U.S. patent application number 09/995321 was filed with the patent office on 2003-02-20 for automated mechanical stress assay for screening cleaning ingredients.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Bechmann, Georg Rudolf Theobald, Cooremans, Steven Paul Georges, Godskesen, Michael, Kjaerulff, Soren.
Application Number | 20030035757 09/995321 |
Document ID | / |
Family ID | 27222463 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030035757 |
Kind Code |
A1 |
Cooremans, Steven Paul Georges ;
et al. |
February 20, 2003 |
Automated mechanical stress assay for screening cleaning
ingredients
Abstract
A method for testing cleaning effect of a compound or
compositions is disclosed. The method comprises: (a) Preparing a
liquid sample of less than 10 ml comprising the test compound, (b)
applying liquid sample to a stained surface, (c) applying
mechanical stress to the stained surface by contacting it with a
body present in the liquid sample, (d) evaluating the cleaning
effect of applying solution and mechanical stress on the stained
surface.
Inventors: |
Cooremans, Steven Paul Georges;
(Buggenhout, BE) ; Bechmann, Georg Rudolf Theobald;
(Wezembeek-Oppem, BE) ; Godskesen, Michael;
(Vedbaek, DK) ; Kjaerulff, Soren; (Vanlose,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27222463 |
Appl. No.: |
09/995321 |
Filed: |
November 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60257068 |
Dec 20, 2000 |
|
|
|
Current U.S.
Class: |
422/82.05 ;
436/164; 73/60.11; 8/137 |
Current CPC
Class: |
G01N 33/34 20130101;
G01N 3/32 20130101; G01N 33/38 20130101; G01N 3/08 20130101; G01N
2013/0275 20130101; G01N 33/44 20130101; G01N 13/00 20130101; G01N
19/00 20130101; G01N 33/46 20130101 |
Class at
Publication: |
422/82.05 ;
436/164; 73/60.11; 8/137 |
International
Class: |
G01N 021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2000 |
DK |
PA 2000 01781 |
Claims
1. A method for testing the cleaning effect of a compound or
compositions containing said compound, said method comprising: (a)
preparing a liquid sample of less than 10 ml comprising said
compound, (b) applying said liquid sample to a stained surface, (c)
applying mechanical stress to said stained surface by contacting
said stained surface with a body present in said liquid sample, (d)
evaluating the cleaning effect of applying solution and mechanical
stress on said stained surface.
2. The method of claim 1, wherein the test compound is selected
from the group consisting of enzymes, enzyme stabilizers, enzyme
inhibitors, enzyme enhancers, enzyme co-factors, builders, builder
systems, bleach systems, bleach activators, metal-containing bleach
catalyst, optical brighteners, nonionic-, anionic-, cationic-,
zwitterionic and amphoteric surfactants, fabric softening agents,
softening clays, clay flocculants, dye-transfer inhibiting agents,
polymeric soil release agents, clay soil removal agents, anti-soil
redeposition agents, polymeric dispersing systems, chelating
agents, alkoxylated polycarboxylates, perfumes, perfume systems,
carrier systems, dyes and pigments, fabric care agents and color
care agents.
3. The method of claim 2, wherein the enzymes are alkaline.
4. The method of claim 1, wherein the liquid sample has a volume
selected from 5-95% of the volume of 3.7 ml, 320 .mu.l, 160 .mu.l,
and 14 .mu.l, respectively.
5. The method of claim 1, wherein the surface is an inorganic
surface selected from metal, ceramic, glass, enamel concrete, rock,
marble, gypsum and composite combinations thereof or an organic
surface selected from plastic, rubber, wood, paper, leather, fur,
paint and fabric.
6. The method of claim 5, wherein the surface is a fabric.
7. The method of claim 6, wherein the fabric is made from natural
plant fibers, animal based fibres or synthetic fibres or
combinations thereof.
8. The method of claim 7, wherein the fabric is woven or
non-woven.
9. The method of claim 8, wherein the fabric is a cellulose
containing fabric selected from textiles and tissues or an animal
based fabric.
10. The method of claim 1, wherein the stain comprises a traceable
compound or composition associated to the surface.
11. The method of claim 10, wherein the stain is a traceable
compound or composition associated to the surface.
12. The method of claim 10, wherein the traceable compound is
selected from light absorbing dyes, fluorescent dyes, radioactive
compounds, reactive compounds and catalysts or activators capable
of performing measurable interaction with substrates.
13. The method of claim 10, wherein the traceable compound is
comprised in a particulate composition, preferably carbon particles
or iron oxide particles.
14. The method of claim 10, wherein the traceable compound is in a
soiling composition.
15. The method of claim 14, wherein the soiling composition is
selected from naturally occurring soiling and processed soilings
thereof.
16. The method of claim 15, wherein the soiling composition is a
natural occurring soiling selected from grass, mud, clay, coffee,
tea, blood, egg, lard and moulds.
17. The method of claim 15, wherein the soiling composition is a
processed naturally occurring soiling selected from butter,
processed meat, dyed lard, oil, make up, spice blends, processed
tomatoes (ketchup or puree), chocolate, ice cream, cacao, baby
food, refined protein compositions, refined polysaccharide
compositions, refined fatty acid compositions, refined triglyceride
compositions.
18. The method of claim 1, wherein the body comprises a metal, more
preferably a ferromagnetic metal, more preferably iron or alloys
thereof.
19. The method of claim 18, wherein the body has a surface
comprising at least one edge or corner.
20. The method of claim 1, wherein the stained surface is placed at
the top or the bottom of the container.
21. The method of claim 20, wherein the stained surface functions
as a cover on an opening in the container.
22. The method of claim 1, wherein the mechanical stress is applied
by moving the body against the surface.
23. The method of claim 22, wherein the liquid sample applied to
the stained surface by moving the body against the surface and
depositing liquid sample adhering to the moving body.
24. The method of claim 23, wherein the body is moved by repeatedly
applying a force to the body.
25. The method of claim 24, wherein the force is an oscillating
force selected from randomly oscillating force and periodically
oscillating force.
26. The method of claim 24 and 25, wherein the force is selected
from magnetic force, electromagnetic force, electrical force,
mechanical force and combinations thereof.
27. The method of claim 26, wherein the force is a magnetic force
applied to a magnetizable body by moving a magnet relative to the
container containing the body.
28. The method of claim 26, wherein the force is a mechanical
vibration force applied to an assembly comprising body, container
and stained fabric.
29. A device suitable for testing cleaning effect of a composition,
said device comprising: (a) at least one container having a volume
of less than 10 ml, preferably less than 2 ml, most preferably less
than 0.2 ml, (b) at least one body capable of moving inside the
container, (c) at least one stained surface, preferably a stained
fabric and (d) means for providing movement of the body relatively
to the stained surface.
30. An assembly suitable for use in the device of claim 29
comprising at least one container and a stained coherent fabric,
wherein the container comprises at least one opening covered with
the stained coherent fabric.
31. The assembly of claim 30, comprising an array of containers,
wherein each container comprises one or more openings and wherein
the stained coherent fabric covers at least one of the openings in
each container.
32. The assembly of claim 31, further comprises least one body in
each container.
33. The assembly of claim 31, wherein the array of containers is a
micro plate selected from 24, 96, 384 or 1536 well micro
plates.
34. The assembly of claim 30, wherein the stained coherent fabric
have a dimension within 0.2-10 cm by 0.2-15 cm, preferably a
dimension enabling the stained fabric to cover all the wells in the
micro plate.
35. The assembly of claim 30, further comprising a support lid
located in the assembly so that the coherent stained fabric is
positioned between the support lid and the container opening.
36. The assembly of claim 31, wherein the interconnecting surface
between containers in an array and the surface of the support lid
facing the fabric and the container opening are unparallel.
37. The assembly of claim 30, further comprising means for
providing mechanical stress to the stained fabric.
38. The assembly of claim 37, wherein the means for providing
mechanical stress comprises a piston-cylinder constructions, which
by movement of the piston cylinder constructions confers
vibrational force to the container.
39. The assembly of claim 37, wherein the means for providing
mechanical stress comprises an electro engine connected to the
container, the engine spinning a mass element, wherein the mass is
heterogeneously distributed around the spinning axis whereby the
spinning of the mass cause a repetitive displacement of mass
conferring vibrational force to the container.
40. The assembly of claim 37, wherein the means for providing
mechanical stress comprises a permanent magnet and means for
providing movement of the magnet relative to a magnetizable body
comprised in the container.
