Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR REDUCING ODOR AND BIOFILM
BACKGROUND
Field of the Invention
[0001] This invention pertains to products and methods for treating
clothing,
textiles or other surfaces to reduce the presence of malodor or to reduce
biofilm.
Background / Description of Related Art
[0002] In the laundry, textile, and personal care industries, significant
challenges remain in reducing odor, especially human body odor in fabrics
including synthetic fabrics such as polyester. Persistent odor in polyester
has
been reported by many users, especially those who engage in strenuous exercise
regularly. Many report that malodor, especially in the axillary regions,
returns
quickly after thorough laundering, and sometimes may not be thoroughly removed
by laundering. Such malodor is sometimes called "perma-odor" or "perma-stink."
We have observed, for example, that some sports apparel may continue to have
symptoms of perma-odor even after treatment with dilute bleach or other harsh
agents that normally might be expected to eliminate microbial sources of odor.
Repeated washing is often ineffective, even with advanced commercial laundry
detergents. Many users simply feel they have to discard such infected
clothing.
There is a long-standing need for means to reduce such perma-odor.
[0003] Without wishing to be bound by theory, we propose that the
recalcitrance of perma-odor is akin to the recalcitrance of bacterial
infections
when a microbial biofilm is present. Biofilms are remarkable adaptations of
bacteria and other microbes such as protists and fungi, including yeasts, in
which
polysaccharides, proteins, DNA, and other materials may be used to create a
protective matrix that can prevent antimicrobials or harsh chemical agents
from
penetrating effectively. In a biofilm, microbes, sometimes from more than one
species, share chemical signals and cooperate to create protective materials
that
help secure them on a solid surface and protect them from external threats,
allowing them to reproduce and thrive. However, there has not yet been a
widespread recognition of the role of biofilms in athletic clothing and
ordinary
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attire, ranging from sports gear to casual wear, office wear, and formal
attire,
including socks, shoes, underwear, shirts, pants, bedding, medical attire, and
so
forth, but especially clothing items in contact with portions of the body such
as the
underarms where sweat commonly occurs. Indeed, a common view is that
conventional washing and drying is likely to prevent biofilms from forming.
See, for
example, Erin Williams, "Bacterial Biofilms: Live Chat with Rob Hull," Nappy
Science Gang blog, October 8, 2016. Our explorations, in contrast, suggest
that
biofilms in clothing may provide a stronghold for generation of perma-odor.
[0004] Biofilms in fabrics may present problems in a variety of other areas
also
in need of improved solutions, and the advances disclosed herein may be
helpful
in those areas. For example, sanitation of clothing, medical apparel, linens,
draperies, carpets, upholstery fabrics, protective apparel, towels, rags, wall
coverings, and other fabrics used in medical care facilities and by healthcare
workers is an increasingly serious problem, as it may be in homes, hotels,
senior
care facilities, aircraft, automobiles, buses, trains, aquatic vessels such as
boats
and submarines, laundromats or public or private laundry facilities, airports,
prisons, schools, and institutions of many kinds. The materials and methods
useful in reducing perma-odor therefore may also be applied in other settings
to
mitigate biofilms and enhance cleaning or odor reduction.
SUMMARY
[0005] It has been discovered that perm a-odor in a variety of textiles can
be
significantly mitigated through treatment with the combination of certain
enzymes
with one or more biofilm attack agents such as N-acetyl cysteine, mixtures of
N-
acetyl cysteine with other agents such as panthenol, or other agents described
herein, applied for an effective period of time, followed by or performed
substantially simultaneously with laundering with a laundry detergent,
treatment
with other soaps and detergents, or simply rinsing with water. Enzymes used in
combination with biofilm attack agents in one aspect are provided as an
enzymatic blend comprising surfactants and optional bacterial spores or live
bacteria. The biofilm attack agents such as N-acetyl cysteine are provided in
a
solution, either with the enzymes or in a separate container, or provided at
least
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partially in solid form such as a capsule, a powder, a tablet, a stick, etc.,
to be
dissolved in a solution before, after or during application to the textile
item.
[0006] In describing various versions and aspects of the methods and
products
disclosed herein, it should be understood that the elements, steps, features,
etc.
of any version or aspect are combinable with any other version or aspect or
collection of versions and aspects unless stated otherwise or clearly
unsuitable.
[0007] Thus, in one aspect, a method is provided for treating a solid
material
such as fibrous material including textiles, items of clothing, woven and
nonwoven
materials or combinations thereof, etc., wherein the material is suspected of
having microbial biofilm matter in one or more regions that may be associated
with persistent odor or other symptoms, or used in an environment or
application
at risk of developing biofilm and/or persistent odor, the method comprising
applying an enzymatic composition to the one or more regions of the solid
material, providing suitable time for the enzymatic mixture to attack biofilm,
and
then washing the textile item, wherein the enzymatic composition comprises:
(a)
water, (b) from 5% to 60% of a surfactant, (c) from 1 A to 20% of an enzyme
mixture comprising at least two of lysozyme, proteinase, amylase, mannanase,
lipase, pectinase, DNAse and cellulase; and (d) from 0.1 A to 10% of N-acetyl
cysteine. The enzymatic composition in some aspects is packaged with indicia
instructing a user to wait at least 5, 10, 15, or 30 minutes between applying
the
enzymatic composition and washing the textile, wherein washing generally
comprises washing in water with a laundry detergent but may comprise rinsing
without use of further detergents. In some aspects, the enzymatic composition
further comprises from 0.01% to 8% by weight of bacterial spores adapted to
become active in response to the presence of contaminants selected from at
least
one of proteins, carbohydrates, lipids, and carbohydrates, the spores then
producing enzymes that attack a portion of said contaminants. The spore concen-
tration in the enzymatic composition may be from 1 x 105 to 5 x 1010 CFU/ml.
[0008] The enzymatic composition in one aspects has two portions, a first
portion comprising an enzyme mixture and a second portion comprising N-acetyl
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cysteine (NAC), further associated with indicia instructing a user to apply
both
portions to the one or more regions of the textile item with persistent odor.
The
enzyme mixture may be in a liquid or comprise granulated enzymes to be
combined with liquid prior to application to a textile. Granules may be
combined
with NAC for treating textiles or washing machines, and may also be combined
with any of allantoin, panthenol or a derivative thereof, a catechin, a
chelant, etc.
[0009] In some aspects, the second portion comprising NAC is in liquid or
powder form separate from the enzymes but adapted to be combined with the
enzymes on the clothing, such as by adding the powder or a solution to the
clothing before, after, or while adding the enzyme mixture and other
components
of the composition. The second portion of the enzymatic composition may
comprise from 1 A to 90% N-acetyl cysteine, optionally from 1 A to 10%
panthenol, and sufficient alkaline agents such that when the second portion of
the
enzymatic composition is combined with enough water at pH 7.0 to bring the
concentration of the N-acetyl cysteine to 1 A, that the pH of the resulting
aqueous
mixture is at least 6Ø Such a composition can be effective in reducing the
amount of biofilm matter present or the surface area with biofilm matter
present in
the textile item, and is also effective in reducing malodor in the textile
item, while
also being substantially free of non-enzymatic bleaching agents.
[0010] Some aspects also comprise visualizing the presence of suspected
biofilm matter using UV light. In such cases, the textile item with malodor
may
have been treated with a suitable dye that fluoresces in UV light to identify
one or
more regions that show relatively high fluorescence in UV light, wherein the
enzymatic composition is applied to at least one of the one or more regions
that
show relatively high fluorescence. Such a dye may comprise any known optical
brightener such as Calcofluor White.
[0011] Through extensive experimental work, possible solutions to reduce or
eliminate perma-odor have been found using materials that are generally safe
and
suitable for use in consumer products and in some cases may even be known as
edible dietary supplements or components of natural edible products, rather
than
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harsh compounds or restricted pharmaceutical compounds. Application of the
pretreatment followed by laundering can be effective in reducing perma-odor in
clothing, and the effect is believed to be achieved at least in part by
undermining
biofilm material that may exist in articles with perma-odor.
[0012] The pretreatment can be applied with a liquid medium that is
sprayed,
poured, wiped, daubed, rolled on, or otherwise transferred to articles of
clothing,
particularly to regions suspected of having malodor. The liquid medium may be
provided to the user in ready-to-use form, or may be provided as a concentrate
such as a liquid, slurry, paste, or solid such as a powder that can be
prepared by
the user through addition of water or through the mixing of two or more
components to create the ready-to-use composition. The pretreatment may
involve comprise two or more physically distinct compositions.
[0013] In one aspect, evidence for the existence of a biofilm is considered
by
the user in applying the pretreatment. Thus, a method of detecting and
mitigating
a microbial infection in an article of clothing comprises: 1) exposing an item
of
clothing one or more times to a solution comprising at least 0.001% of one or
more fluorescent optical brighteners such as Calcofluor White (an optical
brightener believed to be present in many common laundry detergents), 2)
shining UV light on the item of clothing to determine if there is preferential
absorption of optical brighteners in a region of the clothing, 3) treating the
region
with preferential absorption of optical brighteners with an effective amount
of an
enzymatic mixture comprising an effective amount of bacterial spores, one or
more surfactants, and a mix of at least three laundry enzymes, 4) allowing the
enzymatic mixture to reside on the clothing for an effective time, and then 5)
washing the clothing to remove the enzymatic mixture, wherein the treatment
results in reduced fluorescence and/or perma-odor. In a related aspect, the
item
of clothing is also treated with a biofilm attack agent such as NAC and
panthenol.
[0014] In one aspect, the enzymatic mixture is provided as a liquid
concentrate
comprising: (1) surfactant at a concentration of 10% to 55%, 15% to 45%, or
from
15% to 35%, such non-ionic surfactants derived from plant carbohydrates and
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from plant oils such as coconut and palm oil; (2) a mixture of at least 3, 4,
5, or 6
more different classes of enzymes, such as a mix of protease, cellulase,
amylase,
lipase, mannanase and pectinase (pectin lyase), wherein the enzymes are
provided in liquid concentrate form that comprise from 5% to 20% of the mass
of
the mixture (including water), such as from 8% to 20% or 8% to 15%, wherein
the
total protein mass can be from 3% to 15% of the concentrate, the protease mass
from 1% to 5% of the concentrate, and wherein the lipase comprises between 1%
and 25%, or between 1`)/0 and 10%, or between 1`)/0 and 7% of the total enzyme
mass (or, in some versions, the concentrate has less than 0.6% total lipase or
is
substantially lipase free); (3) from 1`)/0 to 10% salts for pH control and
enzyme
stability, such as from 1% to 4% sodium citrate and 1 to 4% sodium
bicarbonate;
(4) optionally an effective amount of a mixture of bacterial spores, such as
Bacillus subtilis marketed by Novozymes Biologicals, Inc., USA or JTech Sales
(Baton Rouge, FL), or the spores described in US Patent Application No.
20190284647, "Spore Containing Granule," published Sept. 19, 2019 by P. Bach.
[0015] In another aspect, the bioenzymatic mixture ready for application to
clothing comprises (1) an effective amount of bacterial spores comprising
between 1 x 105 and 5 x 1019 CFU/ml (colony forming units per ml) of bacillus
spores, more specifically from 1 x 107 and 5 x 109 CFU/ml, such as from 1 x
107 to
8 x 108, (2) a mix of at least three or at least four or at least five laundry
enzymes
from at least three different categories of enzymes collectively having a
total
enzyme concentration from 1`)/0 to 20% and optionally no more than 1.5% or 3%
lipase, (3) at least one surfactant such as non-ionic surfactants having a
concentration from 5% to 40% in the bioenzymatic mixture, (4) at least 15%
water
such as from 10% to 80% water or from 30% to 80% water; (5) optionally from
1`)/0
to 10% of a solvent other than water such as propanediol, 3-phenyl-1-propanol,
(2,2-Dimethy1-1,3-dioxolan-4-yl)methanol (also known as Solketal,
isopropylidene
glycerol, or Augeo Multi Clean), propane diol, propane glycol, propylene
glycol,
glycerin, isopentyldiol, pentylene glycol (in general, any alkyl diol having
from 3 to
9 carbons and a viscosity at 20 C of at least 5 mPa-s and more specifically
any
1,n-alkanediols for n less than 9), 2-methoxy-2-phenylethanol, 2-
phenylethanol,
methyl chavicol, and myristicin aldehyde. Liquid alkyl triols may be
considered
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such as butanetriol. Esters having up to 7 carbons with carboxylic acids
having up
to 8 carbons may be considered, including, for example, neopentyl glycol
diheptanoate and other mono- and diglicerides.
[0016] In one aspect, an item of clothing is treated with a biofilm attack
agent
in a target region suspected of harboring a biofilm, the method comprising:
1) applying an effective amount of a naturally-derived biofilm attack agent to
the
target region, the biofilm attack agent being selected from one or more of N-
acetyl
cysteine, panthenol or a derivative theorof, a catechin, and a biofilm attack
enzyme such as DNase, lysozyme, etc., 2) simultaneously, subsequently, or
previously applying a bioenzymatic mixture comprising bacterial spores
suitable
for attacking typical energy sources for bacteria, one or more surfactants,
and an
enzyme mixture of three of more categories of laundering enzymes selected from
proteases, cellulases, amylases, mannanes, lipases, pectinases, and DNases;
3) providing suitable conditions for the spores to become active (i.e.,
providing
sufficient time such as 1 hour or more or 2 hours or more at a suitable
temperature such as 15 C to 60 C, 18 C to 45 C, or 20 C to 40 C with suitable
moisture such as at least 10% moisture in the item of clothing, or from 10% to
80%, 15% to 50%, or 20% to 50% moisture relative to the dry fiber weight);
4) washing the item of clothing in a washing machine with a detergent and then
drying the item of clothing, wherein the method is effective in reducing the
amount
of biofilm matter present relative to what is possible with washing alone.
[0017] Applicant has found that "biofilm attack agents" (so termed even
though
our understanding of the theory behind the success of these agents may be
incomplete) useful for undermining perma-odor may comprise one or more of:
[0018] 1. N-acetyl cysteine (NAC), a relatively non-toxic medication often
used
to treat overdoses of acetaminophen and to help treat cystic fibrosis in
part by reducing phlegm viscosity, and also known as a commercial
dietary supplement due to its antioxidant properties. The NAC in some
aspects is combined with panthenol or derivatives thereof, optionally
various salts or buffering agents, optional surfactants, and other optional
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agents such as allantoin, cyclodextrin, zeolites, phosphates, chelants,
enzymes, antimicrobials, catechins, bacterial phages and the like,
wherein, without wishing to be bound by theory, NAC is believed to open
up the biofilm at least temporarily in order to allow a second agent to be
more effective, often giving positive results in reduction of perma-odor.
[0019] 2. Lysozyme, pectinase including fruit pectinases and other pectinases
that may optionally have an optimum efficiency under acidic conditions
such as from a pH of 4 to 6.5, and other enzymes capable of attacking
portions of the biofilm matrix or reducing the ability of the biofilm or
bacteria to adhere to a fabric. Such other enzymes may include certain
endoglucans or cellulases, proteinases, amylases, mannanases, DNase,
alginate lyase, F-actin, and bacterial-produced enzymes for lysing
portions of a mature biofilm. Selection of enzymes depends on the
textile, the bacteria creating malodor or biofilm, the environment of the
textile, and the energy sources that are consumed by the bacteria.
[0020] In some aspects, a catechin and particularly epigallocatechin
gallate
(EGCG) and related compounds (epicatechin gallate and epigallocatechin, (¨)-
gallocatechin-3-gallate, (¨)-epigallocatechin-3-gallate, (¨)-catechin-3-
gallate, and
(¨)-epicatechin-3-gallate) can also serve as a biofilm attack agent, but is
not
usually preferred because unwanted staining can occur at elevated pH.
[0021] In one aspect, a laundering composition comprising a biofilm attack
agent is provided for users to apply to an item of clothing suffering from
strong
odor. The composition may be provided as a powder, a liquid concentrate, or
ready-to-use material that may be a liquid, a foam, a paste or slurry, etc.,
and may
be provided in two or more containers for application in two or more steps,
such
as rubbing a paste, applying a foam, spraying a solution or sprinkling a
powder
onto a malodorous region of an article of clothing, followed by further
treatment
with another material such as spraying a solution, applying a foam, rubbing a
paste, sprinkling a powder, etc., followed by rinsing or washing and drying.
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[0022] In one aspect, a method for removing persistent odor is provided
comprising applying an aqueous composition to clothing prior to washing,
comprising from 0.1% to 10% NAC, one or more laundering enzymes at a
concentration of from 0.1% to 7% or from 0.5% to 5% or from 0.6% to 3%, and
from 0.1% to 25% or from 0.5% to 10% of one or more surfactants, at a suitable
pH such as from 2 to 6.7, 7 to 10.5, 6 to 8.5, 4.5 to 8, 3 to 6.5, 2.5 to 5, 7
to 11,8
to 10.5, 7.5 to 10, and so forth. In another aspect, a powder comprising NAC
particles may be applied to an item of clothing that is wetted by an enzymatic
blend optionally also comprising one or more surfactants, panthenol, etc.
[0023] In one aspect, a multi-step method to reduce perma-odor comprises:
a) identifying an article of clothing with persistent odor in one or more
odorous
regions such as the regions adjacent armpits or other high-odor or high-
sweating zone of the human body, or alternatively, displaying evidence of
biofilm material when viewed under UV light,
b) treating the article of clothing by applying a first solution comprising N-
acetyl cysteine (NAC) such that at least 0.1 g, 0.2 g, or 0.3 g of NAC per 50
cm2 area is delivered to the one or more odorous regions; and
c) applying a solution comprising one or more laundry-suitable enzymes, and
optionally one or more species of bacterial spores selected for the ability to
assist in cleaning of textiles by producing one or more laundry-suitable
enzymes in response to the presence of suitable contaminants; such that
both NAC and the enzymes and/or spores are present simultaneously.
(The first and/or second solution may further include surfactants such as
polyalkoxy glycosides, sodium laureth / lauryl sulfate, etc., which may be
applied in yet another step before or after the steps mentioned above.)
[0024] A related product comprises a container comprising NAC particles or
solution and one or more compounds selected from panthenol and derivatives
thereof; zinc, ammonium, or alkali metal (sodium, potassium, magnesium,
calcium, etc.) salts such as chloride salts, carbonate salts, bicarbonate
salts,
citrate salts, formate sates, sulfate salts, phosphate salts, etc.; buffering
agents;
fragrance and/or odor reduction compounds that mitigate the odor of NAC. The
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container may comprise pouches that can be torn or cut open to mix with a
laundry preparation or with water or sprinkled directly on clothing that is
moist or
will be moistened with water and/or laundry preparations such as an enzymatic
blend delivered from a spray bottle or other applicator, etc. The product may
be
associated with indicia that directs users to apply the product to textiles in
combination with enzymatic materials. Enzymatic material may be applied first,
after NAC application, or simultaneously with NAC.
