0.1-1 mg of enzyme protein per liter of wash
liquor.
Lysostaphin may additionally be incorporated in the detergent formulations
disclosed in WO 97/07202 which is hereby incorporated as reference.
The present invention is further illustrated in the following examples, which
are
not in any way intended to limit the scope of the invention as claimed.
EXAMPLES
The chemicals used in the following examples were commercial products of at
least reagent grade.
The Malthus Flexi M2060 instrument is available from Malthus Instruments
Limited, England.
Tryptone Soya Broth (TSB) is available from Oxoid, England.
2s CFU: Colony Forming Units.
EXAMPLE 1
Evaluation of odour in swatches soiled with human axillary sweat and sebum
The odour reducing effect of lysostaphin is tested in a one-cycle washing
trial carried
so out in a thermostated Terg-O-tometer (available from United States Testing
Co, 1415
Park Ave, Hoboken NJ07030).
Experimental conditions:
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Soiling
Swatches of 100% polyester or 100% cotton (10 x 14 cm, previously cleaned by
solvent extraction - hexane for polyester, chloroform for cotton - using a
Soxhlet) are
soiled by applying human male axillary sweat and sebum from the armpits and
upper
body of male runners after extensive exercise. Two swatches (10 x 14 cm) are
used
for each male - one swatch is made of 100% polyester and one is made of 100%
cotton. The left armpit and left part of the upper body is wiped with one
swatch, and
the right armpit and right part of the upper body is wiped with the other
swatch after
~o exercising. This procedure is performed twice before washing. Prior to the
washing,
each swatch is cut into 12 equally sized pieces and distributed between four
Terg-O-
tometer wash beakers. Cotton and polyester textiles are kept apart.
Washing
Washing is done in Terg-O-tometer using phosphate buffer (0.05 M, pH 7.5).
Temperature: 32°C
Lysostaphin dose: 5 mg/L (Sigma L4402)
Wash time: 12 minutes
Wash liquid: 1000 mL per wash beaker
2o Rinsing: 15 minutes in running tap water
Sensory evaluation
After wash the wet swatches are evaluated and "Malodor index (24 hours)" and
"Malodor index (48 hours)" are calculated. The results show that a lower
"Malodor
index (48 hours)" is obtained with the swatches that has been washed with
lysostaphin compared to those that has not been washed with lysostaphin.
EXAMPLE 2
Removal of bacteria from textile by lysostaphin
3o Staphylococcus aureus (ATCC 6538) was grown overnight (TSB; Tryptone Soya
Broth) and inoculated to approximately 103 Colony forming units/ml (CFU/ml) in
diluted TSB (1:1 ). Sterile cotton swatches were inoculated overnight in the
diluted
TSB allowing S.aureus to grow on the textile. The swatches were rinsed in
sterile
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water for 30 seconds and dried in sterile air for 30 minutes. Swatches were
washed
in a beaker with stirring at 32°C for 20 minutes in either phosphate
buffer (0.05 M, pH
7.5) or in a liquid U.S. detergent of a commercial type (0.75 g/liter) with
and without
addition of 5 mg/L Lysostaphin (Sigma L4402). Three swatches were washed in
each
s beaker. After wash all swatches were rinsed in sterile water for 10 minutes
and dried
in sterile air. The number of living S. aureus on the swatches was determined
by
incubation of the swatches in Malthus tubes with CASO medium (Merck 1.05459).
The bactericidal activity was determined by incubation in Malthus. The
detection
times (dt) measured by the Malthus instrument were converted to CFU/swatch by
a
~o calibration curve (Johansen et al. 1999, Methods in Enzymology, vol 310, p.
353-
360). Direct Malthus measurements were used when enumerating total survival
cells.
By the direct measurements, the cell metabolism was determined by conductance
measurements in the growth substrate. The swatches were after enzyme treatment
transferred to the Malthus cell. As cells attached to the textile are growing,
the cell
~s metabolism will change the conductance in the growth medium. When the con-
ductance change is measurable by the Malthus, a detection time (dt) will be
recorded. The dt's were converted to colony counts by use of a calibration
curve
relating CFUlswatch to dt.
2o Results:
Swatch Beaker Average number of S. aureus
on
each swatch
(three swatches washed in each
beaker)
Reference swatches before 8.8 x 10' CFU/swatch
wash
Washed in phosphate buffer 1 2.2 x 10' CFU/swatch
2 7.3 x 106 CFU/swatch
Washed in phosphate buffer 1 4.6 x 10 CFU/swatch
with
lysostaphin (5 mg/L) 2 1.2 x 105 CFU/swatch
3 4.8 x 105 CFU/swatch
Lysostaphin resulted in a reduction of the microbial cell number of
approximately 10~
to 102 CFU/swatch. A reduction in cell number was also determined after
lysostaphin
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treatment in detergent, however, the determination of the exact cell number
was not
possible by using the Malthus. But a delay in outgrowth of the microorganism
was
observed visually from the swatches washed in detergent with lysostaphin, this
delay
corresponds to a lower cell number on the swatches compared to the swatches
washed without lysostaphin.