41. Use of a coherent stained fabric as cover on an array of at
least two, preferably at least 24, more preferably at least 96
containers for testing cleaning ingredients.
42. Use of the assembly of any of the claims 30-40 for testing
cleaning ingredients.
43. A method for testing cleaning effect of a non-cellulolytic
enzymes comprising: (a) Preparing liquid samples comprising the
non-cellulolytic enzyme in an assembly according to any of the
claims 30-40, with the proviso that the container does not contain
a solid body capable of moving inside the container, (b) repeatedly
applying liquid sample to the stained fabric, (c) evaluating the
cleaning effect of applying solution on the stained fabric.
44. The assembly of claim 40, wherein the assembly comprises: (a) a
rotate able horizontal support surface mounted on a vertical axis,
said surface comprising means for fastening the container at a
position different from the rotational centre and (b) a permanent
magnet connected to the axis, enabling variation in the magnetic
field applied to the container upon rotating the support.
45. An assembly comprising at least one engine capable of spinning
at least one heterogeneously distributed mass, said engine rigidly
connected to a rigid holder plate for holding an array of
containers and said engine connected to a rigid lid plate via a
first set of flexible elements and wherein the holder plate and the
lid plate forms a slit for positioning an array of containers and
wherein said holder plate is connected via a second set of flexible
elements to a base construction, said second set of flexible
elements allowing the vibrational movements of the holder plate,
the lid plate, the engine and the first set of flexible
elements.
46. The assembly of claim 45 further comprising means for
compressing the first set of flexible elements enabling insertion
or removal of an array of containers.
47. The assembly of claim 45 further comprising means for
controlling the rotational speed of the engine.
48. The assembly of claim 45 further comprising means for
controlling the temperature of the holder plate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims, under 35 U.S.C. 119, priority of
Danish application no. PA 2000 01781, filed Nov. 27, 2000, and the
benefit of U.S. provisional application No. 60/257,068 filed Dec.
20, 2000, the contents of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to an automated assay for testing
real application performance of cleaning ingredients by testing
them on mechanically stressed surfaces. The invention also relates
to surfaces and other devices adapted for application in the
automated assay and to cleaning ingredient identified by the assay
and subsequently produced.
BACKGROUND OF THE INVENTION
[0003] It has been the object for researchers in the detergent
industry to continuously search for new ingredients for use in
cleaning compositions, which improves the cleaning effects of these
compositions. In the field of surface cleaning, such as textiles of
fabrics most people believe, that the flow of a liquid cleaning
solution also called the washing liquor against and/or through a
surface is sufficient for achieving a good cleaning process and
accordingly that such conditions are suitable when the cleaning
effect of new cleaning ingredients and/or compositions are
evaluated to identify improvements.
[0004] Various artificial methods has over time been developed to
provide tools for simulating a cleaning process and for testing
cleaning effects. However using artificial methods often results in
the finding of promising cleaning ingredients or compositions,
which despite the predictions from the artificial methods does not
provide the expected improvement when applied to real cleaning
processes. Accordingly, in order to test and make reliable
predictions on the cleaning effect of new ingredients or
compositions, the skilled person has had adopt full scale cleaning
processes and real surfaces, such as cleaning fabrics in a washing
machine to provide useful results.
[0005] WO 99/34011 describes a method for assaying wash performance
of new enzymes and/or detergent formulations.
[0006] The published Danish patent application PA 1997 00507
describes an assay for analyzing cellulolytic detergent
enzymes.
SUMMARY OF THE INVENTION
[0007] Our research has shown that, besides contact between washing
liquor and a fabric, the mechanical stress, which in real life
applications is conferred to the fabric, is also of major
importance. However, tests using full scale washing methods are
difficult to automate and has a very limited sampling capacity
rendering these method expensive and laborious. Moreover, some
effects in a cleaning process may be very difficult or even
impossible to assess using full scale washing methods. The present
invention enables us of remedying these limitations by
providing:
[0008] A method for testing cleaning effect of a compound or
compositions thereof said method comprising:
[0009] (a) Preparing a liquid sample of less than 10 ml comprising
the test compound,
[0010] (b) applying liquid sample to a stained surface,
[0011] (c) applying mechanical stress to the stained surface by
contacting it with a body present in the liquid sample,
[0012] (d) evaluating the cleaning effect of applying solution and
mechanical stress on the stained surface.
[0013] As this method is particularly suitable for automated
testing of small volume samples, the invention also relates to a
device developed for testing cleaning effect of a composition, said
device comprising:
[0014] (a) at least one container having a volume of less than 10
ml, preferably less than 2 ml, most preferably less than 0.2
ml,
[0015] (b) at least one body capable of moving inside the
container,
[0016] (c) at least one stained surface and
[0017] (d) means for providing movement of the body relatively to
the stained surface.
[0018] The invention in accordance with these aspects provides
means easily automated for testing of cleaning compositions, means
which, through the use of small test samples less than 10 ml,
preferably less than 2 ml, most preferably less than 0.2 ml, offers
a large sampling capacity and through the element of mechanical
stress offers simulation of the cleaning process comparable to real
life or full scale cleaning processes.
BRIEF DESCRIPTION OF THE TABLES AND DRAWING
[0019] FIG. 1 is a drawing showing the principle of using a
permanent magnet for providing mechanical force to a stained
fabric.
[0020] FIG. 2, shows the results of testing effect of a
xyloglucanase enzyme on a stained fabric in a test method using a
permanent magnet for providing mechanical force to a stained
fabric.
[0021] FIG. 3 is drawings showing cross sections of different types
of assemblies suitable for use in testing effects of cleaning
ingredients.
[0022] FIG. 4 shows a cross section an assemble comprising a micro
plate fitted with a lid and a stained fabric, the assembly being
suitable for use in testing effects of cleaning ingredients.
[0023] FIG. 5 shows an assembly such as given in FIG. 4, mounted on
a device for generating mechanical force.
[0024] FIG. 6 shows the results of testing different protease
enzymes in a commercial 96 well test plate with a single coherent
stained fabric covering all well openings of the commercial plate,
the test plate and stained fabric mounted on the device of FIG. 5
without addition of a movable body to the wells.
[0025] FIG. 7 shows the results of testing different protease
enzymes in the a 96 well custom made test plate assembly according
to FIG. 4 with a single coherent stained fabric covering all well
openings of the plate, the test plate and stained fabric mounted on
the device of FIG. 5 without addition of a movable body to the
wells.
[0026] FIG. 8 shows the cleaning performance of 23 different
pectate lyases and concentration 0.1 ppm (A) and 1 ppm (B) when
tested using the method and system of the invention in terms of
light reflected from stained fabrics cleaned with the pectate
lyases.
[0027] FIG. 9 shows the cleaning performance of 4 different hard
surface cleaning products (3 commercial and 1 experimental)
measured using conventional techniques (B) and the using the method
and system of the present invention (A).
[0028] FIG. 10 shows a schematic cross sections of an assembled
device particularly suitable for carrying out the method of the
invention.
[0029] FIG. 11 shows the difference in cleaning performance of a
commercial detergent when dissolved in soft water (0 mM
Ca.sup.2+/Mg.sup.2+) and in hard water (10 mM Ca.sup.2+/Mg.sup.2+)
against different grass stains (A-F) impregnated on fabric when
tested using the method and system of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The Method
[0031] One object of the invention is to provide a method for
testing the cleaning effect of compounds on a stained surface using
only small sample volumes less than 10 ml while, but which
simulates and is comparable to real cleaning processes performed
e.g. in a washing machine. One challenge in developing such a
method is the implementation of the mechanical stress factor in a
small sample volume system. It is to be understood that the system
is a collection of functional members or tools used to carry out
the method of the invention such as containers, stained surfaces,
bodies or samples, and/or means for sealing, temperature control,
pressure control etc. as described, infra.
[0032] The method we have developed, simulating full scale cleaning
of surfaces, is easily automated and it is possible and even
feasible to employ conventional testing vessels e.g. arrays of
small containers, such as wells of a micro plate. The method and
devices developed for performing the method allows to simulate
cleaning processes on surfaces like fabrics at a micro scale even
using extremely small sample volumes such as below 50 .mu.l. The
evaluation of cleaning effect of compounds such as new enzymes, can
be done using this method at a 50.000 to 500.000 times smaller
quantities than it is required for a full scale cleaning process.