[0025] In one aspect, a laundry pretreatment with a biofilm attack agent is
followed by or simultaneous with treatment by a cleaning composition
comprising
one or more of (1) enzymes effective in stain removal or laundering (e.g.,
proteinases, lipases, amylases, mannanases, cellulases, etc.), (2) a
detergent, (3)
optionally an antimicrobial agent such as a chemical antimicrobial agent or
bacterial phages including bystander phages that may assist in attacking
targeted
microbes, particularly once the biofilm has been undermined to some degree.
[0026] In another aspect, the biofilm attack treatment is followed directly
by
washing such as in a top-loading or front-loading machine in cold, warm, or
hot
water using known laundry detergents such as Tide , Gain , Persil , Arm and
Hammer , and the like. The laundry detergent may comprise a variety of
laundering enzymes and surfactants, chelants, builders, bleaching agents, etc.
Complexing or sequestering agents may be employed such as sodium carbonate
and sodium bicarbonate, and/or chelants such as Dissolvine GL-47-S
(tetrasodium glutamate diacetate). Other chelants may include tetrasodium
dicarboxymethyl glutamate, EDTA, trisodium nitrilotriacetate, ethylenediamine,
glutamic acid, histidine, organic diacids such as malates, polypeptides such
as
phytochelatin, citrates, silicates, polymers of acrylic and maleic acid, PBTC
(2-
phosphonobutane -1,2,4-tricarboxylic acid), VERSENOLTM (Dow Chemical), etc.
Chelating agents may comprise from 0.01 to about 5 wt% of the compositions.
[0027] In one aspect, the method further comprises providing suitable time
for
the biofilm attack agent and/or cleaning composition to be effective, such as
a
dwell time before adding the cleaning composition or a dwell time before
washing
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of at least about 2, 5, 10, 15, 20, 30, or 60, minutes, or 2, 4, 6, or 8
hours, such as
from 3 minutes to 1 hour, 5 minutes to 30 minutes, 3 minutes to 3 hours, 30
minutes to 8 hours, 1 minute to 30 minutes, etc. In one aspect, the user is
provided with the biofilm attack agent and the cleaning agent, with suitable
instructions. In another aspect, the user is provided with the biofilm attack
agent,
the cleaning agent, and a detergent, with instructions.
[0028] In one aspect, a method is provided for reducing perma-odor in an
item
of clothing comprising synthetic fibers, comprising applying a combination of
NAC,
protease, amylase, and lipase in an aqueous solution to a region of the item
of
clothing likely to have odor (e.g., pits, neck region, cuffs, groin and
buttocks area),
and after an effective amount of time, washing the item of clothing, wherein
the
aqueous solution has from 0.1 A to 10% NAC. The aqueous solution may be
formed by premixing NAC with the other ingredients, applying a NAC solution
and
an enzyme blend in separately or simultaneously, or by applying a powder or
slurry of NAC to the item and more fully dissolving the NAC in the solution
compri-
sing the enzymes as it is applied to the item. The pH of the aqueous solution
on
the item of clothing may be from 3 to 10, 4 to 9, 6 to 9, etc. In one aspect,
the
aqueous solution further comprises any one or more of panthenol, pectinase,
cellulase, mannanase, DNAse, cellulase, an antimicrobial, and a chelant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 depicts a shirt with UV-fluorescent biofilm-laden zones in
the
armpits of a shirt being illuminated with UV light.
[0030] Figure 2 depicts spraying a cleaning composition on a biofilm
region.
[0031] Figure 3 depicts a flowchart showing a process in which biofilm
visualization is used to guide treatment of clothing with persistent odor.
[0032] Figures 4A and 4B depict dispensing options, such as a roll-on
applicator (4A) for use on clothing and a pod (4B) for use in a wash cycle.
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[0033] Figure 5 depicts a packaged product with an assembly of a pre-
treatment cleaning composition, a biofilm attack agent, and tablets or
capsules to
be used in a washing machine to reduce biofilm and odor sources.
[0034] Figure 6 depicts another assembly comprising a pre-treatment spray
and a biofilm attack agent that can be added to water to form a biofilm attack
solution, both of which can be used to spray malodorous suspected biofilms.
[0035] Figure 7 depicts a spray bottle containing a biofilm attack agent
inside.
[0036] Figures 10A to 10J depict photos of portions an orange 100%
polyester
shirt, Chanpion brand, that was believed to afflicted with perma-odor, showing
remnants of a possible biofilm visible as a dark region in visible light and
with blue
fluorescence in UV light, especially after treatment with an optical
brightener,
Calcofluor White. Treatments with various biofilm attack agents were applied
resulting in reduction but not complete elimination of the darkened matter and
the
associated fluorescence. Details for these and following photos of clothing
items
are given in the "Further Experimental Work" section below.
[0037] Figures 11A to 11F present photos of a blue polyester sports shirt
showing evidence of fluorescence from biofilm matter in the shirt that can
absorb
optical brighteners.
[0038] Figures 12A to 12S show photos of a gray polyester sports shirt
previously afflicted with perma-odor, showing remnants of a possible biofilm
visible via Calcofluor White fluorescence in UV light.
[0039] Figures 13A and 13B show photos of the right and left pits of a
triathlon
shirt under UV light.
[0040] Figures 14A and 14B are photos of a pit in a men's polyester golf
shirt
having apparent biofilm, in which additional biofilm was grown through
application
of an artificial sweat composition.
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[0041] Figures 15A and 15B show views in UV light of the left and right
pits of
a lace dress with a perma-odor problem, displaying strong fluorescence in the
pit.
[0042] Figures 16A and 16B show views of the pits of the lace dress in UV
light
after treatment with bioenzymatic liquid and a biofilm attack agent.
[0043] Figures 17A and 17B are before-and-after images of a biofilm region
in
the lace dress taken with confocal microscopy with a UV laser and no added
dye.
DETAILED DESCRIPTION
Definitions
[0044] As used herein, "N-acetyl cysteine" unless otherwise specified,
includes
N-acetyl cysteine, N-acetyl-L-cysteine, N-acetyl-D-cysteine, salts thereof
such as
pharmaceutically acceptable salts, and mixtures thereof. As used herein,
derivatives of N-acetyl cysteine include esters, amides, anhydrides, and thio-
esters and thio-ethers of the sulfhydryl moiety. Nonlimiting examples include
methyl N-acetylcysteine, ethyl N-acetylcysteine, stearyl N-acetylcysteine, N-
acetylcysteine methylthioether, N,S-diacetylcysteine, N-acetylcysteine amide,
and
the mixed anhydride of N-acetylcysteine and acetic acid.
[0045] As used herein, "odorants" and "odorous compounds" are the chemical
sources of malodor, which are frequently derived from the action of microbes
on
typically non-odorous compounds in the sweat, sebum, or otherwise on the
human body or in other materials that may be present on fabrics. Odorants may
include 3-methyl-3-sulfanylhexanol (3M3SH), 3-methyl-2-hexenoic acid (3M2H),
and 3-methyl-3-hydroxy-hexanoic acid (HMHA), acetic acid, isovaleric acid, 2-
methyl-butanoic acid, 3-methylbutanoic acid, butanoic acid, (E)-3-methy1-3-
hexenoic acid, ethylbutanoate, (Z)-4-heptenal, (E)-2-nonenal, 2-methoxyphenol
(guaiacol), 4-methyloctanoic acid, sulfanylalkanols and particularly 3M3SH (3-
methy1-3-sulfanylhexan-1-ol), and the steroids androstenone and androstenol.
[0046] As used herein, "synthetic fibers" refer to fibers that are not
obtained
from plant or animal sources such as nylon, polyester, acrylic and polyolefin
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fibers. Further non-limiting examples include modacrylic, Spandex, rayon
(e.g.,
viscose, modal and lyocell), vinyon, saran, vinalon, aramids, PLA, etc.
[0047] As used herein, "optical brighteners" include dyes and related
materials
that fluoresce in UV and/or blue light to enhance the brightness or appearance
of
colors in various fabrics. Optical brighteners often absorb effectively onto
natural
fibers, but less on polyester or other synthetic fibers. They also often
absorb onto
biofilm matter. Typical optical brighteners are frequently stilbene compounds,
particularly anionic diamino stilbene (DAS) or distyryl biphenyl (DSBP)
derivatives,
such as di- and tetra-sulfonated triazole-stilbenes and di-sulfonated stilbene-
biphenyl derivatives. Optical brighteners such as those used in laundry
detergents
may include Calcofluor White (CAS 4404-43-7) or Calcofluor White M2R; C.I.
Fluorescent brightener 260; Fluorescent Brightener FWA-1 (CAS 16090-02-1);
Disodium 4,4'-bis(2-sulfostyryl)biphenyl (CAS 27344-41-8), also known as
Tinopal
CBS-X (BASF, Ludwigshafen, Germany); Uvitex 2B, Phorwite MBBH, 4,4'-
bis(benzoxazoly1)-cis-stilbene, 2,5-bis(benzoxazol-2-y1)thiophene, 4,4'-
diamino-
2,2'-stilbenedisulfonic acid (amsonic acid, CAS 81-11-8), and the like. In
laundry
detergent, optical brighteners may be present at concentrations from about
0.02%
to about 1%. Solutions of optical brighteners used to identify the presence of
biofilm matter may have any effective concentration, such as from about 50 PPM
to 2%, 100 PPM to 1%, 100 PPM to 0.05%, 0.2% to 1%, and the like.
[0048] As used herein, the term "textile" means any material made of
interlacing fibers, including fabrics, carpeting, etc., whether woven or
nonwoven,
or comprising including yarns, yarn intermediates, fibers, and fabrics made of
these materials and related product (garments and other articles). Items made
at
least in part from textiles may include shirts, pants, socks, shoes, hats,
gloves,
underwear, suits, dresses, gowns, face masks, robes, linens, draperies,
upholstery materials, etc.
[0049] As used herein, a "fabric" is a material made through weaving,
knitting,
spreading, crocheting, or bonding of fibers and may be used in production of
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further goods (garments, etc.), and includes both woven and nonwoven materials
and may include knits, felts, multilayered items, composites, denims, yarns,
etc.
[0050] As used herein, "detergent composition" refers to compositions for
removal of undesired compounds from surfaces such as textile surfaces. Such
compositions may be in any suitable product form such as liquid, gel, slurry,
dispersion, powder, solid stick, granulate, paste, or spray compositions. It
may
include liquid and/or solid laundry detergents and fabric detergents and may
comprise one or more enzymes such as hem icellulases, peroxidases, proteases,
cellulases, xylanases, lipases, phospholipases, esterases, cutinases,
pectinases,
mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases,
lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases,
beta-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,
DNase,
chlorophyllases, amylases, perhydrolases, peroxidases, xanthanase and mixtures
thereof. The detergent composition may further comprise ingredients such as
surfactants, builders, chelating agents, bleach system, polymers, fabric
conditioners, foam boosters, suds suppressors, perfume, tarnish inhibitors,
bactericides, fungicides, soil suspending agents, anti-corrosion agents,
enzyme
inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic
enzymes,
oxido reductases, fluorescent dyes, antioxidants, and solubilizers, etc.
[0051] As used herein, a "cleaning composition" can include a detergent
composition for cleaning laundry or other textile material, but can include
cleaning
aids for many surfaces such as bathrooms, kitchens, walls, floors, machinery,
foods, etc. For example, in one version of a laundry cleaning composition
comprising water, NAC at a level of 0.01% to 7%, such as from 0.5% to 2.5%,
can
be combined with 5-30% of a detergent formulation such as Pilot Chemicals'
(Cincinnati, OH) super concentrate blend of anionic, nonionic, and
zwitterionic
surfactants, plus 1-3% each of three or more enzymes, an optional solvent or
stabilizer up to roughly 7% such as propylene glycol or propane diol, chelants
and
builders, various salts and buffering agents such as citrates, chlorides,
carbonates, etc., to assist in stabilizing enzymes. It may also comprise up to
4%
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rheology modifiers such as 1%-2% ACULYN TM 22 Rheology Modifier (Dow
Chemical). Such compositions can be applied to any textile item or solid
surface.
[0052] As used herein, the term "laundering" relates to both household
laundering and industrial laundering and means the process of treating
textiles
with a detergent composition typically comprising surfactants and/or enzymes.
The laundering process can be carried out using a household or industrial
washing machine such as front-loading or top-loading washers, or by hand.
[0053] As used herein, "derivatives of panthenol" may include pantothenic
acid
and salts thereof (e.g., the calcium, sodium, potassium salts, etc.),
pantethine,
pantetheine, and so forth. Panthenol is closely related to its derivative,
pantothenic acid, and pantethine (bis-pantethine or co-enzyme pantethine), a
dimeric form of pantetheine produced from pantothenic acid (vitamin B5) by
addition of cysteamine. Most vitamin B5 supplements are in the form of calcium
pantothenate. However, in one aspect, a composition may be substantially free
of
pantothenic acid while containing panthenol or derivatives thereof. Without
wishing to be bound by theory, panthenol's efficacy against perma-odor and
biofilms in infected fabrics may relate to the uptake of panthenol by microbes
that
need pantothenic acid, wherein the similarity to panthenol "fools" microbes
into
taking up panthenol as if it were a nutrient when it is not. Such a
possibility in
another context is proposed in G.F. Helaly et al., "Dexpanthenol and propolis
extract in combination with local antibiotics for treatment of Staphylococcal
and
Pseudomonal wound infections," Archives of Clinical Microbiology 2/4 (Dec.
2010). If that mechanism is applicable Applicant's results, then panthenol or
its
derivatives substantially free of panthothenic acid may be especially useful.
However, it may also be that panthenol (a.k.a. dexpanthenol) has a softening
effect or other secondary effect on the biofilm of an infected fabric.
[0054] As used herein, "post-treatment" in the context of the washing of
clothing refers to an application of a formulation as described herein to all
or a
portion of an item of clothing after it has been washed. For washing with
water,
the post-treatment may be done prior to drying, after drying, or during
drying.
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[0055] As used herein, "washing" of clothing or other items made from
fabrics
or textiles generally refers to the use of water and typically a cleaning
agent such
as a detergent to remove soil, food, or other agents from the clothing, and
may
comprise the use of an automated washing machine running a programmed
washing cycle comprising combinations of soaking, agitation, rinsing, and
spinning to remove water. However, "washing" may also refer to dry cleaning,
hand cleaning, chemical bleaching, solar or UV bleaching, treatment with
disinfecting or cleansing vapors, etc.
[0056] As used herein, percentages in a composition should be taken as
weight percent values unless otherwise specified. Thus, a suspension said to
comprise 2% of, say, cucurbituril is understood to have 2% cucurbituril
particles
relative to the mass of the suspension itself, including the water or other
liquids.
[0057] As used herein, ranges such as concentration ranges for a compound
may have a lower limit and an upper limit selected from any suitable
concentration
value mentioned for that compound. In aspects where a compound is to be
excluded or kept at a low level, the concentration range may be from zero or
substantially zero (e.g., 0.1%, 0.05%, 0.01%, 0.001%, 100 ppm, 10 ppm, or 1
ppm) to an upper limit of any concentration mentioned herein for that compound
or salts thereof.
N-Acetyl Cysteine
[0058] N-acetyl cysteine (NAC), a derivative of a vital amino acid,
cysteine, is
used as a medical agent to treat overdoses of acetaminophen and to help in
treatment of cystic fibrosis. It is an antioxidant that is also marketed as a
dietary
supplement. We have found that aqueous solutions of NAC can help reduce
perma-odor, either through direct attack or by enhancing the ability of
cleaning
agents such as enzymes or other compounds to clean the surfaces of infected
fibers. NAC has been reported to have success in certain medical environments
against several species of bacteria that can create biofilms.
[0059] The ability of NAC or any other medical agent does not appear to
have
been considered previously in terms of reducing malodor on clothing or in
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affecting the hypothesized biofilm formation that may account for some of the
more intractable issues of odor control that occur for some users and clothing
types. Of course, the usefulness of any compound in one environment against
one particular bacterial species does not indicate success in another
environment,
especially when the target may be different bacteria.
[0060] NAC solutions in the range of 0.01% to 15% or more appear to be
useful in reducing biofilm on fabrics with persistent odor, especially when
combined with or followed by treatment with additional agents such as
panthenol
or derivatives thereof, laundry enzymes or other enzymes, catechins,
detergents,
and various solvents. Suitable formulations may have 0.1% to 13%, 0.5% to 13%,
1% to 10%, 0.3% to 8%, and 0.5% to 6% NAC, such as from 0.7% to 3.5% NAC.
Without wishing to be bound by theory, it is believed that NAC may help
opening
up the biofilm (reducing viscosity or thickness, or increasing permeability of
the
biofilm) to allow the enzymes or other actives to more effectively attack the
food
source for the odor causing bacteria, or to allow antimicrobial agents or
enzymes
to more effectively attack the bacteria, or both.
[0061] NAC can be found in garlic, onion, etc., or derived from natural
materials such as corn, typically through fermentation and extraction. It can
be
derived from cysteine through acetylation, or produced according to the
methods
described in EP090528261, or by Alexander G Zhdanko et al. in "One-step
synthesis of N-acetylcysteine and glutathione derivatives using the Ugi
reaction,"
Tetrahedron 65/24 (June 2009):4692-4702. NAC can be obtained in dietary
supplements such as those of Swanson.com, or from a variety of chemical
suppliers. For some aspects, low-odor NAC such as Ajipure (Swanson Pharm.,
Fargo, North Dakota) or Fluimicil (Zambon Italia S.r.I., Milan, Italy) may be
useful.
[0062] In some aspects, enzymes and/or bacteria or spores may be provided
in a mixture having high ionic strength such as having at least 2% or higher,
5% or
higher, 8% or higher, or 10% or higher, such as from 2% to 20%, 5% to 20%, 8%
to 20%, 10% to 25%, etc., of a salt such as sodium citrate, sodium chloride,
aluminum sulfate, ammonium sulfate, potassium chloride, etc. Citrate ions may
be
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beneficial in some versions, and thus solutions of sodium citrate, potassium
citrate, ammonium citrate, zinc citrate, and the like may be used.
[0063] Other agents can also be considered. Without wishing to be bound by
theory, it is believed that some liquids may be useful as solvents or "biofilm
modification" agents to soften or weaken a biofilm or enhance its permeability
so
agents may be more effective. Such agents may include 2"-hydroxycinnamic acid,
3-methyl-2(5H)-furanone, phenyl propanol such as 3-phenyl-1-propanol, propane
diol, propane glycol, pentylene glycol, DMSO, panthenol, pantothenic acid,
glycerin, 3-methoxyphenylacetic acid, 4"-hydroxyphenylacetic acid, 2-methoxy-2-
phenylethanol, 2-phenylethanol, methyl chavicol (Basil oil) and other
essential
oils, myristicin aldehyde, 3,4-dihydroxybenzoic acid, and isopropylidene
glycerol,
also known as Solketal. They may be present at levels of at least 1 A, 3%, or
5%,
or from 0.5% to 15%, 1% to 8%, 1.5% to 6%, etc.
[0064] EGCG is known as an oral care agent, a probiotic agent for human
consumption, and as an agent with various health benefits. Its potential in
odor
control and especially odor control of fabrics appears to have not yet been
recognized. We have found particularly useful odor control systems can be
produced using aqueous EGCG solutions. EGCG is most commonly available as
an extract of green tea, typically produced in China. Applicant has found EGCG
to
be most useful at 98% purity or above, although levels of at least 80%, 85%7
90%7
92%7 9,0,/o 7
97% and the like can also be used in some versions. EGCG may be
combined with ascorbic acid, citric acid, mandelic acid, lactic acid, etc., to
achieve
a suitable pH to prevent color formation from reactions involving EGCG.