Basically the method provides a predictive system with the
capability to be incorporated in high throughput facilities as it
is established in the pharmaceutical industry. This new tool can
therefore accelerate dramatically the development effort of new and
improved cleaning compounds and/or compositions by providing the
capability to test ingredients at high numbers and of low
quantities.
[0033] The Test Compound
[0034] The purpose of the present invention is to provide means of
identifying compounds or compositions thereof providing improved
cleaning of a stained surface.
[0035] The compound or compositions thereof in the context of the
invention is preferably selected from compounds known to be
incorporated in detergent compositions such as enzymes and/or
enzyme stabilizers, inhibitors, enhancers, co-factors, builders,
builder systems, bleach systems, bleach activators,
metal-containing bleach catalyst, optical brighteners, nonionic-,
anionic-, cationic-, zwitterionic and amphoteric surfactants,
fabric softening agents, softening clays, clay flocculants,
dye-transfer inhibiting agents, polymeric soil release agents, clay
soil removal agents, anti-soil redeposition agents, polymeric
dispersing systems, chelating agents, alkoxylated polycarboxylates,
perfumes, perfume systems, carrier systems, dyes and pigments,
fabric care agents, color care agents and the like. Further, also
compounds which are present in the water used to form a washing
liquor may contain relevant test compounds. Such compounds include
dissolved salts, such as salts of Ca and/or Mg which will determine
the hardness of the water, or salts of carbonate, nitrate,
chloride, sulfate and/or phosphate.
[0036] Typical enzymes used in fabric care and cleaning products
are proteases, lipases, amylases, cellulose hydrolyzing enzymes
such as cellulases, carbohydrases such as mannanases or
pectatlyases, transferases, oxidoreductases and like, preferably
the enzymes are alkaline enzymes characterized by having their
catalytic optimums between pH 7-12, more preferably high alkaline
having their catalytic optimums between pH 9-12.
[0037] The Liquid Sample
[0038] In the present invention a liquid sample is prepared
comprising the compound and/or the composition to be tested. The
sample is preferably an aqueous solution or dispersion of the test
compound or composition, but it may also be non-aqueous of nature,
which may be relevant when testing compounds suitable for use in
non-aqueous cleaning liquids.
[0039] In a preferred embodiment the liquid sample is prepared by
first preparing a base solution and/or dispersion and transferring
predefined amounts hereof to a test vessel or container. The
compound or composition may then be added to the vessels in minute
amounts, preferably in solution. In accordance with the invention,
the liquid sample thus prepared have a volume of less than 10 ml,
preferably less than 2 ml, most preferably less than 0.2 ml, but
the method works with considerably less volume, which is feasible
to increase the sample capacity. Accordingly the sample volume is
comparable to the well volume of commercially available micro
plates. A suitable volume is less than the well volume of a 24 well
micro plate, preferably less than the volume of a well on a 96, 384
or 1536 well plate. Based on the type of micro plate, the volume
can be chosen between 5-95% of the volume of the well, which is 3.7
ml, 320 .mu.l, 160 .mu.l, and 14 .mu.l, respectively.
[0040] The Surface
[0041] Surface materials relevant to be employed in the present
invention are materials used in real life, which are subjected to
cleaning processes. Such materials may be inorganic such as metal,
ceramic, glass, enamel concrete, rock, marble, gypsum or composite
combinations thereof. The material may also be mainly organic by
nature such as plastic, rubber, wood, paper, leather, fur, paint or
fabric. A preferred surface is a fabric. The fabric may be any
fabric made from natural plant fibers, animal based fibres or
synthetic fibres or combinations thereof. The fabric may be woven
or non-woven or soft or stiff. Most preferred fabrics are cellulose
containing fabrics such textiles (woven) and tissues (non woven)
and animal based fabrics such as wool.
[0042] A stained surface may be achieved by employing any
conventional staining techniques associating a stain comprising a
traceable compound or composition to the surface and/or associating
the traceable compound or composition itself to the surface.
[0043] Suitable traceable compounds includes dyes such as light
absorbing or fluorescent dye, radioactive compounds, reactive
compounds, such a catalysts and/or activators capable of performing
measurable interaction with substrates. Such compound may be
associated directly or indirectly to the surface by covalent
bonding, ionic bonding and/or hydrogen bonding.
[0044] The surface may also be stained with a particulate
composition such carbon particles, e.g. carbon black or iron
oxides.
[0045] In a preferred embodiment the surface is stained by a
soiling composition comprising the traceable compound or
composition. Such soiling composition is preferably a naturally
occurring soiling such as grass, mud, clay, coffee, tea, blood,
egg, lard, moulds (damp stained) or the composition may made of
processed naturally occurring soiling, such as butter, processed
meat, dyed lard, oil, make up, spice blends, processed tomatoes
(ketchup or puree), chocolate, ice cream, cacao, baby food and the
like. The soiling may also preferably be a man made composition
comprising compounds selected from refined protein compositions,
refined polysaccharide compositions, refined fatty acid
compositions, refined triglyceride compositions or other refined
biological or non-biological compounds.
[0046] Most fabrics are stained by applying the staining material
as it is or as an aqueous solution onto the fabric surface by
soaking, brushing and/or spraying. The stained fabrics will be
dried before testing. There is also a group of colourless
materials, which bind soil and particles onto surfaces.
Carbohydrates like Guaran Gum, Locust Bean Gum and starch and the
like are belonging to this group of materials, which are attracting
soil materials. One example of preparing stain with such materials
is as follows:
[0047] 1) Dissolving the soil binding material Guar Gum in a
aqueous solution at a level of 0.1 to 1%.
[0048] 2) Applying the solution onto the fabric surface by soaking,
brushing, or spraying onto the surface.
[0049] 3) Removal of the access gum material by washing the coated
fabrics in a commercial washing machine (Miele, short cycle,
40.degree. C.) without detergent.
[0050] 4) Staining of the coated fabrics in a commercial washing
machine (Miele, short cycle, 40.degree. C.) by adding 0.1 to 1% of
clay, carbon black, iron oxide and/or other pigments/particular
soiling materials into the wash machine without detergent.
[0051] Fabric surfaces having a range of different stainings are
commercially available under the trade name EMPA.RTM. swatches
marketed by EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St.
Gallen, Switzerland.
[0052] The Body
[0053] For applying mechanical stress to the surface the present
invention requires a body present in the liquid sample comprising
the test compound. It is necessary that the body has a size and
shape which allows the body to go into the container holding the
liquid sample, so that the body is present in the liquid sample.
Preferably the body has a size and a shape, which allows the body
to move in multiple directions within the container holding the
sample and the body. The size and shape of the body should also
preferably allow the body to be submersed in the sample.
[0054] The body is made of a material, which is preferably
insoluble in the solution. The body is preferably rigid, but it may
also be flexible and it may have a smooth or a rough surface. It is
important that the body material is predominately inert, i.e. that
the body does not chemically interact with the sample or vice
versa. Accordingly the body material is made from durable
materials. Such materials may be inorganic such as metal, ceramic,
glass, enamel concrete, rock, marble, gypsum or composite
combinations thereof or organic such as plastic, rubber or
combinations thereof. Preferably the material is sufficiently
ductile to prevent breakage of the body upon collision with the
stained surface or container walls. The body may also be any
suitable material coated with said inert and ductile material.
[0055] In a preferred embodiment the body comprises a metal, more
preferably a ferromagnetic metal such as iron or alloys thereof or
a composite comprising such metals or alloys. To inhibit corrosion
of metals, the body is preferably coated with a layer of inert
material such as organic polymers, separating the metal(s) from any
outer corrosive media.
[0056] In this case the body may be any suitable nut, bolt, screw,
bed or ball, preferred bodies have a surface comprising at least
one edge or corner like a rectangular body.
[0057] Any shape like spherical, round-flat, disk shape, cube
shape, elongated cube shape, cylindrical round shape, cylindrical
rectangular shape, star shape, hexagonal shape, and like is
suitable which fulfils the requirements for size and shape to fit
into the test container.
[0058] The choice of body material is important for the choice of
method for transferring kinetic energy (movement) to the body. The
size, shape, mass and surface texture is important for the type and
amount of mechanical stress the body is capable of conferring to
the stained surface. Depending on the type of compound to be tested
excellent results have been obtained with 3-5 rectangular stainless
steel bodies (3.times.1.6.times.1.9 mm) per container in a 96 well
plate or stainless steel balls or beads having a diameter of 3 mm
or less.
[0059] Application of Liquid Sample and Mechanical Stress to the
Stained Surface
[0060] In the method of the invention the stained surface must be
placed where the body and the sample solution has access to the
stained surface. Having the sample and the body in a container the
stained surface may be placed in any position from the bottom to
the top of the container. Preferably the stained surface is placed
at the top or the bottom of the container and more preferably the
stained surface functions as a cover on an opening in the
container.
[0061] When the stained surface is placed below sample level in the
container (submersed), the application of the liquid sample to the
stained surface is continuous, while when the stained surface is
placed above sample level in the container application of the
liquid sample to the surface depends on movement of liquid sample
relative to the stained surface to allow liquid to contact the
surface. This may be achieved by movement of the liquid, movement
of the stained surface a combination thereof.
[0062] The mechanical stress may be conferred to the stained
surface by providing movement of the body relative to the stained
surface, which allows the body to collide with the stained surface.
This may be achieved by movement of the body, movement of the
stained surface a combination thereof.
[0063] In the preferred embodiment of the stained surface acting as
a lid on top of the container, the application of sample liquid and
mechanical stress to the stained surface may advantageously be
combined. In this case the mechanical stress may be provided to the
to the stained surface by moving the body and allowing it to
collide with the surface. However, as the body moves from having
contact with the liquid sample, e.g. by being submersed therein,
some sample liquid will adhere to the body and when the body
collide with the stained surface some of liquid sample adhering to
the body will be deposited or applied on the surface. Further,
repeating movement and collision between surface and body will
provide exchange of previously deposited sample and fresh sample,
so that changes in the sample liquid deposited on the stained
surface, e.g. as a result of interaction between stain and sample
liquid, will be equilibrating with un-deposited sample liquid.
Accordingly, by employing this system application of both liquid
sample and mechanical stress may be achieved by moving the body.
This system further solves problems related to having the stained
surface present inside the container, e.g. submersed in the liquid
sample. Firstly, such submersed surfaces has to be cut into very
small pieces to fit into the containers encompassed by the
invention, which may be a tedious procedure, secondly, a submersed
surface may interfere with movement of the body, as stained surface
and body may get entangled with each other inhibiting or changing
the movement pattern, third, the mechanical stress conferred to the
stained surface by collision with the body may occur in an
irregular and non-reproducible manner, introducing increased levels
of noise into the evaluation of the cleaning effect, fourth, if
evaluation of the cleaning effect is includes analyzing the stained
surface this process is difficult to automate because each stained
surface is to handled individually. The second, third and fourth
problem may, however, be somewhat remedied by attaching the stained
surface to the bottom of the container, which may be feasible if
the method for evaluating the cleaning effect of the liquid samples
allows such a setup, e.g. if measurement can be made on the liquid
sample or on the stained surface through the bottom wall of the
container.
[0064] Movement of liquid sample, the body and/or the stained
surface may be provided for by employing a force, preferably an
oscillating force. The term oscillating as used in this context
means that the force is following a directional and amplitudical
repetition scheme, preferably a periodical pattern, in amplitude
and direction. As an example an applied force may repeatedly act
first upwards and then downwards on the body (or any other element
of the invention) and/or it may act first towards one side and the
towards the opposite side and/or from front to back. The force
might be applied in a regular, periodic oscillating manner or
completely in random, non-periodic irregular manner. An oscillating
force also encompasses combinations of these directional forces,
i.e. in altering force patterns such as applying upward and
downwards force for defined period of time followed by applying
side wards forces for period of time followed again by up and
downwards forces and so on. By applying an oscillating force the
collisions between body and stained surface are repeated many times
conferring significant mechanical stress to the stained
surface.
[0065] Various types of forces may be employed to achieve movement
of the liquid sample, the body and/or the stained surface.
[0066] The oscillating force may be mechanical of nature e.g. by
applying external movement to all or selected parts of the system,
which subsequently confers movement to the liquid sample, the body
and/or the stained surface. This may be achieved by subjecting the
system or parts of the system to shaking and/or vibration, e.g. by
vibrating the container holding the liquid sample, the body and the
stained surface. Mechanical forces includes subjecting the system
or parts of the system to motion waves such as sound waves,
preferably of higher frequency such as ultra sonic waves.
[0067] Other suitable forces are forces capable of acting on
selected system elements having special properties. Such forces
include magnetic, electromagnetic, electrostatic and/or electrical
forces. Magnetic or electromagnetic forces for example act only on
magnetizable materials such as ferromagnetic materials. Accordingly
it is possible to employ forces, which acts only on selected
elements of the system. One advantage of applying these types of
forces is that these forces act on susceptible elements though
force fields, which may act on susceptible materials over distance
penetrating non-susceptible materials. Accordingly it is possible
the apply force to a system element, such as the susceptible body,
e.g. through a non-susceptible container wall. As an example a
magnetic force field will act on an ferromagnetic containing body
through the wall of a plastic, glass or non-ferromagnetic metal
container.
[0068] In a preferred embodiment the force includes at least one
force field selected from magnetic field, electromagnetic field,
electrostatic field and electric field.
[0069] In a further preferred embodiment is preferred to employ a
force acting on and moving the body only whereby, as described
supra, both application of liquid sample and mechanical stress to
the stained surface may be achieved independent of the position of
the surface. More preferably the force includes a magnetic or
electromagnetic field and the body comprises a magnetically
susceptible material, such as a ferromagnetic material. It is to be
understood that in this embodiment, the force may also act on
components of the liquid sample on a molecular level, such as ions
or it may act on fixed members of the system elements, but it does
not directly induce movement of the liquid sample, the stained
surface, the container or other elements (such as tables, container
holder etc.) used to carry out the method of the invention.
[0070] The present invention also encompasses employment of
combinations of different force types such as combination of
mechanical force and magnetic force fields.
[0071] In a most preferred embodiment the force is applied to a
magnetizable body by moving a magnet relatively to the container
containing the body, thus altering the magnetic field affecting the
body or the force is a mechanical vibration force applied to an
assembly comprising body, container and stained fabric.
[0072] A suitable system consists of a micro plate and a source of
field force on top of the plate. A modification is an additional
force field at the bottom of micro plate.
[0073] Evaluation of Cleaning Effect
[0074] Evaluation of the cleaning effect of a sample liquid may be
achieved by analyzing the stained surface, the liquid sample or
both after subjecting the stained surface to the liquid sample and
the mechanical stress. A suitable choice of method will depend on
the type of stain, which is used.
[0075] It may be feasible to measure changes of either surface or
the liquid sample or both. Various methods are known to the art and
includes optical methods, optical methods combined with coupled
reaction between staining compounds and indicators, enzyme assays,
physical measurements of altered surface properties chromatographic
or fluorimetric methods, spectroscopic methods, radioactive
labeling methods, immunochemical methods such as ELISA and more. If
the stain is a more or less complex soiling composition it is
usually more feasible to analyse the stained surface e.g. by
measuring the stained surface reflectance of light with suitable
reflectometers or calorimeters.
[0076] The Testing Device
[0077] As described, supra, this invention also relates to a device
suitable for testing cleaning effect of a composition, said device
comprising:
[0078] (a) at least one container having a volume of less than 10
ml, preferably less than 2 ml, most preferably less than 0.2
ml,
[0079] (b) at least one body capable of moving inside the
container,
[0080] (c) at least one stained surface, preferably a stained
fabric and
[0081] (d) means for providing movement of the body relatively to
the stained surface.
[0082] The advantageous features of speed and capacity in testing
compounds provided by the present invention relies to a major
extent on the assembly of equipment making up the testing device.