[0065] In some aspects, the composition may be substantially free of any or
all
of the following or any subset: ethanol, methanol, propanol, alcohols,
alcohols
having 3 or fewer carbons, alcohols having 2 or fewer carbons, glycolic acid,
acetic acid, critic acid, latex, spermicides, Octoxyno1-9, TEA (triethylamine,
a
compound which may contribute to unwanted odor) or derivatives of TEA, TMA
(trimethylamine), ammonia or complexes thereof, amines, polyhydroxy fatty
acids,
polyhydroxy acids, alpha-hydroxy acids having 14 or greater carbons, fatty
acids,
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polyhydroxy fatty acid esters (or polyhydroxy fatty acid derivatives such as
esters,
amides, and alcohols), benzoic acid, parabens, preservatives, perfumes,
artificial
colors, sodium bicarbonate, bicarbonates in solid or ionic form, retinol, or
Retin-A.
"Substantially free" in this context may mean lacking an effective quantity.
For
alcohols and acids this may be taken as less than 0.1%. In some cases, the
concentration may be less than 0.05%.
Other Products
[0066] The
laundry composition may comprise an effective amount of at least
one of: (1) one or more enzymes selected from a protease, a lipase, an
amylase,
a mannanase, a pectinase, a lysozyme, a cellulase, and a DNase; (2) one or
more quaternary amines such as, by way of example, soyethyl morpholinium
ethosulfate, a quat known to be useful in reducing odor; (3) one or more
essential
oils with odor control and antimicrobial efficacy, such as the mix of
essential oils
marketed under as Odor Medicine OF. Concentrate by Odor Medic, LLC
(Minneapolis, MN); and (4) a biological agent such as a bio-enzymatic cleaner
or
deodorizer comprising one or more bacteria strains or one or more microbial
phage strains that can be directly or indirectly effective in attacking
targeted
bacteria species.
Bacterial Spores and Other Microbial Agents
[0067] Bacterias spores used herein may be any of those described in US Patent
No. 9228284, "Mitigation of odor in cleaning machines and cleaning processes,"
issued Jan. 5, 2016 to S.C. Mchatton, et al., particularly the strains of B.
subtilis.
See also US Patent No. 9756862, "Proportioner-ready bioenzymatic concentrated
cleaning product," issued Sept. 12, 2017 to D.A. Cooper et al.
[0068] The spores are obtained from non-pathogenic spore-forming
microorganisms that are capable of reacting with and removing various organic
substances. Such spores can produce extracellular enzymes that may include
protease enzymes, urease enzymes, amylase enzymes, lipase enzymes,
cellulase enzymes, and combinations thereof. Commercially available
concentrated of spores suspended in liquid may be used. Such spore
concentrates may comprise from 1`)/0 to 50% of the compositions described
herein, or from 5% to 30% or from 10% to 25%. The bacillus spores may
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constitute 0.05% to 60% by weight of the spore concentrate, and after blending
with enzymes, surfactants, and other agents to form a concentrate or ready-to-
use
mixture for treating laundry, the bacterial spore concentration may be from
about
0.01% to about 10% or 0.05% to 5%, or from 0.1% to 4%. Alternatively, the
number of colony forming units (CFU) per ml in the concentrate or diluted
mixture
may be 1x105t0 1x1010, 1x105t0 1x109, or 1x106t0 1x108.
[0069] The bacillus spores may have an average particle diameter of about 2-50
microns, such as from about 10 to 45 microns. Bacillus spores are commercially
available in blends in aqueous carriers. Commercially available bacterial
spore
blends include without limitation Freshen Free TM CAN (10X), from Novozymes
Biologicals, Inc.; J-Zyme AB-20XNF of JTech Sales (Baton Rouge, FL), a 20X
liquid concentrate comprising spores from Bacillus subtilis; Bio-Enzymatic
Odor
Eliminator from Ecolab (St. Paul, MN); and Evogen Renew Plus (10X) and
Evogen GT (10X, 20X and 110X), both from Genesis Biosciences, Inc.
(Lawrenceville, GA). Here, the parenthetical notations (10X, 20X, and 110X)
indicate relative concentrations of bacillus spores. Useful spores may include
those made according to the methods described in J. Edward Donnellan, Jr. et
al.,
"Chemically Defined, Synthetic Media for Sporulation and for Germination and
Growth of Bacillus Subtilis," J. Bacteriology, 87/2 (Feb. 1964): 332-336, or
the
spores described in US Patent Appl. No. 20190284647, "Spore Containing
Granule," published Sept. 19, 2019 by P. Bach, which not only describes many
spores but also describes how granulated enzymes capsules can be coated with
bacterial spores in a single dry product, which may be of use in dry
precursors to
the liquid-based mixtures described herein.
[0070] Examples of commercially available bacteria to consider include
Ecolab's Bio-Enzymatic Cleaner, bio-enzymatic "Biologicals" mixes provided by
Novozymes Corp. (Bagsvrd, Denmark), bacterial mixes distributed by Maroon
Group (Boca Raton, FL) such as J-Zyme TM AB-20X NFC distributed by J Tech
Sales (Boca Raton, FL), a variety of Nature's Miracle products from Spectrum
Brands (Middleton, WI), as well as the Bio-Enzymatic Stain & Odor Remover of
Xion Labs (Kissimmee, FL) and the NATURE'S SOLUTION TM Bio-Enzymatic
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Deodorizer/Spotter/Digester of National Chemical Laboratories, Inc.
(Philadelphia,
PA). Also to be considered are bacteria described in US Patent Application
20180305658, "Composition of Bacterial Mixture and Uses Thereof" by M.T.
Glimp, published Oct. 25, 2018. Such bacteria may include, for example,
species
and strains from Nitrospira including Comammox bacteria, Nitrosospira,
Nitrobacter, Nitrosomonas, Nitrosococcus, Lactobacillus; Lactococcus, etc. The
use of bacteria in the systems described herein should also comply with
relevant
health and safety requirements.
[0071] Also see US Patent Publication 20060062742, "Compositions for
reduction of human malodor" by C. Davis, March 23, 2006. Such bacteria, said
to
be useful in reducing human malodor, include bacteria selected from
Lactobacillus
iners, and similar clones. The composition may further comprise Lactobacillus
variants such as crispatus, casei, gasseri, fermentum, amylolyticus,
acidophilus,
jensenii, coleohominis, etc. Other species may include Anaerococcus,
Dialister,
Finegoldia magna, Bifidobacterium, Bacteroides, thetaiotaomicron,
Lachnospiraceae, Leptotrichia, Streptococcus, Comamonas, Aerococcus,
Veil/one/la, Mycoplasma, and Micromonas. Other examples of bacterial
compositions for odor control are described in US Patent Application
20110318289, "Methods and Compositions for Reducing Body Odor," published
by M. Frodyma of Novozymes Corp., Dec. 29, 2011.
[0072] Bacteria or spores may be provided in an aqueous suspension, a gel,
a
foam, a wipe, in microspheres, etc. One vehicle for delivery of beneficial
bacteria
may be microspheres comprised of poly (D.L-lactide-co-glycolide)(PLGA) and
poly(D,L-lactide)(PLA) as described in Goodman, et al, "Microsheres Under In
Vitro Release Conditions," APPS PharmSCiTech, 2003: 4(4) article 50. Other
methods for delivery are described in U.S. Pat. No. 6,509,028 issued to
Williams,
et. al on Jan. 21, 2003.
[0073] In an aqueous suspension, the concentration of bacteria or their
spores
may be 10 billion or more per liter such as form 10 billion to 200 billion per
liter, or
can be about 5 billion or more per liter, 500 million or more per liter, or 50
million
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or more per liter. The bacteria may comprise any number of species such as any
integer from 1 to 1000, such as 1 species, 3 or more species, from 3 to 10,
etc.
Such agents can be present in a cleaning composition or in the biofilm attack
agent, if compatible.
[0074] In some aspects, the cleaning composition may comprise phages that
attack bacteria associated with malodor such as Micrococcus luteus or other
micrococci. Phages are virus-like agents that attack specific strains of
bacteria.
Examples of phages for use in products intended for human use are described in
United States Patent Application 20170157186, "Phage to Treat Bacteria on
Skin," published June 8, 2017 by Jacob Shiach. See also US Application
20110038840, issued Feb. 17, 2011 to L-kuang Chen and Nien-tsung Lin, and US
Application 20170319637, issued Nov. 9, 2017 to F. Pouillot et al. Commercial
phage therapy for nutritional purposes to enhance the bacteria in the GI tract
is
marketed at Flora88.com by Optium, LLC (Provo UT), and similar phages can be
considered for use with the compositions described herein. Another source of
phages for commercial products is Micreos Food Safety B.V. (The Netherlands).
[0075] Phages can be provided in aqueous solution and applied with NAC,
enzymes, flavanols and other agents to attack odorous bacteria or their
biofilms. A
related approach is the use of bystander phage technology, in which a phage
attacks a specific microbe but induces the targeted microbe to release
defensive
antimicrobials in response, which in turn can target a broad spectrum of
nearby
bacteria. Bystander phage therapy is being offered for commercial use through
Brigham Young Univ. from the research of Dr. Sandra Hope, as described by T.
Scott Brady et al., "Bystander Phage Therapy," Antibiotics 7/4 (2018): 105.
Enzymes
[0076] Enzymes, particularly hydrolases, have been used as a tool for
laundering fabrics for decades. Among hydrolases, proteinases, amylases,
cellulases and mannanase are also commonly employed in some products.
Lysozymes, pectinases, and DNases may also be considered.
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[0077] Enzymes may be of any suitable origin, such as vegetable, animal,
bacterial, fungal and yeast origin, and may be modified in various ways and
expressed via host organisms in which the genetic material responsible for the
production of the enzyme has been cloned. The enzymes can be added as single
ingredients (prills, granulates, stabilized liquids, etc. containing one
enzyme) or
as mixtures of two or more enzymes (e.g. cogranulates or blends in solution).
[0078] There are a wide variety of specific enzymes. Numerous powder and
liquid detergents for washing machines, including machines for clothes washing
as well as dish washing, comprise blends of enzymes to provide detergency
effects. Enzymes useful with the formulations described herein may include any
combination of lipases, proteases, amylases, cellulases and mannanases,
pectinases, hem icellulases, peroxidases, lysozymes, xylanases,
phospholipases,
esterases, cutinases, pectinases, laccases, keratanases, reductases, oxidases,
phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases,
pentosanases, malanases, 8-glucanases, arabinosidases, hyaluronidases,
DNAse, chondroitinases, and hexosaminidases, including those described in US
Patent No. 6489279, "Laundry and cleaning compositions containing
xyloglucanase enzymes," issued Dec. 3, 2002 to Convents et al. However, in
some aspects the composition may be substantially free of particular enzymes,
such as substantially free of any particular class of enzymes such as lipase
or
laccase (e.g., having less than 0.2%, less than 0.1% or less than 0.05% by
weight
of the excluded enzyme or enzyme category) or substantially free of the
bacteria
or bacterial spores that produce the excluded enzyme or enzyme category. .
Some lipases may be most effective at low moisture levels, such as from 10% to
40% moisture in fabric, acting primarily at air-water interfaces, and thus the
peak
activity of lipase applied to laundry may occur after washing as the clothing
is air
dried or tumble dried, resulting in some components that may remain in the
clothing until washed again, raising the risk of malodor when lipase
concentration
is too high. Thus, one may limit lipase to less than 25%, 15%, 10%, or 5% of
the
enzymes present, with lower limits of, say, 1%, 3%, 5%, or 10%, when feasible.
Lipases may include those derived from shrimp and other marine life.
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[0079] As used herein, "laundering enzymes" refers to enzymes commonly
incorporated into laundry detergents, both liquid and granulated detergents,
such
as lipase, cellulase, mannanase, protease, pectinase, and amylase. These are
often engineered to be active at an alkaline pH such as from 7 to 9.5 or 7.5
to 8.5
but may individually or collectively be adapted for optimum performance in
other
pH ranges such as from 3 to 12, 3 to 6, 4 to 7, 5 to 8, 6 to 8, 4.5 to 8.5, 5
to 7.6,
3.5 to 6.5, etc.
[0080] Enzymes may be incorporated in a product at levels from 0.01% to 20%
of active enzyme by weight, or from 1% to 15%, 2% to 12%, and the like.
Examples of potentially useful enzymes are disclosed in U.S. Pat. Nos.
5,576,282,
5,728,671 and 5,707,950. Proteases are described in WO 95/30010, WO
95/30011, and WO 95/29979. Peroxidase enzymes are discussed in U.S. Pat.
Nos. 5,576,282; 5,728,671; 5,707,950; 5,817,495; 5,968,883 and in European
Patent application EP No. 96870013.8. A range of enzyme materials for
detergent
compositions are found in WO 9307263 and WO 9307260 to Genencor
International, WO 8908694 to Novo, and U.S. Pat. No. 3,553,139 to McCarty et
al.
Enzyme materials useful for liquid detergent formulations are discussed in
U.S.
Pat. No. 4,261,868, Flora et al, Apr. 14, 1981. Enzymes for use in detergents
can
be stabilized by various techniques discussed in U.S. Pat. No. 3,600,319, EP
199,405 and EP 200,586, and US Pat. No. 3,519,570. Enzymes produced by
bacterial phages may also be employed, including polysaccharide depolymerases
or EPS depolymerases and phage endolysins.
[0081] As used herein, "lysozyme" is a glycoside hydrolase that can be
found
in egg whites of chickens, in human milk and the milk of some mammals. It
catalyzes the hydrolysis can cause lysis of some bacteria. Lysozyme activity
generally increases with increasing temperatures, up to about 60 C, with a pH
range of 6.0-7.0 for best activity. Suitable concentrations of salts such as
sodium
chloride or potassium salts can increase lysis by lysozyme (e.g., a salt
concentration from 0.01 to 7%, 0.1% to 3%, 0.02% to 1.5%, 0.05% to 1%).
Alternatively, the enzyme solution may be substantially salt free, such as
having
an upper limit in concentration of less than 0.1%, less than 0.05%, less than
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0.01%, or less than 0.001% salt; upper limits to be considered may also
include
100 ppm, (parts per million), 10 ppm, 1 ppm, 100 ppb (parts per billion), 10
ppb, 1
ppb, and 10 ppt (parts per trillion). These upper limits can also be applied
to limit
the amount of any optional ingredient or potentially adverse ingredient
described
herein. These limits can be taken as applicable if desired to any known toxin,
pollutant, dye, pharmaceutical compound, and so forth, in order to achieve
such
objectives as meeting safety guidelines, reducing harm to the environment, or
preventing harms to the treated products.
[0082] Proteases (sometimes known as peptidases) may include serine
proteases, which include a serine group in the catalytic center, or metallo
proteases, cysteine proteases (including papain and bromelain), aspartic
proteases, threonine proteases, and the like. 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), such as Subtilisin A (Enzyme Commission or EC number 3.4.21.62),
marketed by Novozymes, an alkaline non-specific serine protease from Bacillus
subtilis that catalyzes the hydrolysis of proteins and peptide amides.
Variants
include subtilisin BPN' (also subtilisin B, subtilopeptidase C, nagarse,
nagarse
proteinase, subtilisin Novo, bacterial proteinase Novo) and subtilisin
Carlsberg
(subtilisin A, subtilopeptidase A, alcalase Novo). Examples of trypsin-like
proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium
protease
described in WO 89/06270 and WO 94/25583. Other examples of useful
proteases are the variants described in WO 92/19729, WO 98/20115, WO
98/20116, and WO 98/34946. Proteinase K may also be considered, but can pose
a threat to keratin and thus to wool.
[0083] Other commercial serine proteases from Novozymes include:
Alcalase , an esterase that hydrolyzes amino esters which include heterocyclic
amino esters; Savinase , which can hydrolyse some esters as well as strained
amides under alkaline conditions; Esperase , an endo-peptidase with a broad
specificity; Neutrase (E.C.3.4.24), a neutral, zinc metallo endo-protease
from
Bacillus amyloliquefaciens that randomly hydrolyses internal peptide bonds and
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also facilitates enzymatic synthesis of oligopeptides, and the related
protease
thermolysin, a zinc dependent metallo endo-protease; rTrypsin (EC 3.4.21.4);
and nattokinase, a protease with 275 amino acid units made by the bacterium
Bacillus subtilis var. natto, manufactured by Daiwa Pharmaceutical (Tokyo),
Contek Life Science Co., Ltd. (Taibei) and the Japanese Nattokinase
Association
organized by Japan Bio Science Laboratory (Osaka). Proteases may also include
the peptidase known as SAPV from the halophilic Virgibacillus natechei sp.
nov.,
strain FarDT.
[0084] Pectinases can include those from any known source such as from
bacteria, fungi and nematodes, and can include the XPect@ series of pectinase
marketed by Novozymes and related Pectinex@ products. Pectinase products are
often provided as a mixture of enzymes that may include pectintranseliminase,
polygalacturonase and pectinesterase and small amounts of hem icellulases and
cellulases. Pectinex@ and several related enzymes are believed to be produced
by a strain of the fungus Aspergillus niger, said to exhibit optimum activity
around
pH 4.5. In some aspects, the pectinase includes one or more pectinases of
Dickeya dadantii.
[0085] Lipases (triacylglycerol acyl-hydrolases, EC 3.1.1.3) are typically
classified as serine hydrolases and act to hydrolyze various lipids, typically
only
when present at an oil-water interface and generally do not hydrolyze
dissolved
substrates in the bulk fluid. Lipases, when used in the compositions described
herein, may be biological or engineered lipases that are extracted from living
organisms or combinations thereof. Microbial sources may include Candida
rugosa, Pseudomonas aeruginosa and other Pseudomonas species, Aeromonas
species, Acinetobacter species, Burkholderia species, Aspergillus oryzae,
Bacillus
cereus, Bacillus coagulans, and other Bacillus species, Penicillium roquefort,
Geortrichum species, etc. Others include M1 Lipase and Lipomax@ (Gist-
Brocades) and Lipolase@ and Lipolase Ultra (Novozymes). Lipase may also be
extracted from arthropods, marine animals and mollusks, including lipase from
the
hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), as described by
S. Kuepethkaewa et al., International Journal of Food Properties 20/4 (2017):
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769-781. Cutinases [EC 3.1.1.50] can be considered as a kind of lipase that
does
not require interfacial activation. See WO 88/09367 (Genencor).
[0086] Cellulases may include any combination of known cellulases including
those used for laundry detergents or proposed for that purpose, including
alkali
stable endoglucanase from alkalothermophilic Thermomonospora sp. (T-EG); the
BioTouchTm cellulases such as BioTouchTm FLX1, DCL, FCL75, Duo 505, and
ROC 250 laundering composition of AB Enzymes (Darmstadt, Germany);
Aspergillus niger cellulase; the acid, neutral, or alkaline cellulases of
Creative
Enzymes (Shirley, NY); the cellulases of US Patent No. 6451063, issued Sept.