Accordingly, an important aspect of the invention is the practical
applicability of the stained surface. In order to achieve the
optimal capacity and speed in the present assay it is, as described
supra, preferred to use a stained surface which functions as a lid
on the container in which the test sample is placed. Within this
embodiment it is further preferred that the stained surface, as a
coherent entity, has a dimension, which makes it capable of acting
as a lid on two or more containers simultaneously. More preferably
the stained surface have a dimension, which makes it capable of
acting as a lid on an array or arrays of containers simultaneously
enables. The embodiment of using a stained surface having a
dimension, which makes it capable of acting as a lid on numerous
test containers is very important indeed not only considering test
capacity but also considering the accuracy of the testing. For many
stained surfaces, such as fabrics, there may be variations between
different entities of the same type of surface. For example
variations in the texture and firmness between different parts of a
cloth, from which stained fabric sub-pieces are made, may cause
variations between fabric sub-pieces in stainability and ability to
interact with test compound(s). However, by choosing a test
assembly which requires that the stained fabric has a certain
dimension, which, as a coherent entity, is capable acting as a lid
for numerous test containers, it is secured that the stained fabric
used to test compounds in each individual container has a lowered
variation in texture, firmness and staining, because it, taken as a
coherent entity must originate from the same part or area of e.g.
the cloth from which it is taken. The term "coherent" in this
context is to be understood in the broadest sense, but preferably
that the fabric is in one coherent woven or non woven piece without
seams or stitches or other fastening means, which may make the
fabric surface less uniform.
[0083] Accordingly, the present invention also relates to an
assembly suitable for use in testing effect of cleaning
ingredients, e.g. in the device as described above, which through a
special combination of containers and stained fabric provide for
high speed and capacity as well as good accuracy in testing
cleaning ingredients, such as enzymes. Accordingly, the present
invention provides an assembly comprising at least one container
and a stained coherent fabric, wherein the container comprises at
least one opening covered and/or lidded with the stained coherent
fabric. The invention also encompasses an assembly comprising a
container and a stained coherent fabric, wherein the container
comprises two oppositely positioned openings and at least one,
preferably both of said openings are covered and/or lidded with a
stained coherent fabric. In one preferred embodiment the assembly
further comprises least one body in each container for providing
mechanical stress to the stained fabric, as described supra,
preferably within the container. In another preferred embodiment
the assembly comprises an array of containers, wherein each
container comprises one or more openings and optionally one or more
bodies and wherein the stained coherent fabric covers and/or lids
at least one of the openings in each container. In a further
preferred embodiment the array of containers is a micro plate
comprising 24, 96, 384 or 1536 containers or wells and the stained
coherent fabric have a preferred dimension within 0.2-10 cm by
0.2-15 cm, preferably a dimension enabling the stained fabric to
cover all the wells in the micro plate.
[0084] The term "cover/lid" in this context is to be understood in
the sense that the stained fabric covers the opening of a container
by being in contact with the edges of the opening.
[0085] Preferably the assembly comprises an additional support lid
or cover located in the assembly so that the fabric is placed
between the support lid and the container opening. The support lid
is preferably made from a material, which is impermeable to the
liquid sample in the container and has a dimension which makes the
support lid rigid enough to support the fabric and prevent the
fabric from moving away from the opening when subjected to
mechanical force. The assembly also preferably comprise means for
fastening the support lid to the container so that the fabric may
act as a sealing gasket. One important feature in this context is
that the contact zone between the fabric and the edge of the
opening is small so that the pressure holding the fabric in contact
with the container opening, when the support lid is fastened to the
container, is sufficient to seal the container, using the fabric as
a gasket. This is important to prevent or inhibit that liquid
sample from one container contaminates, through capillary movement,
an adjacent container. To improve contact pressure between fabric
and container opening by minimizing the contact zone, the edge of
container openings and interconnecting surface between containers
and the side of the support lid facing the fabric are designed so
that they form a line toward each other instead of a surface. This
can be achieved by designing the support lid and containers so that
the interconnecting surface between containers in an array and the
surface of the support lid facing the fabric are essentially
unparallel, i.e. greater or less than about 180.degree.. Examples
of this design is given e.g. in FIG. 3.
[0086] Also other elements may be added to the assembly, such as
devices for providing mechanical force to the body, the sample, the
fabric or a combination, means for controlling temperature, devices
for measuring properties of the stained fabric or the test sample.
For convenience the assembly comprising container, stained fabric
and optionally a support lid is from hereon referred to as the
"primary assembly" and assemblies comprising additional elements
are referred to as "secondary assemblies". Accordingly the present
invention encompasses any test system comprising primary or
secondary assemblies.
[0087] In one embodiment of the invention a secondary assembly
comprises a device for providing mechanical force, the device
preferably comprises a piston in a cylinder construction for
generating vibration or shaking force, which is transferable to the
stained fabric. Preferably the piston device is connected, via a
rigid plate, suitable for mounting and/or fastening the primary
assembly, to the primary assembly. The piston based device is
preferably driven by a pneumatic or hydraulic system, using
compression and/or expansion of air or liquid to move the piston
relative to the cylinder. In a preferred embodiment the cylinder or
the piston is fastened on the rigid plate at the opposite side
(preferably to bottom side) of the primary assembly and this
secondary assembly is connected to a stationary support
construction via an elastic material capable of moderating movement
of this secondary assembly. In this construction mechanical force
in the form of vibration is transferred to the primary assembly
(and to the stained fabric and movable body within the test
container) by the weight displacement and/or acceleration by which
ever of the cylinder or piston, which is not connected to the rigid
plate. This displacement is achieved by moving the piston or
cylinder relatively to the other elements of the secondary assembly
by means of a hydraulic or pneumatic system. Such devices for
providing mechanical force are commercially available e.g. from
Copenhagen Vibrator Product, Denmark.
[0088] An alternative to the piston-cylinder construction is a
device comprising an engine spinning a mass element, such as a
driving wheel, having a heterogeneous mass distribution, said
device connected to the primary device and to a stationary support
construction via a rigid plate. To moderate movement the rigid
plate may be connected to a stationary support via an elastic
material, such as rubber. When the mass element spins, the
heterogeneous mass distribution will cause repetitive displacement
of mass and will confer vibration to the primary assembly and the
container holding the test sample.
[0089] In a particular embodiment the secondary assembly comprise
the element of the secondary assembly depicted in FIG. 10. Here at
least one engine (1) (preferably two electric engines having
opposite spin directions) is rigidly connected to a rigid plate (5)
for holding a primary assembly. The engine(s) spins one or more
heterogeneous mass elements (2) capable of vibrating the rigid
holder plate (5). A rigid lid plate (4) is connected by flexible
elements (3) (preferably one or more springs) to the engine(s) (1)
and the rigid holder plate (5). Between the lid plate (4) and the
holder plate (5) there is a slit (10) for holding a primary
assembly. The width of the slit (10) is dimensioned so that the
flexible elements has to be compressed in order to insert a primary
assembly and so that the flexible elements (3) will press the lid
plate (4) against the container openings of the primary assembly
once inserted into the slit (10). The elements 1-5 are connected to
a base construction (8) through flexible elements (7) (preferably
one or more springs). The secondary assembly comprise means for
compressing the flexible elements (3) holding the rigid lid plate
(4) when the a primary assembly is to be inserted or removed. These
compressing means may in particular be connected to base
construction (8). The means for compressing the flexible elements
(3) may comprise an engine driven gearbox (9) which e.g. via
threaded bolt (12) and a rigid construction (6) may drive one or
more rods (11), e.g. through holes or bushings in the holder plate
(5) against the lid plate (4) whereby the flexible elements (3) are
compressed. The engine(s) (1) may further be connected to means for
controlling the rotation speed e.g. a frequency controller. Still
further, the secondary assembly may comprise means for regulating
the temperature of a primary assembly placed in the slit (10). In a
particular embodiment the whole secondary assembly in encased in an
insulated casing wherein the temperature is controlled by a
thermostatted air heater. Hence the invention provides in this
embodiment: An assembly comprising at least one engine capable of
spinning at least one heterogeneously distributed mass, said engine
rigidly connected to a rigid holder plate for holding an array of
containers and said engine connected to a rigid lid plate via a
first set of flexible elements and wherein the holder plate and the
lid plate forms a slit for positioning an array of containers and
wherein said holder plate is connected via a second set of flexible
elements to a base construction, said second set of flexible
elements allowing the vibrational movements of the holder plate,
the lid plate, the engine and the first set of flexible
elements.
[0090] In another embodiment the assembly comprises a magnetic
device which comprises a permanent magnet or an electromagnet. In
the case of a permanent magnet such as a Neodymium Iron Boran
magnet, the magnetic device also comprise means for providing
movement of the magnet relative to a magnetizable body comprised in
the container of the primary assembly applying an alternating
magnetic force to a magnetizable body present in the container.
[0091] In a specific embodiment this secondary assembly preferably
is a carousel construction comprising
[0092] (a) a horizontal rotatable support surface comprising means
for fastening, in a position different from the rotational center,
a container containing a movable and magnetizable body and a least
one opening covered with the stained coherent fabric,
[0093] (b) a permanent magnet connected to the axis, enabling
variation in the magnetic field applied to the container upon
rotating the support and
[0094] (c) means for rotating either the magnet, the support
surface or both so as to create a relative movement between magnet
and support surface.