17,
2002 to K.A. Clarkson et al.; and the Celluclast and Cellic cellulases of
Novozymes, including Cellic CTec3 HS, a cellulase and hemicellulase complex.
[0087] Mannanases may include Mannaway from Novozymes, Cp-
mannanase marketed by PhylloZyme (Philadelphia, PA) and extracted from
Trichoderma reesei, or mannanase extracted from various leafs as described in
Uma Kumari, "Validation of leaf enzymes in the detergent and textile
industries:
launching of a new platform technology," Plant Biotechnology Journal, 17
(2019):
1167-1182.
[0088] DNase can include deoxyribonuclease and related peptides or enzymes
such as those described in US Patent No. 10,479,981. Also to be considered are
the compositions of WO 06/017816 and the Deoxyribonuclease I (bovine DNase I)
of WO 2009/121183 and the materials of US Patent No. 9675736.
[0089] Various amylases (a and/or [3) can be included such as Termamyl ,
Ban , Fungamyl and Duramyl , from Novo Nordisk. WO 94/02597 describes
cleaning compositions with mutant amylases. See also W094/18314,
W095/10603, U.S. Pat. No. 5,003,257, EP 252,666, WO 91/00353, EP 525,610,
EP 368,341, W095/26397, W095/35382, WO 94/18314 and W096/05295.
[0090] In many aspects, a combination of enzymes is provided, either in
solution or in powdered form. The combination may comprise commercial
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combinations such as Novozymes Leviti mix of protease, cellulase, mannanase,
amylase and pectinase, though such a mix can be assembled from individual
sources. A mix can be fortified with additional individual enzymes such as
lipase
and other forms of protease such as papain, lysozyme, DNAse, and the like.
Antimicrobials
[0091] Antimicrobial agents may be present in any of the solutions used in
the
methods described herein. For example, common preservatives such as
banzalkonium chloride, 1,2-benzisothiazolin-3-one or other isothiazolinones,
methylchloroisothiazolinone/methylisothiazolinone, phenoxyethanol, potassium
sorbate, propylparaben, benzyl alcohol, dehydroacetic acid, or benzoic acid
may
be present that also attack odor causing bacteria. Alternatively or in
addition,
nisin, lysozyme, antimicrobial peptides, etc. may be present. However, in some
aspects, the composition may be substantially free from certain preservatives
such as parabens and/or formaldehyde generators. Antimicrobials may be present
in a composition at a level of 0.01% to 15%, 0.01% to 5%, or 0.05% to 2%,
etc.,
and the NAC or salt or derivative thereof may comprises from 0.1% to 20% of
the
dry or wet mass of the composition. Other agents may include cetylpyridinium
chloride, imidazolidinyl urea, propyl benzoate, sodium benzoate, potassium
sorbate, biguanide, nisin, chitosan derivatives, silver nanoparticles or other
silver-
based compositions capable of releasing silver ions, etc.
[0092] Antimicrobial agents can include the cationic steroidal
antimicrobial
(CSA) compounds described by Paul Savage and D. Leung in US Patent No.
7754705, "Cationic steroid antimicrobial compositions and methods of use,"
issued July 13, 2010; Carl Genberg and Paul Savage, US Patent No. 9603859,
"Methods and products for increasing the rate of healing of tissue wounds,"
issued
March 28, 2017; and Carl Genberg, C.S. Beus, and Paul B. Savage, US Patent
Application 20150374719, "Methods for Treating Fungal Infections," issued Dec.
31, 2015. Any of the compounds described therein such as CSA-13, CSA-25,
CSA-54, CSA-90, CSA-92, CSA-190, CSA 191, and CSA 1921 and the like may
be combined with the compositions described herein. Antimicrobials may have a
concentration of from 0.01% to 1 A, such as from 0.01% to 0.5%, or from 0.02%
to
0.4% by weight. CSA compounds are commercially available from Purishield Life
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Sciences, LLC (Walnut Creek, CA) under the PuriShield , Purifect or Ceragyn
brands. The antimicrobial system may be substantially free of any one or more
of
parabens, formaldehyde donors, halogens, isothiazolinones, and phenoxyethanol.
Surfactants
[0093] A surfactant system can comprise nonionic and/or anionic and/or
cationic and/or ampholytic and/or zwitterionic and/or semi-polar nonionic
surfactants. They may be present at 0.01% to 25% by weight, such as from 0.1%
to 5%, 0.5% to 20%, etc., or otherwise at an effective concentration to enable
the
solution comprising the surfactant to achieve the intended purpose of the
surfactant, which may be, for example, enhancing penetration of the solution
into
a woven or nonwoven fabric or assisting in removing foodstuffs or other
contaminants from a fabric. The surfactant can be formulated to be compatible
with enzyme components.
[0094] Surfactants may be bio-based such as GlucoPure Sugar Surfactants
of Clariant, Spectrapon of Spectrum Chemical (Boca Raton, FL), Glucopon alkyl
polyglycoside surfactants from BASF (Ludwigshafen, Germany), Sucranov SF
from Jarchem (Newark, NJ) comprising a blend of Sodium cocoamph-oacetate,
glycerin, lauryl glucoside, sodium cocoyl glutamate, and sodium lauroyl
lactylate,
or other systems comprising such components as glycosides of fatty acids and
alcohols, polyether glycosidic ionophores and macrocyclic glycosides,
carotenoid
glycosides and isoprenoid glycolipids, biologically active glycosides of
aromatic
metabolites, lipopeptides, biologically active marine and terrestrial alkaloid
glycosides, fatty acid amide glycosides and their analogs and derivatives,
etc..
[0095] Biosurfactants may also include rhamnolipids or fungal extracts such
as
sophorolipids, or combinations of sophorolipids or other biosurfactants with
sodium dodecyl sulfate (SDS) or other surfactants. Sophorolipids may weaken
the
EPS biofilm matrix, assisting in surface-detachment and breakup of the
biofilm.
[0096] Polysorbate surfactants may be used such as Polysorbate 20 (often
known as Tween 20), a nonionic surfactant formed by the ethoxylation of
sorbitan
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before the addition of lauric acid. Polysorbate 40, 60, 65, 80 or other
polysorbate
surfactants may be used with other surfactants. Examples of nonionic, anionic,
cationic, ampholytic, zwitterionic and semi-polar nonionic surfactants are in
U.S.
Pat. Nos. 5,707,950 and 5,576,282,. Another nonionic class of surfactants is
the
TEGITOLTm series of surfactants of Dow Chemical (Midland, MI). Other known
surfactants include alpha olefin sulfonates (AOS), cocamide MEA (CMEA),
cocamidopropyl betatine (CAPB), lauryl alcohol ethoxylates, lauryl amine
oxide,
sodium coco sulfate, sodium lauryl ether sulfate, sodium lauryl sulfate, etc.
Examples include are alkylamidopropyldimethylamine oxides such as
NEOMINOX CPG and NEOMINOX LP of Oxtiteno.
[0097] Anionic surfactants include alkyl alkoxylated sulfate surfactants
that are
water soluble salts or acids of the formula RO(A),S03M wherein R is an
unsubstituted Cio-C24 alkyl or hydroxyalkyl group having a Cio-C24 alkyl
component, A is an ethoxy or propoxy unit, m is greater than zero, typically
between about 0.5 and about 6, and M is H or a cation which can be, for
example,
a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.),
ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as
alkyl propoxylated sulfates BIO-TERGE AS-40 of Stepan Co. (Northfield, IL)
are
also contemplated.
[0098] Cationic surfactants include water-soluble quaternary ammonium
compounds and various compounds with ethylene oxide moieties. The surfactant
may further comprise a co-surfactant selected from the group of primary or
tertiary
amines such as amines. Other amines to be considered include 1-octylamine, 1-
hexylamine, 1-decylamine, 1-dodecylamine, C8-Ciooxypropylamine, N coco 1-
3diam inopropane, coconutalkyldimethylamine, lauryldimethylamine, lauryl
bis(hydroxyethyl)amine, coco bis(hydroxyehtyl)amine, lauryl amine 2 moles
propoxylated, octyl amine 2 moles propoxylated, lauryl
amidopropyldimethylamine, C8-C10 amidopropyldimethylamine and Clo
amidopropyldimethylamine. Also to be considered are n-alkyl amines. Such
amines for use herein may be selected from 1-hexylamine, 1-octylamine, 1-
decylamine and laurylamine. Other primary amines include C8-C10
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oxypropylamine, octyloxypropylamine, 2-ethylhexyl-oxypropylamine, lauryl amido
propylamine and amido propylamine.
Regimens for Odor Control, Including Washing Machine Cleaners
[0099] The
treatments described herein can be used as part of a regimen for
odor control that includes not only applying pre- and post-treatments to
laundered
clothing, but can also be used in conjunction with personal deodorants,
particularly with substantially aluminum-free and zirconium-free deodorants
that
reduce odor by enhancing the skin microbiome under the arms or on any part of
the body where it is applied. Of particular value is use of the compositions
described in US Pat. Application Ser. No. 63/066426, filed Aug. 17, 2020.
[00100] Such a regimen can also include periodic sanitizing of a washing
machine to reduce the risk of microbes being transferred to clothing. In one
aspect, the laundry treatments are packaged with a washing machine treatment
such as a powder, liquid, or tablet that can be placed in a washing machine
and
run in a cycle not to launder clothing but to attack biofilm and microbes in
the
machine. Such a machine cleaner may comprise from 1`)/0 to 80% NAC with
suitable excipients (for tablets), panthenol, detergents, buffering agents,
etc., such
as a tablet comprising NAC, a binder such as magnesium stearate or a starch
compound, sodium carbonate or other salts, catechins such as EGCG, and a
detergent. In a related aspect, a solid cleaning tablet comprises 5-50% NAC,
optionally 1-10% panthenol, optionally 3-20% boric acid or borax, 1-10% sodium
carbonate peroxyhydrate or other peroxides, 1-10% sodium carbonate or other
alkaline salts (sodium bicarbonate, potassium carbonate, sodium hydroxide, and
so forth), and suitable excipients. An effective concentration of quaternary
amines
and other antimicrobials including CSAs can also be employed to attack
microbes.
Surfactants and other agents may also assist. In another aspect, a composition
for the cleaning of biofilm material in washing machines for clothing and/or
dish
washers or other washers comprises: from 1`)/0 to 50% of a biofilm attach
agent
such as NAC, panthenol, and a catechin, such as 3% to 30% NAC and 1% to
25% panthenol; from 1`)/0 to 30% of boric acid, a borate salt such as sodium
borate, or a combination thereof; from 5% to 20% sodium carbonate; from 2% to
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25% sodium carbonate peroxyhydrate; up to 20% of a laundry enzyme or other
enzyme described herein such as an enzyme mix in powder form comprising
protease, cellulase, mannanase and lipase; and up to 25% of an anionic,
cationic,
or nonionic surfactant. In one aspect, all the ingredients are provided in a
single
integral product that may comprise solid and liquid portions embedded in a
water
soluble polymeric film similar to that used in TIDE pods or CASCADE
dishwasher pods, with the liquid portion comprising an enzyme solution, for
example. A 2-step process may also be employed comprising first spraying the
interior of a washing machine with an enzymatic composition comprising enzymes
and surfactants, optionally also comprising bacterial spores and one or more
biofilm attack agents and/or antimicrobials, and after allowing the sprayed-on
enzymatic mixture to reside for a predetermined time such as 1, 2, 4, or 6
hours, a
wash cycle is then started which uses the solid ingredients that may be in the
form
of a solid tablet, capsule, or powder that is added to the interior, or which
may be
in liquid form or both. The water soluble film that may encapsulate all or
part of the
ingredients may comprise polyvinylalcohol (PVA) or a derivative of PVA, and
may
contain from 5 to 20% water when encasing dry ingredients or from 10% to 60%
water when encasing liquids. MonoSol (Merriville, Indiana) is a producer of
such
water-soluble films. Related materials are described in US Patent No.
10443024.
Segregated Materials
[00101] In treating surfaces including textiles, segregated materials may be
provided. For example, one material may be in the form of a powder or tablet
in a
first container such as a plastic pouch, blister pack with a foil seal, a foil
sealed
packet, plastic or glass bottle or tube, a shaker similar to a salt shaker for
shaking
powder onto a surface, and so forth. A tablet may be an effervescent table
that
can be added to a quantity of water or other liquid to rapidly dissolve to
form a
solution. Sealed packets or pouches may be opened by tearing, popping a
blister,
peeling a peelable layer, and so forth. Packets. pouches, or pods may also be
made with a water soluble film such as the films used in detergent "pods" such
as
TIDE Pods or other films such as polyvinyl alcohol films, films made from
starch,
cellulose, or derivatives thereof, etc., including WATERSOL@ film made by
Arrow
Green Technologies (Liverpool, UK) and SOLUBLON@ film from Aicello
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(Toyohashi, Japan). Thus, a pouch or pod containing NAC and/or other biofilm
attack agents and a segregated portion comprising enzymes and surfactants
could simply be dropped into a container of water such as a spray bottle or
other
applicator, and upon dissolving, could be applied to a target region of a
textile
item with both NAC and enzymes.
[00102] One material may be in liquid form such as a concentrate in a bottle
or
pouch or flexible container to be mixed with water. The concentrate may be
provided in a container large enough to accommodate the requisite amount of
added water to turn the concentrate into a normal strength solution, or the
concentrate may be poured or squeezed from a small container such as a
tearable pouch or small rigid container into a larger container that can hold
the
additional water needed for dilution, which may be added before or after the
concentrate is placed inside the larger container. The larger container may be
a
spray bottle or other liquid applicator for spraying, daubing, pouring,
dipping,
wiping, brushing, or otherwise applying the liquid to an item of clothing, a
textile
product, or other surface to be treated. In one aspect, a powder is provided
that
can be mixed with water in a dispensing container such as a spray battle,
sponge
applicator, roll-on, a foam applicator, etc.
[00103] Separation of two or more components of the treatment may be
designed to overcome problems were the components mixed at the time of
manufacture. In some aspects, a first component is at a relatively lower pH
while
a second components is at a higher pH. Each of these components may be
applied to the target substrate in separate steps or together. For example, a
first
component may comprise a formulation that is most effective or most stable at
low
pH, such as a mixture comprising NAC and/or a and/or a fruit pectinase or
other
enzyme having best efficacy at a pH below 7. The pH of the first composition
may
be from 1.5 to 7, from 1.9 to 6.9, from 2 to 6.5, from 2 to 5, and so forth. A
second
composition may have a higher pH such as from 7 to 12, from 7.2 to 11, from 8
to
11, etc. The difference in pH between the first and second compositions may be
at least 0.3, at least 0.5, at least 1.0, or at least 1.5, such as from 0.3 to
5, or from
0.7 to 3.5.
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[00104] The first or second composition may be applied first, followed by
application of the second application, or visa versa. In some aspects, they
may be
applied substantially simultaneously (e.g., via dissolution of the water
soluble film
of a pod having two or more isolated chambers within), or the user may be
directed to wait a period of time between application, such as from as at
least 1
minute, 3 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, an hour, 2
hours, 4 hours, 8 hours, 12 hours, etc. A time range may be provided in the
user
instructions (e.g., in the indicia associated with the product providing
directions for
use) using any of the aforementioned times as a lower limit, with an upper
limit
exceeding the lower limit by about 20%, 40%, 50%, 100%, 150%, 200%, 300%,
500%, or 1000% or more, such as time ranges of 1 to 10 minutes, 2 to 30
minutes, 5 to 60 minutes, 1 to 24 hours, 10 minutes to 1 hour, etc. A
intermediate
action may be recommended between the two applications, such as rinsing the
area being treated following the first treatment, changing the pH of the
treated
region by applying a powder or spray of an agent such as a base or acid such
as
sodium bicarbonate, vinegar, etc.
[00105] For removing biofilm in washing machines, biofilm attack agents such
as a powder comprising NAC with other solid ingredients and a solution
comprising enzymes and detergents may be combined in a unit-dose pouch or
"pod" typically held in a water-soluble film. Single or multi-compartment pods
may
be made as described in US Patent No. 9470638, issued to S. Kalaf, Oct. 18,
2016. Examples are marketed as Tide Pods, All Mighty Pacs, Purex Ultra Packs,
etc. Further details are in US Appl. No. 2010/0192986A1, US Pat. No.
6,995,126,
US Pat. No. 7,125,828, US Pat. No. 7,127,874, US Pat. No. 7,563,757, US Pat.
No. 7,964,549, US 2009/0199877A1, US Pat. No. 6,881,713, US Pat. No.
7,013,623, US Pat. No. 7,528,099, and US Pat. No. 6,727,215. A pod with
biofilm
attack agents may used in a washing machine cycle is run with or without
clothing
items to clean the machine.
Detailed Description of the Drawings
[00106] Unless otherwise stated, all percentages (%) are percentages by
weight.
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[00107] FIG. 1 depicts a shirt 10 having a main body 12, sleeves 20, a collar
18,
a lower hem 16, buttons 14 for fastening, etc., with underarm regions 22.
Depicted
generally in the underarms region 22 is a malodorous biofilm region 24,
present in
both underarm regions 22. A handheld UV lamp 22 with multiple LED UV lights
forming the illuminating face 28 of the lamp 26 is shown shining UV light
toward
an underarm region 22 to assist in visualizing the biofilm region 24. If the
shirt has
been repeatedly washed in a detergent with optical brighteners or has had an
optical brightener solution applied to it followed by rinsing, the
preferential
absorption of the optical brightener onto the biofilm may cause the biofilm
region
24 to fluoresce, even under normal lighting conditions but most clearly
visible in
dim light or darkness as the UV light is applied. When repeated treatments of
compositions described herein are needed, periodically inspecting fluorescence
can assist in gauging progress as biofilm size and fluorescence intensity is
gradually reduced. In some cases, a single treatment may show dramatic change.
[00108] FIG. 2 shows the shirt 10 of FIG. 1 but instead of biofilm regions (24
of
FIG. 1), a wetted region 36 in an underarm region 22 indicates where the
liquid in
a spray bottle 30 is being applied as a spray 38 using a trigger spray
mechanism
32. The liquid may comprise the enzymatic mix of detergents, enzymes, and
optional bacteria or bacterial spores described herein, with or without
additional
biofilm attack agents such as NAC, panthenol, catechins, and/or biofilm
modification agents. After spraying, the user may be instructed to wait about
15 or
30 minutes before washing.
[00109] FIG. 3 is a flowchart 40 for one treatment method. In Step 42, a user
selects a clothing item suspected of having persistent odor and/or a biofilm.
In
Step 44, the user may detect evidence of a biofilm using UV visualization or
other
tools (these may include confocal microscopy, fluorescence microscopy, dye
staining with Crustal Violet, Congo Red, or other suitable dyes, inoculation
of a
sample from the item of clothing, etc.) to guide Step 46, the application of
an
enzymatic mix and/or biofilm attack agents to the locations likely to have
biofilm.
Then in Step 48, the item of clothing is washed and afterwards in Step 50, UV
or
other tools are applied to examine the remaining regions having apparent
biofilm
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for comparison to the previous findings from Step 44, followed by Step 52,
wearing or continuing monitoring and treating the item of clothing in
additional
cycles as needed. The result of the treatments should be not only reduced
biofilm
presence, but reduced malodor.
[00110] FIG. 4A depicts a roll-on dispenser 80 comprising a cap 92 and a
dispenser body 90 containing either (1) the enzymatic mix described herein,
(2)
the enzymatic mix coupled with a biofilm attack agent such as at least one of
NAC, a flavanol solution, and a lysozyme inside (not shown), (3) a biofilm
attack
agent without laundering enzymes in a suitable carrier such as a base of water
and a glycol, diol, or suitable solvent and surfactants or other agents, or
(4) a
freshener comprising at leat one of an odor neutralizer such as cyclodextrin
or
cucurbituril compounds, a soya-based quat, an antibacterial agent, optionally
with
suitable fragrances, or a combination of any of the above, that can be
dispensed
using a roll-on ball 84, held in place with a roller body 86 which fits into
the upper
end of the dispenser body 82. The cap 92 attaches to the dispenser body 90 by
engaging threads 88 thereon when it is twisted in the proper direction. The
roll-on
dispenser 80 can be used to apply a solution to clothing prior to laundering
or
directly onto clothing after laundering or between washes as a freshener. In
some
cases, it may be applied to clothing as the clothing is being worn. The
contents
may be refilled by opening a bottom cap (not shown) or unscrewing the roller
body
86 or other portion of the dispenser 80.
[00111] FIG. 4B depicts a unit-dose pouch or "pod" 92 comprising a water
soluble film 93 which contains first and second segregated portions, 94 and
96,
each of which contain segregated materials, 95 and 97, respectively. First
material 95 may be a solid such as a powder or solid capsule comprising NAC
and optionally other biofilm attack agents, antimicrobials, buffering agents,
cleaning agents such as borax, boric acid, borax, sodium carbonate
peroxyhydrate, etc., and second material 97 may comprise enzymes, surfactants,
bacterial spores, with suitable chelants, solvents, rheology modifiers,
builders, and
the like. The mass of each segregated material may range from 3 g to 200 g or
greater, as needed. One or more pods may be placed in a washing machine such
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as a clothes washer, dish washer, or other device and run with or without the
normal contents to help reduce sources of biofilm.
[00112] FIG. 5 depicts a combined assembly 100 for combination in a single
package (not shown) of a first container 102 with a cleaner composition compri-
sing at least one of NAC, a suspension of bacteria or spores such as Bacillus
subtiliis, two or more laundering enzymes, panthenol, at least 5% surfactant
such
as from 10% to 70% alkyl glycosides, and a second container 104 comprising a
biofilm attack agent and/or a biofilm modification agent. The contents of the
second container 104 may be a liquid, a slurry, a paste, a powder, capsules,
etc.,
and may comprise a desiccant. The assembly 100 also comprises a blister pack
106 or other packaging system (film or paper packets, pouches, or sachets,
sprinkle capsules, ordinary capsules, tablets such as effervescent tablets in
a tube
or foil pack, etc.) for providing solid material such as tablets or units of
powder.
Three tablets are shown here as 108A, 108B, and 108C with a blister pack
backing 110 holding them together. The tablets or units 108A, 108B, 108C can
be
detached from the blister pack 106 and placed in or emptied into a washing
machine to run a sanitizing cycle, in which the antimicrobial ingredients of
the
tablets 108A, 108B, 108C can be effective in reducing microbes dwelling in the
washing machine (not shown). Alternatively, the tablets may be added to a
liquid
or other mixture such as a spray made from the cleaner composition of the
first
container 102 or a biofilm attack preparation made from the biofilm attack
powder
of the second container 104 to enhance their function, or may be added to a
washing machine for use in a wash cycle in which articles of clothing therein
have
been pre-treated with the cleaner composition of the first container 102 or
the
biofilm attack agent of the second container 104.
[00113] The assembly 100 may also include a deodorant or antiperspirant
product (not shown) designed to enhance the skin microbiome to reduce body
odor, particularly one with synergistic benefits with the other components,
such as
one comprising a mandelic acid composition having at least 0.5% mandelic acid
in
a cream, stick, roll-on, wipe, spray, or other format, such as LUME
Deodorant.
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[00114] FIG. 6 depicts an assembly 120 for a two-part system. A first
container
122, a laundry pre-treatment spray, holds an enzymatic mix comprising one or
more surfactants, optional bacteria or bacterial spores such as Bacillus
subtilis,
and a mixture of three or more classes of enzymes comprising protease and at
least two of lipase, amylase, cellulase, mannanase, and pectinase having a
total
protein content of at least 1 A such as from 1 A to 15% or from 2% to 10% or
from
1 A to 8%. The first container 122 is in the form of a trigger spray bottle
having a
trigger spray mechanism 124 with a trigger 126 operable by one or more fingers
and a spray nozzle 128 such as an adjustable nozzle with multiple settings
(e.g.,
jet, spray, on, off) which may have a foaming screen over the nozzle to
promote
foam formation which in some cases may help reduce the risk of creating minute
aerosol droplets of enzymes that might be inhaled. Also shown is a packet 140
142 containing a powder 144 that may comprise NAC and/or other biofilm attack
agents. The packet 140 may be torn open and emptied into a second container
152 or, when made from a water soluble film, may simply be placed in the
second
container 152 with water, where it will dissolve and can be applied via the
trigger
spray mechanism 154 with its trigger 156 and nozzle 158 in liquid
communication
with the contents of the second container 152. The biofilm attack powder may
comprise at least 10% NAC such as from 10% to 80% NAC or substantially pure
NAC only. It may further comprise from 0.1 A to 50% panthenol such as from 1 A
to 35%, 1 A to 25%, 1 A to 10% or 1 A to 5% panthenol or derivatives thereof.
It
may further comprise agents to adjust pH, ionic strength, viscosity, wetting
angle,
rheology, aroma, etc., such as metal salts including magnesium hydroxide,
magnesium oxide, magnesium sulfate or citrate, magnesium acetate or chloride,
sodium chloride or citrate, sodium bicarbonate or carbonate, potassium
chloride
or hydroxide, etc., wherein the metal salts may comprise from 0.05% to 20% of
the composition. In one aspect, the composition may comprise from 0.05 to 15%
each or from 0.1 A to 10% each or from 0.3% to 6% each of one or more
magnesium salts and one or more sodium salts. The pH of the biofilm attack
solution when diluted according to instructions may be from 2 to 10, such as
an
acidic formulation with a pH range of from 2.5 to 6.5, from 3 to 5.5, or from
3 to
4.8, or less than 4.5. Other pH ranges contemplated include from 3 to 9, 4 to
9, 5
to 9, 6 to 9 and 7 to 9, or from 3.5 to 8.5 or 4 to 9. In another version, the
biofilm
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attack composition 144 comprises caffeine, such as from 0.5% to 12%, or from
1`)/0 to 5% caffeine. Caffeine may have an inhibitory effect on biofilm
formation.
[00115] For aspects in which a solid material such as an NAC-containing
mixture is combined with water to make a biofilm attack agent, the packaging
may
comprise loose powder in a container that is scooped or metered to be combined
with water in a container. Alternatively, solid particles may be combined in a
tablet
that can be dissolved in water, including an effervescent tablet that with NAC
and
a carbon-dioxide releasing material such as sodium carbonate. In another
aspect,
the solid particles may be provided in capsules with water-soluble shells that
can
be dropped into water to form a biofilm attack solution. In a related aspect,
capsule shells can be discarded rather than dissolved. "Sprinkle capsules"
(not
shown) may be used in which large capsules can be gripped and twisted to cause
separation and release of contents. Such capsules are described in US Patent
No. 10610490. They may be made of insoluble material such as polypropylene
(PP), PET, high density poly ethylene material, metal, aluminum, and glass.
[00116] Figure 7 depicts a spray bottle 180 containing a biofilm attack agent
shown as a powder 144 inside a packet 140. The packet 140 is made from a
water soluble film 194 such that when water is added to the spray bottle 180,
the
film 194 dissolves allowing the internal powder 144 to also dissolve and turn
the
water into a biofilm attack solution (not shown). The bottle 180 comprises a
spray
bottle body 182, a cap 184 having a nozzle 186 and a spray button 196, from
which descends a dip tube connector 190 joined to a dip tube 192 descending
into
the interior of the spray bottle body 182, providing fluid communication
between
any fluid (not shown) inside the spray bottle body 182 with the spray nozzle
186,
wherein depressing the spray button 196 causes pumping of the liquid through
the
dip tube 192 to the nozzle 186. The remaining figures are described below.
FURTHER DETAILED DESCRIPTION
Sprays and Dispensers
[00117] Aqueous solutions described herein can be applied with a variety of
spray, including aerosol sprays driven by a propellant such as butane; pump
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sprays driven by manual spray pumps with levers, squeezable handles, push
buttons, or other systems. Micron-sized spray droplets or larger may be useful
in
reducing the production of aerosols that can increase human inhalation of the
spray. Coarse sprays can be useful in this regard. Mesh layers may be placed
over a nozzle to induce foaming and reduce the risk of fine aerosol droplets
comprising enzymes. Liquid dispensers can also be used to deliver small,
controlled quantities of liquid without creating aerosol droplets. Examples
include
known liquid pumps, airless pumps, soap dispensers, etc., including those
described in US Patent No. 9248462.
[00118] A variety of dispenser forms may be used, such as roller ball (roll-
on)
type bottles, such as those described in US Patent No. 10206479. Bottles with
sponge or perforated tops may be used, such as tops with silicone, rubber,
neoprene, or various thermoplastic elastomers or thermoset elastomers. These
may be used to provide scrubbing action during or after application of a
bioenzymatic liquid or slurry composition. Thus, a molded scrubbing unit
comprising elastomeric or non-elastomeric elevated nubs, bristles, or other
elements may be used, and the scrubbing unit may have one or more holes to
allow delivery of the cleaning composition during scrubbing or application of
friction. Examples of related scrubbing units include the elastomeric nubs on
top
of the OxiClean TM MaxForce TM Gel Stick marketed by Church & Dwight Co.
(Ewing, New Jersey) and the Cosmogen Maxi Squeeze'n Scrub body and face
scrubbing product with elastomeric bristles manufactured by Cosmogen (Paris),
believed to be related US Patent Appl. No. 20100028070. Containers with
applicator heads for dispensing the contents may also be provided with on/off
features to seal or open the container, including the use of twist caps or
other
means to open or close a dispenser, as in US Patent No. 8573875.
[00119] An ultraviolet LED or other UV light may be combined with the
applicator, integral with or separate from the applicator, to provide UV light
(e.g.,
with a wavelength from 340 to 410 nm) to assist in visualizing biofilm that
may be
fluorescent due to absorbing optical brighteners from typical laundry
detergents.
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Directions for Use
[00120] Indicia placed on or otherwise associated with packaging may inform
users of the benefits of the product, call attention to the relationship
between odor
on clothing, bacteria on the clothing, as well as possible relationships to
bacteria
on the skin and bacteria that may be present in washing machines or other
locations that may affect clothing. Indicia may also provide guidelines for a
regimen that can result in long-lasting reduction in odor on clothing and the
body,
including steps to take to treat clothing before laundering, steps to treat
clothing
after laundering or between washes, steps to take to treat armpits or other
parts of
the human body with products such as LUME Deodorant for Underarms and
Private Parts, and steps to take to mitigate bacteria in washing machines or
other
sources that may influence bacteria and odor on clothing and/or the human
body.
[00121] Indicia may be placed on the packaging material holding a container of
a composition such as an outer cardboard box, or may be placed on the
container
that directly holds the composition (e.g., a squeezable tube, a plastic or
glass jar,
a spray bottle, a foam dispenser, a tube of wipes, etc.). Alternatively or in
addition,
instructions for use may be associated with the product in a variety of ways
other
that directly printing on a package. The instructions may be provided on
printed
material that is distributed with the product but physically detached
therefrom, or
may be on a website or other information source that is associated with the
product (e.g., accessible via a QR code, barcode, RFID tag, or URL printed on
the
package). Information about the product and its use may also be approved in
promotional media such as in television commercials promoting the product.
[00122] An example of such indicia could be: "Apply the Pre-Treatment Spray to
the most odorous parts of clothing (e.g., the armpit area) before tossing into
a
hamper, leaving at least 30 minutes before laundering for best results. If
odor
persists, use the Between Washes Freshener Spray to treat the smelly regions
and allow to dry for about 5 minutes before wearing. To fight odorous bacteria
residing in low-temperature washing machines, run a cycle once a week with the
Germ Foe TM Washing Machine Tablet with warm or hot water (if available).
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Meanwhile, don't forget to use Lume Deodorant for Underarms and Private
Parts on your body regularly to reduce bacterial sources of malodor."
FURTHER EXPERIMENTAL WORK
Enzymatic Sprays
[00123] Several enzymatic solutions were made: First was an enzymatic blend
labeled El, comprised a buffered solution of Novozymes enzymes for laundry
detergent in a buffered solution with surfactants and bacterial spores from J-
Zyme TM AB-20X NFC distributed by J Tech Sales (Boca Raton, FL), said to
employ spores from Nozozymes. The solution comprised about 20% J-Zyme
which is said to have about 1.1 x 109 CFU/ml of bacterial spores. This
consisted
substantially of water, a probiotic bacteria blend believed to comprise
Bacillus
subtilis spores; enzymes from Novozymes including protease, amylase, pectate
lyase, mannanase, 2 types of cellulase, and lipase; alkypolyglucoside from
sugar
feedstock, sodium citrate, sodium bicarbonate, 1,3 propanediol from natural
feedstock, probiotic bacteria blend, and preservative (0.1 A of a blend of
methylchloroisothiazolinone and methylisothiazolinone). Total enzyme concen-
tration was about 2% by weight. Optimum activity is at a pH of about 7-8.
[00124] E2: A blend similar to El but without lipase and with the addition of
a
gentle quat, soyaethyl morpholinium ethosulfate. Ingredients included
naturally
derived surfactants (from sugar), probiotic bacteria, an enzyme blend
containing
protease, amylase, pectate lyase, mannanase and cellulases (no lipase); a
solvent system made from naturally derived glycerin that also served as an
odor
control agent, and soyethyl morpholinium ethosulfate. The concentration of the
quat was about 0.5% and the enzyme concentration was about 2%.
[00125] E3: a blend made from a mix of enzymes, with a total of 5% enzymes
comprising pectinase, amylase, mannanase, protease, lipase and cellulase. The
solution comprised 20% glucopon-like surfactant from 100% biobased alkyl
polyglycosides, sodium citrate and sodium bicarbonate for buffering to a pH in
the
7-8 range, propanediol, a mix of bacterial spores approved for bio-enzymatic
cleaning from a 10X concentrate comprising Bacillus subtilis spores, a solvent
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system derived from naturally derived glycerin and as an odor control agent,
and
a suitable preservative known to be compatible with the bacterial spore mix.
[00126] E4C: This blend is a 4:1 concentrate intended upon dilution to give a
solution similar to E3, but with slightly reduced surfactant levels. Upon 4:1
dilution,
the concentrated E4C solution was diluted to normal strength and dubbed E4D.
[00127] E6C is another 4:1 concentrate intended upon dilution to give a
solution
similar to E3, but with slightly reduced surfactant levels to facilitate the
concentrate form and less lipase. This concentrated enzyme blend has about 15%
liquid enzyme mixtures comprising pectinase, amylase, mannanase, protease,
lipase and cellulase (the liquid enzyme mixtures themselves are estimated to
have roughly 40 to 60% protein), about 30% surfactants comprising biobased
alkyl polyglycosides, salts such as sodium citrate and sodium bicarbonate,
propanediol, a mix of bacterial spores approved for bio-enzymatic cleaning
from a
10X concentrate, a solvent system derived from naturally derived glycerin, and
a
suitable preservative known to be compatible with the bacterial spore mix.
Upon
dilution (3 parts water to 1 part E6C) the result is E6D ("D" indicates
"diluted").
[00128] EN2: 11.1 ml of KOH 0.1M solution was combined with 11.8 g of 1.8%
NAC solution at pH 3.0, giving a pH of 4.85. Then 20.4 g of this solution was
combined with 39 g of NIC solution, giving a pH of 7.00
[00129] Other enzymes used included:
= Pectinase from Phygene Biotechnology Co (Fuzhou,Chna), product
PH1561, activity > 500u/mg, CAS 9032-7501.
= Pectinase in Kitchen Alchemy Pectinex Ultra SP-L solution from
Modernist Pantry, LLC (Eliot, Maine).
= Pectinase powder ("pectic enzyme"), L.D. Carlson Co. (Kent, Ohio).
= Alpha-amylase from BOSF (1,4-alpha-D-glucan glucanohydrolase),
10kU/g, CAS 9000-90-2, EC 232-565-6, powder form.
= Amylase powder, BSG (Shakopee, Minnesota), product 10019.
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= Papain, Phygene Biotechnology Co (Fuzhou,Chna), product PH9028,
activity > 800u/mg, CAS 9001-73-4.
= Papain tablets, Beazyme brand, MCM (Malaysia Chemical Company,
Kuala Lumpur), 150,000 USP, purchased in Kuala Lumpur, Malaysia.
= Lipase (triacylglycerol acylhydrolase) from Candida rugosa, Ekear Co.
(Shanghai, China), product P0114, CAS 9001-62-1
= Cellulase, Phygene Biotechnology Co (Fuzhou,Chna), product PH9018,
activity > 400u/mg, CAS 9012-54-8.
= Cellulase powder from Heshibi Biotech, China, activity 100,000u/g.
= Cellulase powder, Henan Wan Bang Ind. Co. (Henan Province, China).
= Cellulase powder, Zhejiang Yiruo Biotech (Zhejiang Province, China).
= Cellulase powder, Shandong Longda Biotech (Shandon Province, China).
= Cellulase powder, Yin brand (China).
= Lysozyme from egg whites, Bomei Biotech, activity > 20,000u/mg.
= Lysozyme chloride, Homecare Noflux brand, 90 mg per tablet, purchased
in Kuala Lumpur, Malaysia.
= E-Zyme Troche lysozyme chloride tablets, 200 mg each, from AV
Manufacturing S/B (Malaysia) purchased in Kota Kinabalu, Malaysia.
= NattoEnzym: nattokinase powder purchased in Hanoi, Vietnam marketed
by DHG Pharam (Can Tho City, Vietnam), made from nattokinase from the
Japanese Nattokinase Association (Osaka, Japan).
Other Enzyme Solutions
[00130] AmylaseA: BSG amylase, 1.137 g and BOST amylase, 0.717 g, were
stirred unto 28.0 ml of water.
[00131] CellulaseA: 1.1 g of Shandong Longda cellulase powder and 0.45 g of
Phygene cellulase powder were mixed into 22 ml of water.
[00132] CellulaseB: 1.3 g of Heshibi cellulase powder + 29 ml water.
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[00133] CellulaseC: 1.8 g of Wanbang cellulase, 0.38 g of Phygene cellulase,
1.10 g of Yin cellulase, and 1 g of Heshibi cellulase were mixed on 53.5 ml
water.