[0095] The magnet is preferably placed in a fixed position so that
the support surface is the only movable part and so that the test
container(s) upon rotation is allowed to come into sufficient
proximity of the magnet so that a magnetic field is applied to the
body in the test container, which is sufficient to move the body
within the container towards the magnet.
[0096] The invention also relates to the use of a coherent stained
fabric as cover and/or lid on an array of at least two, preferably
at least 24, more preferably at least 96 containers having a volume
of less than 10 ml for testing cleaning ingredients.
[0097] Special Method Embodiments
[0098] Although for most enzymes acting directly on the cellulose
in textile, severe mechanical stress of the stained surface is
important, we have also found that when testing cleaning effect of
non cellulolytic enzymes such as protease, lipase amylase and
oxidoreductase on stained fabric, the mechanical stress provided by
repeatedly moving the sample liquid against the fabric surprisingly
gives test results which is very similar to results obtained in
real laundry processes. This may be achieved by shaking or
vibrating containers holding the liquid samples and moving the
liquid against the stained fabric. Combined with the finding that a
coherent stained fabric may be used to test liquid samples in more
than one container simultaneously by using the fabric as a lid and
seal on the containers (see description of the primary assemblies,
supra) these findings provide a powerful tool for automated testing
of these enzymes. Accordingly, the present invention also relates
to a method for testing cleaning effect of a non-cellulolytic
enzymes said method comprising:
[0099] (a) Preparing liquid samples comprising the non-cellulolytic
enzyme in an assembly according to any of the claims 30-40, with
the proviso that the container does not contain a solid body
capable of moving inside the container,
[0100] (b) repeatedly applying liquid sample to the stained
fabric,
[0101] (c) evaluating the cleaning effect of applying solution on
the stained fabric.
[0102] This embodiment of course also encompasses within step (a)
use of all primary assemblies as described, supra for testing
cleaning ingredients.
EXAMPLES
Example 1
Evaluation of Cleaning Effects of Mechanical Stress in a Model
Washing Machine
[0103] Effect of Mechanical Stress on Stain Removal Performance
[0104] Light reflectance values of fabrics having different stains
washed in a Launder-O-Meter at low and at high mechanical
conditions.
[0105] Preparation
[0106] Stained fabrics were obtained from Equest Market Research
Limited, Equest House, Greencroft Industrial Park, Annfield Plain,
Stanley, Co. Durham, DH9 7YB, England, a Launder-O-Meter with 500
ml beakers was obtained from Roaches England LTD, Washtec and a
liquid detergent Ariel Futur was used.
[0107] Test Procedure
[0108] A wash solution of 200 ml city water with 1.3 ml Ariel Futur
liquid detergent and 5 stained fabrics (4.times.4 cm) was added to
each beaker. In beakers wherein low mechanical stress were to be
tested no bodies were added, while in beakers wherein high
mechanical stress were to be tested were added 10 Teflon coated
star magnets (24 mm diameter) and 30 steel nuts (25 mm diameter).
The stained fabric were washed in the Launder-O-meter for 30
minutes wash at 40.degree. C. with or without.
[0109] Evaluation of Cleaning Effect
[0110] Light reflectance values of the stained fabrics were
measures by a Spectrafash 500 equipment from "datacolor
international".
1 chocolate L-values burnt beef make-up grass/mud ice-cream
original stain 42.9 59.2 54.1 64.1 washed with low 57.4 63.5 72.8
74.1 mechanical stress washed with high 76.1 81.0 84.2 88.1
mechanical stress clean fabric 92.8 92.8 92.8 92.8 noise level
about +/- 1.5 units
[0111] From this experiment it can be observed that mechanical
stress to the stained fabric has a significant effect on the
cleaning effect of the detergent ingredients.
Example 2
[0112] Testing System 1
[0113] A test device was developed according to FIG. 1, wherein 1
is a movable permanent Neodymium Iron Boran magnet
(20.times.2.5.times.1.3 cm) with 12200 Gauss strength; 2=a stained
fabric; 3 are wells of a 96 well micro plate (350 ul volume size);
4=liquid sample; 5 are magnetizable rectangular stainless steel
bodies (3.times.1.6.times.1.9 mm) which by movement of the magnet
and application of magnetic force jumps up, rolls and scrapes
against the stained fabric while simultaneously depositing liquid
sample on the fabric.
Example 3
[0114] Testing Carbohydrase
[0115] The test device of example 2 was used to test a carbohydrate
hydrolyzing enzyme. A knitted cotton fabric coated with tamarind
seed flour and carbon black particles was used as stained fabric,
which was cut to a dimension fitting a 96 well micro plate.
[0116] Preparation
[0117] 150 ul wash liquor containing 1 mg Ariel Futur liquid
detergent and 4 rectangular stainless steel bodies
(3.times.1.6.times.1.9 mm) was added to each well. Every second
array wells also contained 1 ppm of a Xyloglucanase from Bacillus
licheniformis.
[0118] Process
[0119] The micro plate was heated to 40.degree. C. in a water bath
and after assembling the stained fabric to the top of the filled
micro plate, a strong magnet was moved every two seconds over the
stained fabric for 30 minutes.
[0120] Result
[0121] Differences in cleaning effect between wells containing the
enzyme and wells in which the enzyme is absent was visually
detectable as shown in FIG. 2, wherein "0" denotes no enzyme and
"1" denotes presence of 1 ppm of enzyme in the test solution. This
experiment also shows the benefits of using one coherent stained
fabric covering all 96 wells of the micro plate, because although
all 96 results are to be measured from this stained fabric, the
fabric has a size which is much more easily handled in the
measuring procedure, than if 96 individual pieces of stained fabric
were to be measured.
Example 4
Correlation of Performance in Test Method Versus Real Wash
Procedures
[0122] In this example the correlation between real wash
performance and using the method of the invention was evaluated for
two different enzymes.
[0123] Enzyme cleaning effect using the method of the invention was
evaluated as described in example 3 using 1 ppm enzymes levels and
8 replica samples per enzyme. The two different enzymes were A:
Xyloglucanase from Bacillus licheniformis and B: Xyloglucanase from
Bacillus firmus. The cleaning effect was evaluated by measuring the
light reflectance (L-values) of the portions of stained fabric
subjected to the test solutions and the mechanical stress. A
Spectrafash 500 equipment from "Datacolor International" was
used.
[0124] Enzyme cleaning effect using a real washing process was
evaluated by washing Tamarind seed flour/carbon black stained
fabric (5.times.5 cm swatches) in a Miele W715 washing machine, at
40.degree. C., short cycle together with 0.5 kg realistic items.
The same levels and types of liquid detergent and enzyme were used
and for each enzyme 8 replica washes were preformed. The cleaning
effect was evaluated by measuring L-values of the stained fabric
after wash.
[0125] Results
[0126] The results as given in the table below shows and excellent
correlation between using the method and device of the invention
and realistic washing processes
2 L-values of treated stains Enzyme micro plate system (SD) washing
machine (SD) A 62.2 0.7 65.2 1.6 B 57.0 0.5 58.9 0.7 no enzyme 54.3
0.9 50.0 1.5
Example 5
Container Design 1
[0127] A primary assembly suitable for use in testing cleaning
ingredients was prepared as shown in FIG. 3(A), wherein (1) are
wells of a micro plate having connecting parts between wells which
are lowered as compared to the edges of the wells; (2) is a lid
having an essentially plane surface facing the micro plate well;
(3) the contact zone between the lid and the well, wherein the
fabric, when inserted between (1) and (2), is compressed to form a
gasket seal. This assembly provides an excellent seal and greatly
prevents or inhibits the liquid sample from one well to diffuse or
migrate to a neighboring well. This assembly also provides that the
stained fabric surface accessible to liquid sample and mechanical
stress is at least the same as the area of the opening of the
well.