[00134] CellAmyIA: 12.19 g of 2% NAC at pH 6.14 was combined with 5.2 g of
2% NAC at pH 9.17, 17.69 g of CellulaseC, 4 g of 2% NAC at pH 6.4, a few
grains
of citric acid to bring the pH from 9.5 to 8.82, then 0.55 g BOSF amylase
powder.
[00135] LysoA: 1 tablet of E-Zyme lysozyme chloride (200 mg) + 8.5 ml water.
[00136] LysoPap: Grind one table of Homecare Noflux lysozyme chloride (90
mg of lysozyme) with one tablet of MCM Beazyme papain into 12 ml of water.
[00137] LysoB: Pulverize 2 tablets of E-zyme Troche lysozyme chloride (200 mg
each) and dissolve in 30 ml water.
[00138] PAPA: 1.05 g of Phygene papain was combined with 21.7 ml of water.
[00139] PAPB: 1.3 tablets of MCM papain were ground and dissolved into 15 ml
of water. Some residual solids remained after heating. The slurry was passed
through a fine cloth to filter out some solids. 13 g of solution were
obtained.
[00140] PAPC: 5.65 g of papain from Pangbo Enzymes (Nanning Pangbo Biol.
Eng. Co.), 10,000 U/g, was combined with 53 ml of water. (p.52)
[00141] PANNAC: 33 ml of 3.6% NAC, pH 4.9, mixed with 0.643 g panthenol.
[00142] PANNAC2: 3.6 g of NAC and 1.65 g of panthenol powder were
combined in 108 ml of water, with 2.1 g of NaHCO3 added to reach a pH of 4.7.
[00143] NattoNAC: 0.6 g of commercial nattokinase powder was purchased in
Hanoi, Vietnam under the brand name of NattoEnzym marketed by DHG Pharam
(Can Tho City, Vietnam), made from nattokinase from the Japanese Nattokinase
Association made by Japan Bio Science Laboratory (Osaka, Japan). Capsules
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with 0.6 g of powder, said to have 670 FU (fibrin units, a measure of activity
based
on fibrinolytic activity) per capsule, were used.
Biofilm Attack (BA) Agents
[00144] Pretreatments to attack biofilm were made as follows:
[00145] NAC-AL: To test the interaction of allantoin with NAC, 0.75 g of NAC
were combined with 0.24 g allantoin in 46.5 g hot water. The pH was 3.16. The
characteristic sulfur odor of NAC appeared to be absent, suggesting that
allantoin
may be useful in reducing the odor of NAC solution.
[00146] EGCD-1: 1.20 g 98% EGCG powder (N&R Industries, Xian, China) was
combined with 0.8 g ascorbic acid, 0.53 g citric acid, 1.51 g of hydroxypropyl
beta-
cyclodextrin, in 128 ml of water, heated to about 40 C and stirred. Similar is
EGCD-2: 1.72 g of EGCG powder with 1.03 g of ascorbic acid powder, 0.86 g of
citric acid, and 0.70 g of hydroxypropyl beta-cyclodextrin in 118 ml of water.
[00147] EGCD-A: 27.8 ml of EGCD-1 solution was mixed with 10 ml 70%
ethanol. Similar is EGCD-B: 29 ml of EGCD-2 solution are withdrawn and
combined with 10 ml of 70 wt% ethanol and put in a spray bottle to give spray
EGCD-B. This displayed excellent color stability after multiple weeks.
NAC Solutions:
[00148] NAC powder was dissolved distilled water to give a 2.1% strength
solution, a 1% solution, and a 20% solution. One NAC solution at 1.4%
concentration was adjusted with citric acid and sodium carbonate to achieve a
pH
of 3Ø A NAC solution at 2% was made from 3.9 g of NAC in 185 ml of water.
1.0
g NAC plus 15 ml of 0.1 M KOH solution was prepared with 35 ml water, with a
pH of 3.55. Na2CO3 was then added (0.18 g) to bring the pH to 7.8. Another 2%
NAC solution was prepared with KOH added to give a pH of 8.19 in 51 ml of
water, to which another 0.22 g NAC was added to bring the pH down to 4.33.
Adding 6 ml of 0.1M KOH solution brought the pH to 6.4. Another 2% NAC
solution was prepared with KOH added to give a pH of 6.4.
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[00149] EGCG-NAC solution was made by combining 1.40 g of EGCG powder
with 1.69 g of NAC in 50 ml of water, and placed in a 100 ml spray bottle.
[00150] 28.1 ml of N IC (Naturally It's Clean()) enzyme solution was mixed
with
0.36 g NAC and 0.16 g sodium carbonate to give a pH of 8.37. This was adjusted
by adding 0.04 g NAC to give a pH of 8.08. This is labeled 1.4% NAC in NIC.
[00151] A 2.1% NAC solution at pH 4.0 was made using 1.51 g of Biotal NAC,
0.648 g NaHCO3, and 71 ml of water.
Pectinase Solutions
[00152] PNAC1: 0.2 g of BOSF pectinase powder (believed to be a fruit
pectinase best suited for operation around a pH of 4 to 5, unlike typical
laundry
detergent pectinases which are engineered for higher pH solutions such as from
7
to 9) having an activity of 10 kU/g was combined with 0.41 g NAC powder and
0.13 g sodium bicarbonate. The powder was prepared and mixed, and placed in
dry form into a sealed 100 ml spray bottle. After a period of time, distilled
water
was added, 57 ml. The mix dissolved rapidly at 22 C. The pH was 6.7. To better
optimize performance of the pectinase, 0.21 g of NAC was further added to the
solution plus 0.32 g ascorbic acid, bringing the pH to 4.6. This was spray
PNAC1.
[00153] PNAC2: In 55.7 ml of water, 0.22 g of pectinase powder from BOSF
was added with 0.44 g of NAC powder to form a pectinase-NAC solution having a
pH of 3.2. Then in 12.65 ml of water, 0.26 g sodium bicarbonate was added. 4.5
ml of this solution was added to the pectinase-NAC solution, bringing the pH
to
4.71. This was adjusted by adding 0.06 g NAC, giving a pH of 4.08. This was
put
into a 100 ml spray bottle and labeled PNAC2.
[00154] PNAC3: 0.6 g NAC was combined with 0.5 g BOSF pectinase powder
with 0.13 g sodium bicarbonate and 0.1 g ascorbic acid. The powder mix was
then
combined with 60 ml of water (references to water are generally to distilled
water
unless indicated otherwise). The pH was 4.31, believed to be suitable for the
fruit
pectinase used, but generally too low for typical laundry enzymes.
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[00155] PNAC4: 1.14 g NAC powder was combined with 0.88 g of pectinase
powder (BOSF polygactouronase, product G0200, CAS 9032-75-1, EC 232-885-
6, > 10kU/g), 0.08 g citric acid powder, and 0.5 g NaHCO3 in 106 ml of water,
resulting in a solution with a pH of 4.38, generally suitable for fruit
pectinases.
[00156] PNAC5: Combine 1.256 g NAC with 1.033 BOSF pectinase
(polygalacturonase) into 106 ml g water.
[00157] PMIX1: 0.174 g of Phygene pectinase, 0.802 g of Pectinex solution,
and 24.5 ml of water were combined to create PMIX1 solution.
[00158] Lysozyme Solutions: Three E-Zyme Troche lysozyme chloride tablets,
200 mg each, from AV Mfg. (Malaysia) bought in Kota Kinabalu, Malaysia, were
ground and dissolved in 51 ml of water to form 1.2% lysozyme solution, LYS1.
Steps Toward Perma-Odor
[00159] An effort was made to artificially create persistent odor problems in
several shirts, including the following shirts purchased at second-hand store
in
Shanghai:
= Shirt RD1: a red 100% polyester sports shirt under the Voit brand
= Shirt RW1: a striped shirt made from 75% cotton and 25% polyester.
= Shirt Dec1: A 100% polyester men's top made by Decathlon (China).
= Shirt Ml: Maroon shirt with 47.5% Modal, 47.5% cotton, and 5% spandex.
= Shirt BS1: a black casual short-sleeved shirt made from 100% polyester
(purchased new in Shanghai but worn for four years prior to this work).
[00160] Two malodor sprays were created to add malodor and a biological load
in an attempt to infect clothing with malodor sources formed from mixtures of
odorous French cheeses, meat extracts, soy broth, etc. Several shirts with
persistent odor were eventually brewed with such mixtures applied to the
shirts for
prolonged times. Once persistent odor was detected (odor that remained even
after washing with commercial laundry detergents). The primary treatment was
application of about 0.5-1.2 g of bacteria-rich Fourme D'Ambert cheese total
to
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the both armpits followed by application of a solution with meat extract, and
keeping the moistened shirt in a plastic bag for several hours. After several
such
treatments, Dec1 developed persistent malodor in both pits after washing. Then
the right pit was sprayed with 1.6 g of 1.8% NAC solution (pH about 2) and
washed with a standard wash cycle requiring 78 minutes in a Siemens front-
loading washer, using Bright Blue Moon liquid laundry detergent, a Chinese
enzymatic detergent. Although the detergent had a fragrance, after washing,
the
left pit appeared to have no malodor nor fragrance, while the right pit
manifested
fragrance. After air drying, the left pit still had no sign of malodor, while
the right
pit had some odor. This suggested that NAC can be effective when used in
combination with other agents such as enzymes. It is also believed that
biofilm
material provides a substrate that can more readily retain many fragrances
during
washing relative to synthetic fibers alone. Thus, a reduction in retained
fragrance
after washing may be a sign of successful reduction of biofilm matter.
UK Triathlon Shirt (TR1)
[00161] A volunteer triathlon runner from the United Kingdom provided a 100%
polyester shirt suffering from persistent odor believed to be a prime example
of
perma-odor and a possible biofilm infection. The shirt, code named TR1,
received
in a triathlon event in 2013, had been worn periodically for heavy exercise
for six
years and was about to be discarded because of strong odor, even after
washing,
that would become strong after relatively short periods of exercising, unlike
new
shirts. The shirt was received after exercise, with both pits manifesting odor
levels
of about 5 on a scale of 0 to 5. The left pit was treated with Naturally It's
Clean
(NIC) Laundry Spray by Enzyme Solutions (Garrett, IN)alone, with 5.9 g
applied.
The right pit was treated with a similar amount of blend of NIC with 1% NAC at
pH
7.00. After five minutes, the shirt was rinsed in warm water at about 40 C and
then washed in a standard cycle with room-temperature water with Unilever
Comfort brand laundry detergent (Asia).
[00162] After drying the washed shirt, the right pit was substantially free of
odor
and fragrance, while the left pit manifested fragrance, again suggesting that
attacking biofilm can reduce fragrance retention clothing made from synthetic
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fibers. After Applicant exercised vigorously while wearing the shirt, the
right pit
had very little odor while the left pit had rapidly developed
uncharacteristically
intense odor. It seems the left pit suffered from perma-odor in which odor
rapidly
develops, while odor was mitigated in the right pit from the NAC treatment.
[00163] The shirt was washed in a full cycle with Bright Blue Moon detergent.
The left pit had slight fragrance while the right pit did not have readily
detectable
fragrance. After air drying, the left pit fragrance level was at about 1,
while the
right pit remained at a 0 rating. One tester detected both malodor and
fragrance in
the left pit, estimating the odor level at about 1. After two more hours, the
right pit
appeared to have some residual odor while the left pit odor was difficult to
detect.
After an exercise session, the left pit developed strong odor, a level of
about 3,
while the right pit had mild odor, about 0.5 or 1 (nearly no odor).
[00164] The shirt was again washed with Comfort brand detergent. Both pits
smelled acceptable (essentially no malodor). Then, after another exercise
session
similar to each of the two previous sessions with this shirt, followed by 1
hour of
walking, the left pit had strong malodor as it did previously, at a level of
about 3.5
or 4, while the right pit had much less malodor, at a level of about 2.
[00165] Now to treat the left pit, which appeared to have a perma-odor problem
possibly from a biofilm, a combination of pectinase and NAC was tried. 8 g of
PNAC2 spray was applied to the left pit and allowed to sit for 15 minutes at
about
22 C. The shirt was then washed with Comfort detergent in a fast cycle.
[00166] After drying, the shirt was worn for exercise similar to previous
sessions
and the pits were wetted with sweat, as usual. However, this time, there was
relatively low odor in both pits. The high odor levels created previously in
the left
pit prior to treatment with biofilm-attack agents did not occur this time, and
the two
pits were substantially similar in odor levels (around 1). This suggests that
the
treatment was successful in reducing the source of the perma-odor.
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Neon Orange Champion Shirt
[00167] A neon orange sports top for women made under the Champion
brand, code named CH1, a semi-fitted L/G, 100% polyester shirt had been in
regular use for exercise for 5 years and had symptoms of perma-odor. Slight
odor
would still generally be present after washing, would become strong after one
exercise session. Treatments with EGCD-1 solution at low pH (added citric and
acetic acid) followed by treatment with E2 showed some reduction of odor in
the
left pit, but odor still returned after exercise. Further trials were
conducted in
Borneo, Malaysia, after first finding NAC at a pharmacy in Kuala Lumpur and
hypothesizing that NAC might assist in removing biofilm in clothing. Nova
brand
N-Acetyl cysteine powder in 300 mg capsules (Nova Laboratories, Sepang,
Selangor, Malaysia) was purchased from Sunlight Pharmacy in Kota Kinabalu,
Malaysia. Each capsule contained 300 mg of acetyl cysteine and 70 mg of other
materials, believed to primarily be gelatin. 1.97 g of NAC powder removed from
the Nova brand capsules was stirred into 52 ml of water to form a 3.2% NAC
solution, slightly cloudy, which was applied to the right pit area of the neon
orange
shirt, with 2 g of NAC solution being applied to both the outside and inside
surfaces of the right side over a roughly circular area about 12 cm in
diameter.
After two minutes of dwell time, the right pit was sprayed with N IC enzyme
solution, with 2.33 g on the exterior surface and 2.5 g on the interior
surface.
[00168] The left pit of the neon orange shirt was treated with NIC solution
only,
with 3.4 g applied to the outer surface and 4 g applied to the inner surface,
for a
total of 7.4 g on the pit. After five minutes, the entire shirt was handwashed
in
warm, soapy water using a clear shampoo provided by a local hotel. After air
drying, the right pit, which previously smelled worse than the left, now
smelled
better than the left. Both smelled better than before washing, but there was
residual malodor in the left pit.
[00169] A second biofilm-attack treatment was then applied to the right pit. A
solution of NAC from an effervescent NAC tablet with 600 mg of NAC, also
purchased in a Malaysian pharmacy, was made by dissolving the tablet in 100 ml
of water. 8 ml of this solution was then applied to the right pit to
substantially
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saturate it. Then 5.3 ml of solution EGCD-A was applied to the right pit and
allowed to sit for 20 minutes before handwashing and air drying.
[00170] After an exercise session, it was observed that the right pit
continued to
smell better than the left pit. The same tendency applied to the shirt after
being
stored for 48 hours at room temperature, even though the odor intensity had
increased over this time period, with the left pit exhibiting an odor
intensity of
about 4 to 5 (0 to 5 scale), while the right pit was rated at about 2 to 3.
[00171] The right pit was then treated again. First the right pit and sleeve
were
moistened with 21 g of 2% NAC solution made from 100 ml of water and 2 g of
NAC powder extracted from Swanson's 600 mg capsules of N-acetyl cysteine
(Swanson Health Products, Fargo, ND) which also contain gelatin (capsule
shell)
and magnesium stearate. Then 5.94 g of EGCD-A solution was applied to the
moistened right pit area. After 10 minutes, the wetted region was sprayed with
7 g
of NIC solution and rinsed after about 10 minutes and handwashed with laundry
detergent and warm water. After drying, the right pit had no odor, neither
malodor
nor fragrance, while the left pit exhibited both malodor and fragrance.
[00172] After further washing and two exercise sessions, the right pit still
smelled better than the left, but both have made progress in terms of
decreased
odor levels previously experienced after one session of exercise. It may be
that
both the EGCG treatment and the NAC treatment (and possibly the NAC plus
EGCG treatment) have helped reduce the impact of a biofilm in this shirt. As
odor
developed, it was observed that the treatments (NAC + EGCG) appeared to make
the shirt display longer lasting odor reduction when treated with a freshener
after
exercising such as Oderase TM from AqDot (Cambridge, UK). After exercising and
washing, the right pit of the shirt could still develop odor after exercise,
but not as
intensely as before, while the left pit had strong odor, even after being
treated with
fresheners comprising cucurbituril and also Febreze Free (Procter & Gamble).
[00173] Now the left pit was treated with a biofilm attack protocol. 10 g of a
2%
NAC solution was applied to the pit and allowed to sit for 10 minutes, after
which 9
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g of N IC was sprayed on, sitting for 15 minutes, whereupon the shirt was
rinsed
by hand and then washed with commercial laundry detergent (Bright Blue Moon).
[00174] After further exercise, the right pit was still superior to the left
pit (odor
rating of about 1 in the right and 2 to 3 in the left). To further treat the
left pit, the
lysozyme solution LYS1 was applied, with 7.3 g of solution applied over an
area of
about 10 cm x 8 cm around the left pit. This sat for 20 minutes, then 5 g of N
IC
was applied. After a 5-minute wait, the shirt was placed in a washing machine
and
washed. Following subsequent exercise, the left pit still had mild odor,
though
reduced in comparison with previous states while the right pit had very little
odor.
After several more hours of sitting, the two pits seems roughly equivalent
when
tested again, both rated at about 2 on a scale of 0 to 5.
[00175] The right pit was then treated with 3.11 g of EGCD-B spray, followed
by
3.46 g of 1`)/0 NAC spray at a PH of 6.4. The left pit was treated with 2% NAC
at a
pH of 9.17, 3.6 g applied, followed by treatment with 1% NAC at a pH of 7.8,
2.68
g applied. Then N IC was applied to the right pit, 2.0 g, and1.76 g N IC to
the left
pit. The shirt was hand washed in warm water with laundry soap and air dried.
After an exercise session (a jog of 3 to 5 km is typical for the exercise
sessions
here), the right pit smelled better than the left. Perhaps the elevated pH NAC
solutions are less effective than the low pH solutions in opening or attacking
the
biofilm. The left pit was treated with EGCD-B spray, about 3 gm. After 3-
hours,
both pits were sprayed with 2% NAC solution, 6 g on the left and 7.5 g on the
right. After 5 minutes, the shirt was rinsed in warm water and air dried.
After two
exercise sessions, the pits had an odor level of about 5. The left pit was
treated
with N IC, 1.48 g, while the right pit was treated with EN2, 1.44 g, and air
dried.
The odor in the right pit was estimated at 3 on a scale of 0 to 5, while the
left pit
had a level of about 3.5. A second evaluator gave scores of about 2 for each
pit.
[00176] The pH 3.0 NAC solution was then applied to the right pit, 2.4 g, and
after 5 minutes, the shirt was washed in a short cycle with Bright Blue Moon
brand
detergent. After washing, both pits had slight odor, about 0.5 on a scale of 0
to 5.
After another exercise session, both pits had an odor level of about 4. The
right pit
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was then treated with PNAC3 comprising pectinase. 7.3 g of PNAC3 was applied
to the pit and surrounding region, saturating the pit area. After washing, the
shirt
was again evaluated following exercise. Both pits had low odor. But where odor
existed, it appeared to be correlated with slightly darkened zones in the
pits,
believed to be staining associated with a prior biofilm where deposits of
polysachharides, proteins, and other biofilm matter may have provided a
platform
for absorption of dyes or dyed particles. The darkened areas remained
following
the treatments with enzymes that the shirt has received, though the intensity
of
the darkened regions has declined.
[00177] Steps were then taken to reduce the darkened color regions in the
pits.
The inner right pit was treated with 3.6g of PNAC4 and allowed to sit for 5
minutes. Then 0.9 g of E2 was applied to that spot. After 2 more minutes, 1.4
g of
N IC was applied, and finally 1.4 g of 2.1% NAC was applied. The shirt was
then
washed with Comfort detergent (1.5 ounces of detergent used in a full cycle
at
40 C requiring slightly over one hour), and then air dried. After further
exercise
sessions, the shirt had odor levels of 1-2 in the right pit and 0.2-1 in the
left. The
left was then treated with 4.65 g of PNAC4, seeking to further eliminate the
residual staining in the pit. The shirt was then washed using Comfort brand
detergent. After further exercise, the dark stain region on the inner right
put was
treated with PNAC4, saturating with 3.6 g of spray. After 5 minutes, 0.9 g of
spray
E2 was applied. After two more minutes, 1.4 g of N IC spray was applied to the
pit
and finally 1.4g of 2.1% NAC were applied. The shirt was then washed with 1.5
oz
of Comfort detergent in a full cycle at 40 C. After 5 more exercise sessions
without washing, the pits now had strong malodor with an odor level of about
5.
The right pit was treated with 2% NAC solution and washed. After exercise, the
odor in the right pit was significantly reduced relative to the left pit.
Additional Shirts: Series K
[00178] A blue Decathlon sports top, KB1, 100% polyester and essentially the
same as the neon orange shirt above except for color, had also developed
strong
odor through repeated exercise and was a possible perma-odor candidate which
still had malodor after washing. The left pit was treated with EGCD-1, with
6.3 g
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applied. It sat for 10 minutes, then the shirt was rinsed and washed. After
air
drying, the treated pit smelled much better.
Additional Shirts, Series AA-AC
[00179] Several shirts from an athletic female volunteer were obtained,
including:
= Shirt AA, a pink Forever 21 shirt believed to be made from cotton and
polyester with relatively stronger odor in the right pit after prior washing.
= Shirt AB, a Downeast Basics "Wonder Tee" made from 95% cotton and 5%
spandex, a brand said by some customers to have pronounced odor
issues, perhaps due to surface sizing chemistry. After washing, both the
right and left pits had malodor.
[00180] Treatments of 2.1% NAC solution were applied. For shirt AA, 12.7 g
total was applied across both pits and adjoining shoulder area. For shirt AB,
8.2 g
was applied to the right pit and shoulder area, leaving the left pit
untreated. For
shirt AC, 8.5 g was applied to the right pit. After about 10 minutes, each
shirt was
then treated with Naturally It's Clean (N IC) enzyme spray for laundry. For
shirt AA,
a total of 11 g of spray was applied to the previously wetted areas. Further,
for the
right pit only, 2 g of E2 bio-enzymatic spray was applied. For shirt AB, 3.3.
g of
NIC was applied to the right pit followed by 5.4 g of LPS1 also to the right
pit. After
about 15 minutes of dwell time, the shirts were washed with a standard cycle
using Bright Blue Moon laundry detergent. In each case, treated pits smelled
better than before and smelled better than the untreated pits or the pits
treated
without NAC. Following exercise, the results were mixed. Shirt AB was reported
to
smell better in general. Perhaps the enzymes in the detergent and the added
enzymes and NAC present in the wash from the 3 treated shirts being washed
contributed to effective odor reduction for both pits on shirt AB. But for
shirt AA,
after one work day the right pit was reported to smell again, while the left
pit
remained smelling fresh. For this shirt, additional treatment with NAC may be
needed to achieve more complete odor reduction in the right pit.
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Perma-Odor Candidates: Series L
[00181] Exercise clothes from a heavy exerciser (male) were provided for
further testing. Several items of clothing appeared to have perma-odor, for
even
after washing some residual odor remained, and the user had noted that malodor
returns swiftly during or after exercise unlike the way the clothing behaved
when it
was relatively new. The clothing appeared to be suitable candidates for a
perma-
odor problems that may be due to a biofilm. Several approaches were tried.
[00182] Three items were involved: a red polyester shirt (shirt RL), a white
polyester Lintrel shirt (shirt WL), and a blue polyester sports shirt (shirt
BL). There
were also three pairs of washable children's shoes that had serious odor
issues
that had not been removed with previous washing, including soaking in a
solution
made from Oxi-Clean detergent with bleaching agents.
[00183] Initial treatments includes the use of E2 spray (about 3 g) on the
right
pit of RL, and a similar amount of E2 spray on the right pit of the white
shirt WL.
After sitting overnight to allow bacterial spores to activate and then being
laundered with a washing machine and laundry detergent, the treated pit of the
white shirt smelled significantly better than the other pit, while the pits on
shirt RL
both smelled about the same with some persistent odor still present. The blue
shirt, BL, had both pits smelling acceptable after the treatment and wash.
[00184] An antimicrobial agent, PureShield wound care spray, was applied to
each of the right shoes among two of the pairs, followed later by washing.
After
drying, it was observed that this treatment did not appear to have any effect.
Treatments with EGCG and NAC
[00185] Recognizing that biofilm may be present in some of the shirts, a new
round was conducted aimed at reducing the impact of potential biofilm. A three-
step program was implemented in some cases involving treatment with NAC, then
acidic EGCG solution, followed by treatment with enzymes. The hypothesis was
that the biofilm might be weakened or opened by the NAC and EGCG, allowing
the enzyme solution to more effectively remove materials that may have been
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previously deposited or protected by the biofilm and perhaps help reduce the
foothold of bacteria in the clothing. Testing showed reduction in odor after
washing and later reduced odor after further sweating during exercise.
[00186] In the first trial following the initial treatments described above,
2.1%
NAC solution was applied to the right pit of each of the shirts RL, BL, and
WL,
bringing the pits to saturation. 12 g were applied to RL, 12 g to WL, and 13.5
g to
BL. Then RL was further treated with 5.5 g of EGCD-B solution on the right
pit. BL
was treated with 6.6 g of EGCD-2 solution on the right pit, and WL was treated
with 8.7 g of EGCD-B on the right pit. After about 5 more minutes, shirt RL
had
5.0 g of N IC applied to the right pit. Shirt WL received 6.3 g of N IC
applied to the
right pit. Shirt RL was then washed in a regular wash cycle, while shirts WL
and
BL were hand rinsed in warm water. The shirts were air dried and worn. When
the
owner later reported the results, it was determined that shirts treated with
NAC
solution had significantly reduced odor after washing, and after exercise, the
odor
in the treated pit would generally be less than in the untreated pit.
[00187] In a subsequent trial, after washing and exercise, shirt RL was
reported
to have an odor level of about 2 (scale of 0 to 5) in both pits, while shirt
BL had an
odor level of about 2 in the left and 1 in the right pit. Shirt WL had very
little odor
and was reported as being significantly better than it was prior to treatment.
Shirt
RL was treated in the right pit with 9.1 g of 2% NAC at a pH of 7.8. (Prior to
treatment, odor level was estimated to be about 0.5 in the right pit and 1 in
the
left.) After about 11 minutes, N IC enzyme spray was applied with 5.8 g on the
right pit and 10 g on the left and the shirt was washed. While the residual
odor in
the shirt appeared to have been eliminated, after heavy exercise, shirt RD was
reported to have developed strong odor on both sides. It was speculated that a
different enzyme treatment might be helpful. Thus, the right pit and
surrounding
area was treated with 15 g of PNAC3 and after about one hour was washed using
liquid Tide detergent. The owner reported substantial improvement in the
shirt.
[00188] Shirt WL, manifesting odor levels of about 2 in the left pit and 0 in
the
right (following exercise) before treatment was treated with 4.8 g of 2% NAC
(pH
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7.8) in the left pit, then after 2 minutes, 6.5 g of NIC enzyme spray was
applied.
After 10 minutes, another 5 g of NIC spray was applied. After washing as usual
with liquid Tide detergent and exercising with the shirt, the owner reported
substantial improvement in both pits. After exercising, however, the right pit
had
slightly more odor than the left, so an additional treatment was conducted
aimed
at the right pit, which was treated with PNAC3 (2.2 g) applied to the center
of the
pit, and then PNAC1 (7g) applied more broadly to the pit and sleeve. The left
pit
was treated with NIC only, 4 g. The shirt was then washed. The owner noted
that
the odor problems of the past had been essentially overcome, and the shirt
could
now be used for exercise without the residual odor that had previously been
present after washing.
[00189] For shirt BL, the left pit was treated with 1.4% NAC in NIC solution,
with
9.67 g applied, and after about 30 minutes the shirt was washed using liquid
Tide laundry detergent. Following further use during exercise, the owner
reported substantial improvement. However, the left pit had slightly stronger
malodor. The left pit was then treated with PNAC1, with 11 g applied to a
broad
area around the pit followed by 1.9 g of PNAC3 in the pit area itself. The
shirt was
them washed after about an hour. After exercise, the owner reported
significant
improvement with no residual odor. Odor no longer rapidly returned after
exercise.
Other Shirts: Series XH
[00190] A 100% cotton blue "Superman" T-shirt, was reported to have perma-
odor by an athletic adult male. The shirt was treated in the left pit only
with 5.6 g
of PNAC4 sprayed onto the pit area, followed by washing with Purex Free
detergent in a fast wash cycle. The owner, not knowing that only the left pit
had
been treated, later reported that there was substantial improvement in the
left pit.
FLUORESCENCE AND DYE TESTING:
[00191] In the photographs discussed in this section, the following numbering
system is used: 200 represents an item of clothing, 202 represents a stain or
darkened spot on a fabric or other visible biofilm candidate, 204 represents a
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fluorescing region, 206 represents a region having diminished fluorescence
following a treatment, 208 denotes a boundary marker for the a treatment zone
(e.g., a rubber band or other object denoting the area to be treated or a
circle
drawn on the figure), and 210 denotes a treatment zone where particular
compounds will be applied to reduce a biofilm or for other objectives. Initial
tests
with dyes explored the use of crystal violet to detect biofilms in textiles.
Unfortunately, even with polyester, the dye was too strongly absorbed to
readily
distinguish biofilm from fibers themselves.
[00192] Three UV lamps were used in testing fluorescence in potential
biofilms.
These included a Lightfe UV301D lamp providing a beam at 365 nm, a UVBeast
V3 lamp operating at 395 nm, and a UV Nova 108-LED UV lamp at 395 nm.
[00193] In one approach, biofilm candidates were stained using Calcofluor
White M2R dye, a fluorescent brightener purchased from Phyto Technology
Laboratories (Shawnee Mission, Kansas), CAS No. 4404-43-7. A solution was
prepared of 0.074% Calcofluor white in water, and given the name CF1.
[00194] In examining fluorescence, it was eventually noted that both an SLR
and an iPhone camera could not easily capture the fine details of fluorescence
when using any of the UV lamps available for this study, probably because the
fluorescence, including some background fluorescence, may have interfered with
the camera's visible light operations. Such images often required enhancement
(increasing contrast to around 30% and decreasing brightness to around -15%,
for
example) to show the fluorescent regions clearly visible to the naked eye,
though
sometimes the enhancement resulted in non-fluorescent zones also appearing as
bright as the fluorescent zones in black and white images. However, that
better
images could be obtained by placing a yellow UV-absorbing lens from UV safety
glasses over the eye of the camera. A rubber band could hold it in place.
Shirt BD1 (Blue Danskin Shirt)
[00195] A blue Danskin shirt, BD1, was sprayed with CF1 on both pits, about
4.8 g each, then immersed in warm water for 1 minute. UV examination revealed
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little fluorescence after rinsing. After removing from immersion, the still
wet shirt
was further treated with 7.5 g CF1 to the right pit and 11.6 g to the left
pit. The
shirt was rinsed again. Fluorescence was still present in portions of the
pits. For
example, FIG. 8A shows fluorescent regions 204 in the right pit where the
optical
brightener adhered, photographed with an iPhone 6 Plus camera using a UV-
absorbing yellow filer over the lens to block out some of the purple
fluorescence of
the shirt itself, while FIG. 8B, which shows a fluorescent region 204 in the
left pit,
was photographed without a filter, making it more difficult to get a
meaningful
image. FIG. 8C shows the fluorescent zone 204 also present in FIG. 8B, but
with
better clarity. BD1 had oblong patches of fluorescence about 2 cm wide and 6
cm
long in the left pit and less in the right pit, apart from the above-mentioned
fluorescent region 204 of FIG. 8B and 8C on the sleeve about 9 cm from the
center of the pit. FIG. 80 is another view of the left pit taken without the
UV filter
and requiring increased contrast to make the fluorescent region 204 more
visible
(it was plainly visible to the naked eye).
[00196] After further exercise in shirt BD1, bringing both puts to an odor
level of
about 3, shirt BD1 was treated with 5.2 g of 2.1% NAC (pH 4) in the right pit,
followed by 2.9 g of E3C. The left pit was untreated. The shirt was washed
with
Purex@ detergent. After washing and drying, the right pit appeared to have an
odor level of 0 while some residual odor remained in the left pit at a level
of about
1 (scale of 1 to 5). The fluorescent zones were slightly decreased in
intensity.
[00197] Further treatments were conducted in BD1. 5.2 g of 2.1% NAC at pH
6.4 was applied to the right pit followed by 3.0 g of E3D. The left pit was
wetted
3.9 g of water followed by 3.28 g of E3D. The shirt was put in a plastic bag
and
kept at about 33 C for 2 hours, then washed in a full cycle at 40 C (78
minutes)
with liquid Tide detergent. Fluorescent zones remained as revealed through
use
of a UV Beast V3 lamp (the default lamp used herein; exceptions with a
different
lamp will be noted), though the intensity may have decreased slightly. Shirt
BD1
was then treated in the right pit only with multiple agents in this order, all
applied
via spray: 2.51 g of 2% NAC at pH 9, 1.54 g of LysoB, 1.26 g of CellulaseC,
1.305
g of AmylaseA, 2.38 g of PAPA, 1.06 g of PNAC5, 1.37 g of PMIX1, and 1.21 g of
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E3D. The short was kept at about 35 C for about one hour, then further treated
with about 0.5 ml of Melaluca brand Lite Brite detergent that was rubbed in
with
warm water as the shirt was then rubbed and immersed into a tub of warm water,
followed by washing with liquid Tide in a short cycle at 30 C. Fluorescent
zones
were somewhat visible after the rinse, but following the wash cycle, the
treated
fluorescent zones were largely removed in the right pit.
[00198] The untreated left pit still retained a fluorescent spot at the
outside of a
yellowish region in the blue shirt that did not fluoresce. The left pit was
now
treated. Focused on the yellow zone and the adjacent fluorescent patch, 2.89 g
of
PAPA and 2.11 g of E3D were applied and rubbed into the treated area. The
shirt
was then rolled up and outer layers were wetted with about 50 ml of water. The
shirt was placed in a bowl set in a metal pan with about 5 cm deep of hot
water in
the bottom, intended to help heat the environment and keep the shirt at a
relatively stable temperature with a lid over the contents. The shirt in this
environment was initially at a temperature of about 48 C to 40 C for the
initial
hour or so, followed by reheating about 2 hours later. The shirt stayed in the
container overnight, with heating again in the morning bringing the
temperature to
about 40 C. Shirt BD1 was then washed with Tide liquid detergent in a short
cycle and then the left pit area was visualized in UV light. The fluorescent
region
was still present, though perhaps slightly weaker. In visible light, it was
apparent
that the previously noted yellow region was reduced in size and intensity.
[00199] In hopes of repeating the removal of the white fluorescence that was
seen in the right pit, the left pit was then further treated with a similar
mix to the
previous mix given to the right pit. In this order, the applied compounds were
3.5 g
of NAC 2.1% at pH 9, 1.32 g of LysoB and 0.50 g of LysoB2, 1.3 g of
CellulaseC,
1.18 g of AmylaseA, 1.58 g of PAPA, 1.0 g of PNAC5, 1.16 g of PM IX1, and 1.6
g
of E3D rubbed into the fabric. This was stored in a covered pan with a bowl
with
some hot water inside the pan to keep the temperature at about 47 C to 40 C
for
about 70 minutes. About 0.5 g of Lemon Brite dish detergent was then applied
to
the treated region and rubbed into the shirt, then rinsed out in warm water.
The
targeted fluorescent zone was still present, though apparently slightly
weaker,
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while the yellowish zone had been largely removed. The result for the treated
left
pit is shown in FIG. 8E showing the fluorescent region 204. The shirt was then
washed in a full cycle with Tide liquid detergent at 40 C. FIG. 8F shows the
result for the treated left pit. These results in FIGS. 8E and 8F still show
the
fluorescent patch 204, but its intensity to the naked eye was significantly
reduced.
Fluorescent Testing with a Gray Perma-Odor Sports Shirt
[00200] An athletic female who exercises almost daily reported that her
polyester sports top showed symptoms of perm a-odor following extensive use.
This shirt, code named RA1, was a 100% polyester gray Melange Jersey knit
shirt
from Academy, Ltd. (Katy, Texas), made in Kenya. It exhibited strong
fluorescence as is in both pits, with no need to treat with Calcofluor. FIG.
9A
shows a photo of the right pit under UV light, and FIG. 9B shows the left pit
under
UV light. After turning the shirt inside out, FIG. 9C shows a close-up of the
right pit
under UV light without a UV filter over the camera lens. The result with the
filter in
place is shown in FIG. 90 for the right pit for comparison, and for the left
pit in
FIG. 9E. Fluorescence is visible on the inside and outside. A close-up of the
left
pit, right-side out, is shown in FIG. 9F.
[00201] It is believed that biofilm formation in the pits had created regions
capable of firmly retaining optical brightening agents from typical laundry
detergents. The shape, size, and position of the fluorescent zones were
entirely
consistent with biofilm regions formed by bacteria interacting with sweat from
the
armpit of an active exerciser. The fluorescent zones includes cuff regions of
the
short sleeves near the pits and occupied the center of the pits but centered
slightly away from the center of the pit, shifted slightly toward the front of
the body,
consistent with the a slight forward lean during jogging or other exercise
that
would allow the sweat to be inclined toward the front of the body. The
intensity of
the fluorescence was relatively high and seemed unaffected by ordinary
washing.
Several treatments were attempted to find ways to reduce the fluorescent
zones.