Example 6
Container Design 2
[0128] Another primary assembly suitable for use in testing
cleaning ingredients was prepared as shown in FIG. 3(B), wherein
(1) are wells of a micro plate having connecting parts between
wells which are lowered as compared to the edges of the wells; (2)
is a lid having elevations fitting into depressions formed around
the wells in the micro plate; (3) is the contact zone between the
lid and the micro plate well, wherein the fabric, when inserted
between (1) and (2), is compressed to form a gasket seal. This
assembly provides a slightly poorer sealing effect than the
assembly of example 5, but has the advantage that the micro plate
part of the contact zone is subjected to less wear upon use and has
increased durability. Also the this assembly provides for the
stained fabric surface accessible to liquid sample and mechanical
stress being at least the same as the inner area of the well
opening.
Example 7
Container Design 3
[0129] Another primary assembly suitable for use in testing
cleaning ingredients was prepared as shown in FIG. 3(C), wherein
(1) are wells of a micro plate having connecting parts between
wells which are lowered as compared to the edges of the wells; (2)
is a lid having elevations fitting into the opening of the well
(like a cork) and to depressions formed around the well in the
micro plate; (3) is the contact zone between the lid and the micro
plate well, wherein the fabric, when inserted between (1) and (2),
is compressed to form a gasket seal. This assembly provides a
slightly poorer seal than the assembly of example 5, but as in
example 6 possesses increased durability because the micro plate
part of the contact zone is subjected to less wear upon use.
Example 8
Container Design 4
[0130] Another primary assembly suitable for use in testing
cleaning ingredients was prepared as shown in FIG. 3(D), wherein
(1) are wells of a micro plate having connecting parts between
wells which are heightened as compared to the edges of the wells;
(2) is a lid having elevations fitting into the opening of the
wells (like a cork); (3) is the contact zone between the lid and
the micro plate well, wherein the fabric, when inserted between (1)
and (2), is compressed to form a gasket seal.
Example 9
Container Design 5
[0131] Another primary assembly suitable for use in testing
cleaning ingredients was prepared as shown in FIG. 3E, which is
similar to the assembly of example 6, with exception that the
elevations of the lid forms curves instead of straight tilted
lines. This provides an increased durability.
Example 10
Container Design 6
[0132] An another primary assembly suitable for use in testing
cleaning ingredients was prepared as shown in FIG. 3F, which is
similar to the assembly of example 8, with exception that the
elevations of the lid forms curves instead of straight tilted
lines. This provides an increased durability.
Example 11
Container Design 7
[0133] An another primary assembly suitable for use in testing
cleaning ingredients was prepared as shown in FIG. 4, which is
similar to the assembly of example 6, with the exception that this
assembly was made in the standard format of a 96 well plate with
standard size of wells and distances between wells. Accordingly,
this assembly will fit into standard equipment developed for this
format. (1) is the support lid, (2) is the stained fabric and (3)
is the micro plate.
Example 12
Testing System 2
[0134] A secondary assembly which further comprises a device for
providing mechanical force to the stained fabric was prepared as
shown in FIG. 5. In this secondary assembly a primary assembly of
the type described in example 11 was fastened to a NTK 15 or NTK 25
type pneumatic flask vibrator available from Copenhagen Vibrator
Products, Industrivej 15, 4652 H.ang.rlev, Denmark. This
construction is showed in FIG. 5, wherein the primary assembly (1)
is mounted with bolts (9) on the top side of a rigid plate (2) and
a cylinder (3) is mounted on the down side of the rigid plate (2),
The cylinder (3) surround a piston (4) with mass elements (5),
capable of moving in the longitudinal direction of the cylinder (3)
by applying compressed air to valves or apertures (6) in the
cylinder. This rigid plate (2) is rested on a stationary support
(7) via rubber connectors (8) which is capable of moderating the
vibrating force.
Example 13
Effect of Mechanical Stress
[0135] Five different microbially derived xyloglucanases; A, B, C,
D, E and one cellulase F were tested with (+) and without (-) using
steel beads to provide mechanical stress using the test plate of
example 11 fitted in the assembly of example 12. Each enzyme were
diluted to give a final concentration of {fraction (1/16)}, 1/8,
1/4, 1/2, 1, and 2 ppm in the 96 well plate containing 6.7 g/l
Ariel Color liquid detergent in 15.degree. dH H.sub.2O in a final
volume of 160 .PI.. Eight repeats of each dilutions were carried
out. A fabric stained with tamarind gum and carbon black was used.
Each well in which high mechanical stress were to be tested
contained 3.times.2 mm steel beads, wells wherein low mechanical
stress where to be tested contained no steel beads. Using this
construct the stained fabric was which was washed for 30 min at
26.degree. C. under vibration by the vibrating device. The cleaning
results were evaluated by measuring light reflectance of the
cleaned fabric.
3 Results Enzyme dose Enzyme .mu.g/ml A- A+ B- B+ C- C+ D- D+ E- E+
F- F+ 0 1718 1882 1694 1880 1884 1919 1654 2012 1552 2105 1648 1929
0.0313 1671 2003 1679 2025 1670 2015 1925 1830 1836 2116 1793 1940
0.0625 1745 1992 1859 2215 1620 2046 1650 1970 1746 1977 1706 1924
0.125 1808 2168 1914 2105 1605 1903 1746 2056 1686 1989 1698 1946
0.25 1741 2343 1693 2321 1814 1978 1805 2106 1775 2256 1687 1997
0.5 1862 2524 1867 2381 1799 2224 1794 2186 1607 2312 1714 1848 1
1884 2639 1804 2468 1784 2086 1808 2127 1830 2156 1747 1836 2 1815
2424 1743 2320 1768 2190 1712 2234 1693 2298 1685 1833
[0136] The results shows that for each enzyme treatment with the
steel beads increases removal of stain, similar to results
obtainable for real washing processes. Also a ranking of the
different enzyme efficiency could be was deducted from the results,
rendering enzyme A the most efficient enzyme.
Example 14
Test of Non-cellulolytic Enzymes with No Body to Induce Mechanical
Stress
[0137] In order to show the applicability of the system to other
enzymes a series of proteases were tested using the assembly of
example 12 using both commercial 96 well test plate and the custom
made test plate of example 11. The proteases were mixed in amounts
of 0, 5, 10, 20 and 40 nanomoles per liter to samples of a
commercial OMO color detergent dissolved in 15.degree. dH water
yielding a pH of about 10.4 in the solution. The test samples were
transferred to the wells of the test plates, and all well openings
in each test plate were subsequently covered by one single coherent
EMPA 117 stained test fabric and a lid. The wells of the test plate
did not contain a body for providing mechanical force.
[0138] The test plates were then mounted on the assembly of example
12 and vibrated at 30.degree. C., and after this treatment each
part of the test fabric corresponding to single samples was
evaluated. For both plates a clear indication of samples having a
high protease concentration was observed, so that samples having
higher protease concentrations had removed more stain from the
fabric in an area corresponding to the circumferences of the wells.
However, for the commercial plate it was evident that the test
samples had spread beyond the area of the stained fabric which was
circumferenced by the edge of the well (i.e. the area of the
opening of the well) so that stain removal was also seen outside
the areas of the stained fabric directly exposed to the test
samples. Accordingly, the test samples were leaking from the wells
or migrated through capillary effects to areas of the stained test
fabric not directly covering the opening of the wells. This
phenomenon obscured the results of the test. The results of using a
stained fabric with a commercial 96 well test plate can be seen in
FIG. 6. In FIG. 6 A-D represents different proteases tested at
different concentrations.
[0139] Using the custom made test plate, the test samples were
effectively confined to act only on the area of the stained fabric
covering the opening of the well. Consequently very clear cleaning
results were obtained, where the effect of different concentrations
could easily be identified. Moreover it was possible to visually
determine that protease D had better cleaning performance than
protease A. This result also show that for proteases and other
non-cellulose hydrolyzing enzymes application of mechanical force
by means of a solid body colliding with the stained fabric is not
necessary for these enzymes. Moreover, this method and system
generated results which are comparable to the performance of the
test enzyme under real washing conditions those obtainable with a
real washing procedure.
Example 15
Screening for Pectate Lyases with Improved Stain Release
Properties
[0140] 23 different pectate lysases were tested for their ability
to remove pectate stain from fabric using a test devise which was a
carousel construction comprising
[0141] (a) a horizontal rotatable support disc comprising means for
fastening, in a position different from the rotational centre, of 4
96-well microplates sealed with stained fabric. Each well contained
in addition to liquid sample 5 solid magnetic implements for
providing mechanical stress.
[0142] (b) a fixed permanent magnet which was positioned to enable
passing the microplates under the magnet by rotating the support
disc in sufficient proximity to cause the magnetic implements in
the wells to be attracted by the magnet and to collide with the
stained fabric.