[00202] Shirt RA1 was then treated with E3D, 9.2 g in the right pit and the
left pit
first treated with 2.1% NAC, 4.54 g, then 10.7 g of E3D. The initially dry
shirt was
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then misted with about 40 g of water and placed in a plastic bag and kept at
about
33 C for 2 hours, then washed in a short cycle at 30 C with Purex Dirt Lift
Action Free and Clear detergent. After washing, the shirt was examined under
UV light it was noted that the central region of the left pit showed
significant
reduction in fluorescence, giving a donut-shaped ring of fluorescence with a
central void about 5 cm in diameter and an outer diameter of about 12 cm in
diameter. See FIG. 9G, which was taken without the UV filter in place. The
region
with diminished fluorescent 208 is adjacent the more highly fluorescent region
204. The diminished fluorescence is clearer to the naked eye than in images
converted to black and white. The right pit (not shown) still appeared bright
and
lacked the reduction seen in the left, but it may have been slightly reduced
also.
[00203] A second treatment was applied to the left pit of RA1 as 14 g of PNAC4
was applied to the pit and surrounding area. The shirt was heated to about 33
C
for 10 minutes. Then E3D was applied to both pits and adjacent areas, 9.6 g
for
the left and 9.9 g for the right. The shirt was them kept warm for about 10
more
minutes then washed again in a short cycle. The washed shirt was exposed to UV
light and a slight reduction in intensity was seen in both pits, but the
glowing
regions persisted with much the same size and shape they had prior to this
wash
cycle. The left pit under UV light is shown in FIG. 9H, taken without a UV
filter.
[00204] The washed and dried shirt was now used to test different treatments
applied to three sections of the major fluorescent area of the left pit, as
shown
under UV light in FIGS. 91, 9J, and 9K, showing upper, middle, and lower
treatment zones 210, each marked by a respective boundary marker 208. The
entire left pit and sleeve area was treated with 6.1 g of NAC spray, 2.1 A at
pH
4.0, followed by individual treatments in the treatment zones.
[00205] In the upper treatment zone shown in FIG. 91, relatively high in the
fluorescent area (toward the shoulder region), the treatment zone 210 (roughly
a
5-cm diameter circle) was treated with amylase solution, but was first sprayed
with 1.76 g of 2% NAC at pH 9.17 to raise the pH to about 7, confirmed with pH
testing paper, and dropwise application by pipette of 2.6 g of AmylaseA
solution.
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[00206] The lower treatment zone shown in FIG. 9K, received 2.45 g of PMIX1
dropwise by pipette to apply a mix of pectinases to a roughly 5 cm diameter
circle
in a lower fluorescent zone in the pit. FIG. 9K shows the boundary marker 208
(here a rubber band) marking the upper treatment zone 210 for pectinase.
Treatment zone 210 contains portions of the fluorescent zone 204 in the left
pit.
[00207] In FIG. 9J, the middle treatment zone 210 between the above-
mentioned two zones, received treatment with papain and lysozyme. 1.7 ml of
LysoPap solution was pipetted onto the oval treatment zone 210 about 5 cm wide
and 3 cm tall. Then the right pit was prepared by spraying about 2 g of 2.15%
NAC solution at a pH of 6.4 over the lower half of the fluorescent pit zone
(not
shown), then 2.3 ml of CellulaseA solution was applied by pipette to that
region.
Then 1.0 g of E3D was sprayed onto the treated area of the right pit and 1.6 g
of
E3D was sprayed into the treated regions of the left pit. After 10 minutes,
the shirt
was rinsed in 1 liter of hot water (about 45 C) containing 1 g of Lemon Brite
dish
detergent. The shirt was gently agitated by hand for about 1 minute and
rinsed.
[00208] The treated regions were then rinsed in warm water and wrung to
partial dryness. It was observed that the region of the left pit that had been
treated
with LysoPap solution (lysozyme and papain), the middle zone of FIG. 9J, had
reduced fluorescence. The other regions looked much the same as before, though
may have had slightly reduced intensity. The lower left pit was now treated
with
0.76 g of 2.15% NAC solution at pH 6.4, and then 1.1 ml of PAPB solution were
applied via pipette, plus 1.0 ml of AmylaseA, all in a 5-cm diameter circles
over a
fluorescent zone. After 5 minutes, another 0.3 ml of PAPB solution was applied
followed by 0.1 g of Lemon Brite dish detergent in the targeted spot, which
was
then rubbed in. The fluorescence seemed only slightly attenuated in both
cases.
[00209] Now two treatment zones in the left pit were considered, both circles
about 5 cm in diameters, as shown with UV light in FIG. 9L, showing a first
treatment zone 210A and a second treatment zone 210B. The first treatment zone
210A was on the seam between the sleeve and the shirt in the pit area, and the
second 210B was centered about 9 cm lower than the seem toward the adjacent
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side of the garment. In the second treatment zone 210B, 1.6 g of 2.15% NAC (pH
6.4) was applied, then 1.856 g of LysoA solution was applied by pipette
uniformly
in the section. In the first treatment zone 210A, 1.7 g of LysoA solution and
then
0.75 ml of PAPB were applied by pipette, followed by spraying 1.0 g of E3D.
The
shirt after these applications beut before washing is shown in FIG. 9M, taken
with
the UV filter About 3 minutes later, 0.4 gm of Lemon Brite dish detergent was
applied roughly uniformly to the two sections and rubbed in. The shirt was
then
rinsed in warm water. Reduced fluorescence was observed in the second
treatment zone 210B of the left pit. See FIG. 9N, taken with the UV filter.
[00210] Now 2.2 g of 2.15% NAC was applied to the lower half of the
fluorescent zone in the left pit, and 1.3 g of LysoA solution was the glowing
portions on the cuff of the sleeve, with 1.6 g of 2.1% NAC at pH 6.4 and 0.3 g
of
Lemon Brite detergent over the sections. In the lower part of the left pit
area, 2.3
g of LysoB were applied, with 1.67 g of PAPB and 2.58 g of CellulaseB, using a
pipette. The shirt was kept at 32 C for about 10 minutes in a plastic bag. The
bag
was then removed and the treated region was further provided with 7.7 g of
PNAC5 (fluorescing regions: the lower left pit and the left sleeve cuff
region).To
raise the pH, a solution at pH 9.89 was prepared from 1.56 g sodium carbonate
and 1.0 g NaHCO3 in 91 ml of water. 13 g of this solution were applied to the
treated regions and the shirt was returned to the plastic bag and kept at
about
32 C for several hours. After rinsing, fluorescence was slightly less but the
dimensions of the fluorescing areas were substantially the same.
[00211] The left pit was treated again with 3.76 g of 2.15% NAC at pH 6.4
applied to the lower half of the left pit along with 2.16 g of 2.15% NAC at pH
9.17.
The upper half of the left pit was sprayed with 5 g of water. Then 5.6 g of
CellulaseC solution was applied dropwise to both the upper and lower left pit,
along with 1.94 g of AmylaseA. The shirt was kept at about 28 C for 30
minutes,
then rinsed with a 1`)/0 solution of Comfort brand laundry detergent.
Residual
fluorescence was visible, but declined more strongly in the lower half of the
pit.
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[00212] In a further test, 12 g of CellAmylA was applied by pipette to the
entire
fluorescing area of the lift pit and sleeve. After 5 minutes, 2.5 g of E3D was
applied to the fluorescing areas of the left side. Then 1.4 g of 2%NAC at pH
6.4
and 2.25 g of PAPA solution was applied by pipette to the upper portion of the
glowing zone, as shown in FIG. 90 under UV light and with the UV filter in
place,
also showing the state of fluorescence in the left pit with significant
decline
compared to early states and a prominent central region of diminished
fluorescence 206, more visible in color to the naked eye. The shirt was put in
a
bag and held at about 33 for 1 hour. Slightly reduced fluorescence was
observed.
[00213] In one series, the effect of high sodium citrate concentration was
tested.
Based on a speculative hypothesis regarding high ionic strength and citrate
ions
in particular, a 12% solution of sodium citrate was prepared and 7 g was
applied
dropwise to the remaining fluorescent zone of the left pit and to a previously
untreated fluorescent spot near the center of the shirt several inches below
the
neckline, followed by spraying 1.72 g of E3D over the treated regions. This
was
kept at about 30 C for 3 hours, then rubbed with 0.3 g Lemon Brite detergent
and
rinsed in warm water. Water was then wrung out by wringing the shirt rolled up
in
a dry towel, and the treated area was visualized. The previously treated pits
showed only very slight improvement at best, but the previously untreated
fluorescent spot had been significantly reduced in brightness and appeared to
be
slightly smaller in extent. A reddish fluorescent zone that was adjacent the
more
central blue fluorescent zone, possibly from a newly incubated biofilm during
a
period of illness in which the shirt was worn, also showed significant
reduction.
[00214] The right pit, whose fluorescence was much brighter than the left pit
since the right pit had received relatively fewer biofilm busting treatments,
was
now treated. Two zones in the right pit were defined, a lower and an upper,
both
with strong fluorescence and both about 5 cm in diameter. Each received about
2.3 g of the 12% citrate solution. The upper received no further treatment
agents,
while the lower zone was sprayed with 1.86 g of E3D. The shirt was then
incubated at about 40-45 C for two hours, and then washed and rinsed as
described for the left pit above. Examination in UV light showed only little
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reduction in fluorescence. The benefit of the citrate treatment may be most
useful
for biofilms that have not been treated multiple times, though it is also
believed to
have an impact in disrupting living bacteria to hinder further biofilm
formation.
[00215] In another test, a fluorescent zone on the cuff of the left arm
closest to
the left pit was treated with nattokinase and NAC by applying 2 g of the
NattoNAC
solution, waiting 5 minutes, and then applying 1.0 g of E4D solution. This was
kept at 21-22 C for about 4 hours, with about 0.7 g of moisture added again
after
4 hours to keep the cuff moist to best permit bacterial spores to be
effective.
The Triathlon Shirt, TR1
[00216] The triathlon shirt TR1 was examined. Since initial treatments, it had
been worn many days without washing and the pits had a cheesy smell. CF1 was
applied to both pits, 6.48 g to the left and 4.45 g to the right. In a test
spot
elsewhere on TR1, it was observed that the Calcofluor White dye persisted
after
rinsing with water, but could be substantially removed with the aid of a
surfactant.
A laundry cycle (short cycle, 38 minutes at 30 C) was run with shirt TR1 and
Dec1. Based on UV visualization, the optical brightener washed out of TR1
except
in the pits, suggesting strong attachment, perhaps due to biofilm material.
[00217] FIGS. 10A and 10B show pits of shirt TR1 under UV light from the
Lightfe mini-UV lamp with the UV filter in place. This image was taken after
the
initial perma-odor problem had been overcome with biofilm buster treatments
previously described. Since that time, it had been worn during roughly a dozen
exercise sessions and many hours of wear to try to revive the biofilm problem.
UV
visualization shows what may be biofilm material.
Artificial Sweat Trials
[00218] Several shirts were treated with an artificial sweat composition,
bringing
together several approaches in past research. Human sweat comprises a variety
of lipids such as squalene, urea, cholesterol, and several fatty acids,
proteins and
amino acids, sugars, salts, and other agents. Initial efforts involved a
concentrate
with many components 2 or more times more abundant than occurs in sweat, but
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effort to grow biofilm with the concentrate were poor, perhaps because the
sodium
level was too high. Then a non-concentrated mixture was prepared, labeled AS-
B.
A 400-ml batch was prepared. An aqueous phase was first prepared with 1.50 g
NaCI, 1.3 g lactic acid, 0.6 g urea, 1.2 g glycerin, 1.2 g of peptone (a
source of
amino acids), 0.25 g of a Centrum@ MultiGummies vitamin pill for adults (a
product of Pfizer, New York, NY) to provide some of the vitamin and mineral
nutrients in sweat, and 2.0 g of honey (Chinese vetch honey) to provide
glucose
and other carbohydrates, and the mixture was blended into 30 ml of water and
then heated to boiling (in part to denature any enzymes in the honey). Then
1.1 g
of squalene was stirred in. The pH was 6.5. An oil phase was then prepared
with
0.65 g of liquid lecitihin, 3.5 g of oleic acid, 0.405 g of palm itic acid,
0.006 g of
ascorbic acid, 0.438 g of stearic acid, 0.008 g of vitamin E, 0.79 g of jojoba
oil, 0.
405 g of cholesterol, which was heated and blended with 8.6 g of the watery
extract of soft Japanese tofu (tofu on cloth in a woven reed basket, through
which
the watery extract drains). This was then blended vigorously with the water-
phase
solution as water was added to reach a total volume of 400 ml and labeled AS-
B.
Several shirts were treated with various doses of AS-B. The triathlon shirt
(TR1)
described above was treated, after the tests already described, with 3.5 g of
AS-B
in each pit and kept at about 27 C for 24 hours. Changes in fluorescence were
difficult to assess; it is unclear if the trial succeeded.
[00219] More visible success came after considering the tenacity and heat
resistance of some microbes in dry biofilms. It was hypothesized that better
visible
biofilm growth in a shirt might occur if the biofilm is allowed to dry, be
remoistened, and dried again several times before finally being washed and
dried.
For that experiment, a men's polyester Walter Hagen brand long-sleeved golf
shirt, black in color, was purchased at a Goodwill store in Appleton,
Wisconsin
that showed fluorescence in both pits under UV light, most strongly in the
right pit,
a shown in FIG. 11A, where a rubber band serves as a boundary marker 208 for
the treatment zone 210 about to be sprayed with artificial sweat spray AS-B.
The
initial fluorescent region 204A is visible in this image taken through a UV
filter by
an iPhone 11 camera. Both pits had been treated with about 3.7 g each of E7D
spray, wrapped in plastic, and allowed to site overnight before being washed
with
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TIDE liquid detergent and tumble dried. The fluorescent zone in the right pit
appeared slightly weaker and was still prominent. Then the AS-B artificial
sweat
solution was applied to the right pit, at the edge of the existing biofilm
area over
an -8 cm square area with about 1.5 to 1.8 g of AS-B applied per application,
and
also applied to a non-fluorescent region at the lower hem directly below the
right
pits with a dose of about 1.2 to 1.5 g per application over about a 6 cm
square
area, with applications occurring 5 times spaced apart by 6 to 12 hours over a
3
day period, and generally maintained around 24 C while wrapped in a plastic
bag,
with added moisture throughout the shirt (about 40 g added moisture) to reduce
wicking and premature drying. After the first 4 treatments, the shirt was
treated
again but allowed to air dry without being wrapped in a plastic bag. Thus, any
biofilm would experience repeated cycles of wetness and dryness, with the goal
of
creating a "hardened" dry biofilm. After the last drying stage, the shirt was
hand-
washed for 10 minutes in 3 liters of warm water with 8 g of added TIDE Simply
Clean laundry detergent, a detergent believed to contain Calcofluor White.
After
rinsing and tumble drying, the shirt 200 was examined in UV light, as shown in
FIG. 11B and in addition to the initial fluorescent region 204A, was seen to
have a
new fluorescent region 204B that may represent the growth of new biofilm
material capable of absorbing optical brightener. Here the approximate
treatment
area 210 is shown as a circle drawn on the image serving as a boundary marker
208. The new fluorescent region 204B had developed outward and over the
rightmost edge of the initial fluorescent region 204A, with the new
fluorescent
region 204B displaying stronger fluorescence than the original biofilm region
204A. However, the region that had been sprayed with AS-B on the hem, where
no biofilm had been established before, did not show visible biofilm growth
when
observed in UV light. Thus, without wishing to be bound by theory, at least in
this
case it is believed that rapid biofilm growth with the AS-B artificial sweat
mixture
and perhaps with real sweat may be most successful when an active biofilm is
already present that can rapidly exploit the nutrients provided in the
artificial sweat
to grow and expand in size.
Confocal and Fluorescent Microscopy
[00220] To further examine potential biofilm zones in various clothing items
with
a history of perma-odor, work was carried out with confocal and fluorescent
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microscopy at the NanoCenter at the University of Minnesota using a Nikon C2
Confocal microscope operated with Nikon Elements software. The microscope
functions as a manual inverted microscope, a fluorescence-enabled microscope,
or a confocal microscope system, depending on preference. Further details are
provided at http://apps.mnc.umn.edu/pub/pdf/equipment/nikon_confocal_sop.pdf.
For observing fluorescent regions in shirts, rather than cutting and mounting
samples, the shirts were preserved by measuring them in situ while stretched
across the measurement space. For confocal microscopy, the UV laser at 405 nm
wavelength was used, while for fluorescent microscopy, the UV fluorescence was
observed from a widefield white light observed through a DAPI filter cube was
used to see the resulting blue fluorescence (this filters the light to an
excitation
band of 340-380nm, and then filters the emission band to 435-485nm). In both
cases, no dyes were added to the material, but the inherent fluorescence in
the
clothing, believed to be due to optical brighteners, was relied on.
[00221] FIGS. 12A and 12B show the left and right pits in UV light,
respectively,
of a lace dress provided by a subject who complained of persistent odor that
would not wash out from the dress. The washed dress 200 was examined under
UV light and prominent fluorescent zones 204 in the arm pit areas were
observed,
with typical characteristics: the were biased toward the front of the body and
in the
pits, consistent with typical sweat patterns in clothing. The fluorescent
zones 204
were examined with both fluorescent and confocal microscopy (discussed below).
The pits of the dress were then treated. The right pit was treated with 0.7 g
of E6C
with added moisture present in that portion of the dress resulting in roughly
a 2:1
dilution. For the left pit, a pretreatment was applied by spraying 2 g of
PANNAC
onto the biofilm region and adjacent fabric. After 5 minutes, 1.54 g of E6D
was
applied to the same region. With moisture present in the dress, it is
estimated that
the effective dilution of the E6C solution was about 3:1. The dress was
wrapped in
a plastic bag and kept at about 27 C for 11 hours, then washed by hand in warm
water with GAIN detergent. Surprisingly, as shown in FIGS. 13A and 13B for
the
left and right pits, respectively, in UV light after treatment, the
fluorescent matter in
both pits was almost completely eliminated, with only a small region (about 2%
of
the original extent) fluorescing in the left pit, but nothing visible in the
right pit.
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[00222] FIGS. 14A and 14B show before and after confocal microscopy views
of what was the original fluorescent region in the left pit, using the same
settings
for laser intensity, dwell time, and other settings of the microscope with the
UV
laser. FIG. 14A shows the fluorescent matter prior to the bioenzymatic
treatment,
showing the presence of fibrous yarns 220 with numerous fluorescent "islands"
222 on the fibers, and occasionally larger patches 224 where fluorescent
matter
seems to bridge multiple fibers. What was the fluorescent region is again
shown
after the bioenzymatic treatment in FIG. 14B, where a few lone islands 222 of
fluorescence remain, but overall showing much less presence of apparent
biofilm
matter. The bioenzymatic spray was effective, but was somewhat more effective
when coupled with the NAC plus panthenol mixture, the PANNAC spray.
REMARKS
[00223] Having described aspects of the invention in detail, it will be
apparent
that modifications and variations are possible without departing from the
scope of
aspects of the invention as defined in the appended claims. As various changes
could be made in the above compositions, products, and methods without
departing from the scope of aspects of the invention, it is intended that all
matter
contained in the description be interpreted as illustrative, not in a limiting
sense.
[00224] Portions of this work were conducted in the Minnesota Nano Center,
which is supported by the National Science Foundation through the National
Nano
Coordinated Infrastructure Network under Award Number ECCS-1542202.
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