[0143] (c) an electric engine for rotating the support disk at a
constant rate
[0144] Samples of pectate lyases (8 replicas) at two different
concentrations (1 ppm and 0.1 ppm) were mixed, in each well, with
150 .mu.l of a commercial detergent dissolved in city water (2.5
mmol/l hardness). Knitted cotton fabrics impregnated with a citrus
peel based pectate stain was positioned over the microplates
covering all the wells and the fabric was fixed using a lid. A
washing process was now simulated by rotating the support disk at a
constant speed so that the microplates continuously passed closely
under the magnet, whereby the magnetic implements were lifted
towards the magnet thereby colliding with the fabric sealing the
wells. This process was continued for 30 minutes at 40.degree.
C.
[0145] After completing the simulated washing process the light
reflectance of stained fabrics were measured as an indication of
how much of the stain which had been cleaned off by each pectate
lyase. The results were inserted in a plot (FIG. 8), where the
pectate lyases were plotted at the X-axis (numbers 1-23) and the
relative reflectance was plotted at the Y-axis. In the plot higher
relative reflectance means higher cleaning performance of the
pectate lyase. In the plot series A indicates high pectate lyase
concentration in washing liquor (1 ppm) and series B indicates low
concentration of pectate lyase (0.1 ppm).
[0146] The results clearly shows that some pectate lyases are
significantly better than others in removing citrus peel stains.
Pectate lyase 1 is clearly best at low dose while pectate lyase 3
is clearly best at high dose. The results also shows, in
conjunction with examples on the present invention yielding results
very close to those obtained in real washing processes, screening
procedures, involving huge amounts of samples, can be made using
the method of the present invention, which would be extremely
demanding, if not impossible, should the screening have been made
using real washing procedures.
Example 16
Screening for Effects of Water Hardness in Different Washing
Conditions
[0147] This example shows that the methods and systems of the
present invention also is very useful for testing and screening of
compounds present in a washing liquor, which do not possess
cleaning properties in themselves, but which interferes in the
washing process by affecting the action of active cleaning
compounds and that the methods and systems of the present invention
generates result fully comparable with those obtained in a
commercial washing machine.
[0148] In this example it was tested how two different levels of
water hardness (0 mM and 8 mM) affected the washing process at
European washing condition in the method of the invention, at North
American washing conditions in the method of the invention and at
European conditions in a commercial washing machine.
[0149] For the European conditions washing liquors containing 4 g/l
of a commercial European liquid detergent was prepared with water
of low and high hardness.
[0150] For the North American conditions a washing liquor
containing 1.2 g/l of a commercial North American liquid detergent
was prepared with water of low and high hardness.
[0151] 8 different stains were prepared on knitted cotton
fabrics.
[0152] For European conditions the stained fabrics were tested both
by the method of the invention as in example 15 (96 well
microplates in the carousel system) and in a commercial European
washing machine (Miele) at 40.degree. C. and a 30 minutes wash
time. For the North American conditions the stained fabrics were
tested by the method of the invention as in example 15 at
32.degree. C. and a 12 minutes wash time. 12 replicas were made of
each sample to estimate experimental errors.
[0153] The cleaning effect of each sample was evaluated by
measuring the light reflectance of the stained fabric after
cleaning.
[0154] The results showed that under European conditions the method
of the present invention generates the same or very similar stain
removal profiles as obtained in a full-scale European wash
machine.
[0155] The results also showed that use of the present invention
enables quantitative prediction of differences in cleaning
performance between European conditions and North American
conditions. The results showed substantial differences for some
stains between North American versus European conditions. Overall,
the system proved to be fully functional also for screening other
compounds than those directly involved in the cleaning process such
as salts, builders, polymers and the like levels.
Example 17
Screening Cleaning Compositions for Hard Surface Cleaning
[0156] This example shows that the methods and systems of the
present invention also is very useful for testing and screening of
improved compounds or compositions used for cleaning hard surfaces
and that the method and system of the present invention generates
result fully comparable with those obtained in internationally
recognized test methods.
[0157] In this example the cleaning effect of 3 different
commercially available cleaning compositions agents and one new
composition were tested in the methods of the present invention and
the recognized "wet-abrasion-scrub-test" conventionally used for
testing such compositions.
[0158] In the Wet-Abrasion-Scrub Tester four separate sponges with
cleaning composition are scrubbing in a controlled manner over four
soiled hard surfaces. The stain contains a mixture of grease and
particular dirt, which have been heat treated to get a hard,
tenacious soil on the hard surface. The sponges with the cleaning
composition are moving periodically over the soiled hard surfaces
until the tiles are clean and the numbers of strokes are counted.
These numbers are used to calculate the Cleaning Index, which is
always referring to a benchmark or reference product. However this
approach is a very tedious, labor and time intensive procedure.
[0159] For the Wet-Abrasion-Scrub test stainless steel metal plates
were soiled with an oil mix and Carbon Black based soiling. The
cleaning test was executed with four plates at room temperature
(20.degree. C.) in 2 minutes. The strokes were counted and
calculated with "Mr. Proper All Purpose Cleaner" as the
reference.
[0160] For the test according to the present invention stainless
steel metal plates were cut to the size of a 96 well micro-plate
and soiled with an oil mix and Carbon Black based soiling. All 96
wells were filled with 150 ml of the cleaning compositions (14
replicates). A test was conducted as in example 15 at room
temperature (20.degree. C.) in 2 minutes.
[0161] Assessment of the cleaning result was achieved by measuring
light reflectance (via image scan and calibrated against L scale,
wherein the pixel density gave information about stain
removal).
[0162] The results were inserted in a plot (FIG. 9), where the
cleaning compositions were plotted at the X-axis (1=AJAX all
purpose cleaner, 2=Mr. Proper all purpose cleaner, 3=new cleaning
composition and 4=Mr. Proper W3-spray with bleach) and the cleaning
effect was plotted at the Y-axis. In the plot series A indicates
results from the method of the invention and B indicates results
obtained with the wet-abrasion-scrub-test.
[0163] The results clearly shows both systems that some cleaning
compositions are more efficient than others in removing the
selected soiling. The results also shows that results of the method
and system of the present invention are fully comparable to those
of the far more tedious, laborious and time intensive
wet-abrasion-scrub procedure and that the method of the present
invention may advantageously replace the conventional method.
Example 18
Testing Effect of Water Hardness on Cleaning Performance of
Protease Enzymes
[0164] Following example 16 the method of the present invention was
used to evaluate the effect of water hardness in the washing liquor
on the removal of different grass stains impregnated on fabric.
[0165] 5 in-house stained fabric, impregnated with 5 different
grass stains and 2 commercially available grass stained fabric
(EMPA Switzerland) were tested.
[0166] Two washing liquors were prepared by dissolving 4 g/l of a
commercial detergent in either soft water (0 mM
Ca.sup.2+/Mg.sup.2+) or hard water (10 mM Ca.sup.2+/Mg.sup.2+).
[0167] 96 well microplates were filled with 150 ml wash solution
prepared from either hard or soft water in 8 replicates. grass
stained fabrics were fitted to the microplates. The stained fabrics
were treated with the wash solutions for 30 minutes at 40.degree.
C. as in example 15 using the carousel system. Assessment of the
stain removal was done by measuring the light reflectance of the
cleaned fabric by image scanning calibrated against L scale.
[0168] The results were inserted in a plot (FIG. 11), wherein the
fabric having different grass stains were plotted on the X-axis
(A=in house made grass stain 1, B=in house made grass stain 2, =in
house made grass stain 3, D=in house made grass stain 4, E=in house
made grass stain 5, F=commercial grass stain EMPA 164). The
reflectance of cleaned fabric (0 mM hardness) minus reflectance of
cleaned fabric (10 mM hardness) were plotted on the Y-axis. The
results indicates that the water hardness have a tremendous effect
on the cleaning performance of the detergent on grass stains, while
it is different from different stain types. In FIG. 10 higher
values means more lowering of the performance at high water
hardness. Such results easily obtainable by the method and system
of the invention is important when developing improved detergent
compositions.
[0169] Below graph shows the difference in SRI values between 0 and
10 mM water hardness. It seems the stain removal inhibition starts
at a range of 1.5 to 2.5 mM, probably due to the insufficient
masking of free divalent ions above this range. Pictures of some
stains are shown in Appendix 2.
* * * * *